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Oncoprotein DJ-1 interacts with mTOR complexes to effect transcription factor Hif1α-dependent expression of collagen I (α2) during renal fibrosis

Open AccessPublished:July 10, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102246
      Proximal tubular epithelial cells respond to transforming growth factor β (TGFβ) to synthesize collagen I (α2) during renal fibrosis. The oncoprotein DJ-1 has previously been shown to promote tumorigenesis and prevent apoptosis of dopaminergic neurons; however, its role in fibrosis signaling is unclear. Here, we show TGFβ-stimulation increased expression of DJ-1, which promoted noncanonical mTORC1 and mTORC2 activities. We show DJ-1 augmented the phosphorylation/activation of PKCβII, a direct substrate of mTORC2. In addition, coimmunoprecipitation experiments revealed association of DJ-1 with Raptor and Rictor, exclusive subunits of mTORC1 and mTORC2, respectively, as well as with mTOR kinase. Interestingly, siRNAs against DJ-1 blocked TGFβ-stimulated expression of collagen I (α2), while expression of DJ-1 increased expression of this protein. In addition, expression of dominant negative PKCβII and siRNAs against PKCβII significantly inhibited TGFβ-induced collagen I (α2) expression. In fact, constitutively active PKCβII abrogated the effect of siRNAs against DJ-1, suggesting a role of PKCβII downstream of this oncoprotein. Moreover, we demonstrate expression of collagen I (α2) stimulated by DJ-1 and its target PKCβII is dependent on the transcription factor hypoxia-inducible factor 1α (Hif1α). Finally, we show in the renal cortex of diabetic rats that increased TGFβ was associated with enhanced expression of DJ-1 and activation of mTOR and PKCβII, concomitant with increased Hif1α and collagen I (α2). Overall, we identified that DJ-1 affects TGFβ-induced expression of collagen I (α2) via an mTOR-, PKCβII-, and Hif1α-dependent mechanism to regulate renal fibrosis.

      Keywords

      Abbreviations:

      CKD (chronic kidney disease), Hif1α (hypoxia-inducible factor 1α), IP (immunoprecipitation), PDGF (platelet-derived growth factor), TGFβ (transforming growth factor β), UUO (unilateral ureteral obstruction)
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      ).We have established the role of mTOR in renal cell hypertrophy and matrix protein expansion, two pathological features associated with renal fibrosis (
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      ).
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      ). These groups of enzymes fall in the AGC superfamily of protein kinases and are classified into three subfamilies, classical (α, β1, βII, and γ), novel (δ, ε, η, and θ), and atypical (ζ and λ/ι). The role of different PKC isoforms has been extensively studied in renal fibrosis in the context of diabetic kidney disease (
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      ). Although various isoforms have been shown to be activated in renal cells by the fibrotic stimuli such as hyperglycemia, PKCβII plays an important role (
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      ).
      DJ-1 was identified as a ras-cooperating oncogene (
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      ). Later, homozygous deletion and missense mutations were found in DJ-1 gene, which cause aggregation of the protein resulting in early onset of autosomal recessive Parkinson’s disease (
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      ). DJ-1 is a ubiquitously expressed homodimeric protein, which shows significant structural similarity with the bacterial protease Pfp1/PH1704. However, due to an occluded and distorted catalytic site, DJ-1 does not possess any protease activity (
      • Kahle P.J.
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      ). In contrast, DJ-1 has weak glyoxalase II activity to detoxify reactive carbonyl species (
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      Human DJ-1 and its homologs are novel glyoxalases.
      ). More recently, it has been shown to have deglycase activity to repair glycation damage in proteins and nucleic acids (
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      Parkinsonism-associated protein DJ-1/Park7 is a major protein deglycase that repairs methylglyoxal- and glyoxal-glycated cysteine, arginine, and lysine residues.
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      ). High sensitivity of its Cys-106 residue for oxidation protects the neurons from oxidative stress. Thus, it serves as an antiapoptotic protein in the neurons of Parkinson’s disease patients although excessive oxidation of Cys-106 render this protein inactive (
      • Canet-Aviles R.M.
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      ,
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      ). Apart from its function in neuronal cells, its role in spermatogenesis and fertilization, where it cooperates with androgen receptor, have been reported (
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      • Iwamoto T.
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      ). As DJ-1 was originally discovered as an oncogene and since oncogene-mediated biological activities include activated serine/threonine kinases that lead to proliferation of cells during tumorigenesis, it is possible that DJ-1 may interact with the oncogenic kinases. Also, mTOR acts as an oncogenic kinase to drive tumorigenesis (
      • Saxton R.A.
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      mTOR signaling in growth, metabolism, and disease.
      ,
      • Zoncu R.
      • Efeyan A.
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      • Fantus D.
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      • Huber T.B.
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      Roles of mTOR complexes in the kidney: Implications for renal disease and transplantation.
      ). In fact, we and others have shown previously that mTOR contributes to the pathogenic function of TGFβ in renal cells (
      • Das F.
      • Ghosh-Choudhury N.
      • Bera A.
      • Dey N.
      • Abboud H.E.
      • Kasinath B.S.
      • et al.
      Transforming growth factor beta integrates Smad 3 to mechanistic target of rapamycin complexes to arrest deptor abundance for glomerular mesangial cell hypertrophy.
      ,
      • Maity S.
      • Das F.
      • Kasinath B.S.
      • Ghosh-Choudhury N.
      • Ghosh Choudhury G.
      TGFbeta acts through PDGFRbeta to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression.
      ,
      • Das F.
      • Ghosh-Choudhury N.
      • Mahimainathan L.
      • Venkatesan B.
      • Feliers D.
      • Riley D.J.
      • et al.
      Raptor-rictor axis in TGFbeta-induced protein synthesis.
      ,
      • Dey N.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Choudhury G.G.
      TGFbeta-stimulated microRNA-21 utilizes PTEN to orchestrate AKT/mTORC1 signaling for mesangial cell hypertrophy and matrix expansion.
      ). The direct role of DJ-1 in activation of this kinase to affect downstream signaling for renal fibrosis has not been investigated. In the present study, we examined how TGFβ activates mTOR via DJ-1. Also, we determined the role of DJ-1 in activation of PKCβII downstream of mTORC2 in mediating the expression of fibrotic protein collagen I (α2).

      Results

      TGFβ increases the expression of DJ-1

      Renal proximal tubular epithelial cells respond to TGFβ to drive fibrosis in the kidney. Although DJ-1 is a ubiquitous protein, its expression in brain and cancer tissues have been mainly studied in association with early-onset Parkinson’s disease and malignancy (
      • Bonifati V.
      • Rizzu P.
      • van Baren M.J.
      • Schaap O.
      • Breedveld G.J.
      • Krieger E.
      • et al.
      Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism.
      ,
      • Kim R.H.
      • Peters M.
      • Jang Y.
      • Shi W.
      • Pintilie M.
      • Fletcher G.C.
      • et al.
      DJ-1, a novel regulator of the tumor suppressor PTEN.
      ). To initiate a systematic study on the role of DJ-1 in renal fibrosis, human proximal tubular epithelial cells were exposed to TGFβ. Immunoblot analysis of the lysates revealed a time-dependent increase in the expression of DJ-1 protein (Fig. 1A and Fig. S1A). Similarly, expression of DJ-1 mRNA was increased in response to TGFβ, suggesting DJ-1 expression may be regulated at the transcriptional level (Fig. 1B). Further immunoblot analysis revealed that TGFβ significantly stimulated the expression of DJ-1 protein in a sustained manner till 96 h (Fig. S1, B and C). Since TGFβ transmits signal via its type I receptor–mediated phosphorylation of Smad 3, we tested the effect of pharmacologic blockade of TGFβ receptor I by SB 431542 (SB). SB blocked TGFβ-stimulated DJ-1 expression along with inhibition of Smad 3 phosphorylation (Fig. 1C, Fig. S1D and Fig. 1D, Fig. S1E). To test whether Smad 3 regulates DJ-1 expression, proximal tubular epithelial cells were transfected with siRNAs against Smad 3 prior to exposure to TGFβ. Downregulation of Smad 3 abrogated TGFβ-induced DJ-1 expression (Fig. 1E and Fig. S1F). To confirm these observations in human proximal tubular epithelial cells, we performed experiments in mouse proximal tubular epithelial cells. TGFβ significantly increased DJ-1 in mouse proximal tubular epithelial cells in a sustained manner till 96 h (Fig. S2, A–D). Similar to the results in human proximal tubular epithelial cells, SB, which inhibits Smad 3 phosphorylation, and siSmad 3 blocked TGFβ-induced DJ-1 expression (Fig. S2, E–J). These results demonstrate involvement of the canonical TGFβ receptor I signaling for increased DJ-1 expression.
      Figure thumbnail gr1
      Figure 1TGFβ increased DJ-1 expression in Smad 3–dependent manner. A and B, serum-starved human proximal tubular epithelial cells were incubated with 2 ng/ml TGFβ for the indicated periods. C and D, cells were treated with 5 μM SB for 1 h prior to incubation with 2 ng/ml TGFβ for 24 h. E, cells were transfected with siRNAs against Smad 3 or scrambled RNA prior to incubation with 2 ng/ml TGFβ for 24 h. In panels (A) and (C–E), cell lysates were immunoblotted with indicated antibodies to determine the expression of each protein. Molecular weight markers are shown on the left margins. Representative blots from three independent experiments are shown. Quantification and significance of these data are shown in , A and D–F. In panel (B), expression of DJ-1 mRNA was determined. Total RNAs were prepared and used for real tie RT-PCR to detect DJ-1 and GAPDH mRNAs as described in the Experimental procedures. Mean ± SD of triplicate measurements is shown. ∗p < 0.0002 versus 0 h. TGFβ, transforming growth factor β.

      DJ-1 regulates TGFβ-stimulated mTORC1 and mTORC2 activities

      We and others have shown a role of mTOR in renal fibrosis (
      • Maity S.
      • Das F.
      • Kasinath B.S.
      • Ghosh-Choudhury N.
      • Ghosh Choudhury G.
      TGFbeta acts through PDGFRbeta to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression.
      ,
      • Das F.
      • Ghosh-Choudhury N.
      • Mahimainathan L.
      • Venkatesan B.
      • Feliers D.
      • Riley D.J.
      • et al.
      Raptor-rictor axis in TGFbeta-induced protein synthesis.
      ,
      • Eid A.A.
      • Ford B.M.
      • Bhandary B.
      • Cavagliery R.
      • Block K.
      • Barnes J.L.
      • et al.
      Mammalian target of rapamycin regulates Nox4-mediated podocyte Depletion in diabetic renal injury.
      ,
      • Inoki K.
      Role of TSC-mTOR pathway in diabetic nephropathy.
      ,
      • Grahammer F.
      • Wanner N.
      • Huber T.B.
      mTOR controls kidney epithelia in health and disease.
      ). mTOR exists in two complexes with different substrate specificities (
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling in growth control and disease.
      ). Activation of mTORC1 phosphorylates its downstream substrate S6 kinase at Thr-389 and serves as a measure of mTORC1 activation (
      • Saxton R.A.
      • Sabatini D.M.
      mTOR signaling in growth, metabolism, and disease.
      ). TGFβ increased rapid and sustained phosphorylation of S6 kinase initiating at 15 min of stimulation, indicating activation of mTORC1 (Fig. 2A, Fig. S3A and Fig. 2B, Fig. S3B). To test whether DJ-1 regulates mTORC1 activation, we used siRNAs against this protein. Downregulation of DJ-1 blocked TGFβ-stimulated phosphorylation of S6 kinase (Fig. 2C, Fig. S3C). mTORC1-mediated phosphorylation of S6 kinase increases its activity toward rps6 (
      • Saxton R.A.
      • Sabatini D.M.
      mTOR signaling in growth, metabolism, and disease.
      ,
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling in growth control and disease.
      ). TGFβ increased the phosphorylation of rps6, which was inhibited by siRNAs against DJ-1 (Fig. 2D, Fig. S3D). Conversely, when we transfected a vector to express FLAG-tagged DJ-1, it increased the phosphorylation of S6 kinase and its substrate rps6 similar to TGFβ (Fig. 2E, Fig. S3E and Fig. 2F, Fig. S3F).
      Figure thumbnail gr2
      Figure 2DJ-1 regulates TGFβ-stimulated mTORC1 activity. A and B, serum-starved human proximal tubular epithelial cells were incubated with 2 ng/ml TGFβ for indicated periods. C and D, cells were transfected with siRNAs against DJ-1 or scrambled RNA prior to incubation with 2 ng/ml TGFβ for 24 h. E and F, cells were transfected with a vector encoding FLAG-tagged DJ-1 or control vector prior to incubation with 2 ng/ml TGFβ for 24 h. Cell lysates were immunoblotted with the indicated antibodies to determine the expression of each protein. Representative of three independent experiments is shown. Quantification and significance of these data are shown in , A–F. TGFβ, transforming growth factor β.
      mTORC2 phosphorylates Akt and PKCβII at the hydrophobic motif sites Ser-473 and Ser-660, respectively, resulting in their activation (
      • Sarbassov D.D.
      • Guertin D.A.
      • Ali S.M.
      • Sabatini D.M.
      Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.
      ). To determine activation of mTORC2, we examined phosphorylation of these two proteins at these sites. TGFβ rapidly increased the phosphorylation of these two kinases at their mTORC2 phosphorylation sites (Akt Ser-473 and PKCβII Ser-660) starting at 15 min of stimulation (Fig. 3A, Fig. S4A and Fig. 3B, Fig. S4B). Also, TGFβ enhanced the phosphorylation of Akt at Thr-308 (Fig. 3A, Fig. S4A, right panel). Prolonged incubation with TGFβ increased the phosphorylation of Akt and PKCβII in a sustained manner (Fig. 3C, Fig. S4C and Fig. 3D, Fig. S4D). In determining the role of DJ-1 in this process, as shown in Fig. 4, A and B, siRNA-mediated downregulation of DJ-1 inhibited the phosphorylation of Akt and PKCβII, respectively (Fig. S5, A and B). Phosphorylation of Akt and PKCβII at these sites increases their kinase activities (
      • Manning B.D.
      • Toker A.
      AKT/PKB signaling: navigating the Network.
      ,
      • Newton A.C.
      Protein kinase C: Perfectly balanced.
      ). To test their kinase activities, we examined the phosphorylation of their two respective endogenous substrates GSK3β at Ser-9 and myristoylated alanine rich PKC substrate (MARCKS) at Ser-152/156, which elicit many effects of Akt and PKCβII, respectively (
      • Mariappan M.M.
      • Shetty M.
      • Sataranatarajan K.
      • Choudhury G.G.
      • Kasinath B.S.
      Glycogen synthase kinase 3beta is a novel regulator of high glucose- and high insulin-induced extracellular matrix protein synthesis in renal proximal tubular epithelial cells.
      ,
      • Wang Y.
      • Zhou Q.
      • Wu B.
      • Zhou H.
      • Zhang X.
      • Jiang W.
      • et al.
      Propofol induces excessive vasodilation of aortic rings by inhibiting protein kinase Cbeta2 and theta in spontaneously hypertensive rats.
      ,
      • Morash S.C.
      • Douglas D.
      • McMaster C.R.
      • Cook H.W.
      • Byers D.M.
      Expression of MARCKS effector domain mutants alters phospholipase D activity and cytoskeletal morphology of SK-N-MC neuroblastoma cells.
      ). TGFβ significantly increased the phosphorylation of GSK3β and MARCKS (Fig. 4C, Figs. S5C and Fig. 4D and Fig. S5D). Blockade of DJ-1 expression by siRNAs inhibited the TGFβ-induced phosphorylation of GSK3β and MARCKS (Fig. 4C, Fig. S5C and Fig. 4D and Fig. S5D). To confirm these observations, DJ-1 was overexpressed in the proximal tubular epithelial cells. Increased expression of DJ-1 significantly enhanced the phosphorylation of Akt and PKCβII at their hydrophobic motif sites, resulting in phosphorylation of their substrates GSK3β and MARCKS similar to TGFβ (Fig. 4, E–H and Fig. S5, E–H). Together, these results demonstrate that DJ-1 mediates TGFβ-stimulated mTORC1 and mTORC2 activation in proximal tubular epithelial cells.
      Figure thumbnail gr3
      Figure 3TGFβ stimulates mTORC2 activity in human proximal tubular epithelial cells. Serum-starved cells were incubated with 2 ng/ml TGFβ for the indicated periods of time in panels (AD). The cell lysates were immunoblotted with antibodies to detect the indicated proteins. Representative of three independent experiments is shown. Quantification and significance of these data are shown in , A–D. TGFβ, transforming growth factor β.
      Figure thumbnail gr4
      Figure 4DJ-1 mediates TGFβ-stimulated mTORC2 activity. A–D, human proximal tubular epithelial cells were transfected with siRNAs against DJ-1 or scrambled RNA prior to incubation with 2 ng/ml TGFβ for 24 h EH, the cells were transfected with vector-encoding FLAG-tagged DJ-1 or control vector prior to incubation with 2 ng/ml TGFβ for 24 h. The cell lysates were immunoblotted with antibodies to detect the indicated proteins. Representative of 3 to 5 independent experiments is shown. Quantification and significance of these data are shown in A and H. TGFβ, transforming growth factor β.

      DJ-1 forms complex with mTORC1 and mTORC2

      DJ-1 contains multiple domains that can interact with other proteins to modulate their functions (
      • Jin W.
      Novel Insights into PARK7 (DJ-1), a potential anti-cancer therapeutic target, and implications for cancer progression.
      ). Thus, one possible mechanism by which DJ-1 promotes the activities of mTORC1 and mTORC2 is by association with these complexes. To address this hypothesis, proximal tubular epithelial cells were transfected with vectors expressing FLAG DJ-1 and Myc Raptor to examine mTORC1. Immunoprecipitation of the cell lysates with the FLAG antibody followed by immunoblotting with anti-Myc showed association of DJ-1 with raptor (Fig. 5A, Fig. S6A). Reciprocal immunoprecipitation and immunoblotting confirmed complex formation between raptor and DJ-1 (Fig. 5B, Fig. S6B). Similarly, when lysates of cells transfected with FLAG DJ-1 and Myc Rictor were used for FLAG immunoprecipitation followed by anti-Myc immunoblotting, we detected association of rictor with DJ-1 (Fig. 5C, Fig. S6C). Reciprocal immunoprecipitation and immunoblotting confirmed this observation (Fig. 5D, Fig. S6D). These results suggest that association of DJ-1 with both mTOR complexes may regulate their activities.
      Figure thumbnail gr5
      Figure 5DJ-1 association with mTORC1 and mTORC2. A and B, FLAG-tagged DJ-1 and Myc-tagged raptor were cotransfected into human proximal tubular epithelial cells. The cell lysates were immunoprecipitated with antibodies against FLAG (panel A) and Myc (panel B) or nonimmune IgG. The immunoprecipitates were immunoblotted with anti-Myc and anti-FLAG antibodies, respectively. C and D, FLAG-tagged DJ-1 and Myc-tagged rictor were cotransfected into human proximal tubular epithelial cells. The cell lysates were immunoprecipitated with antibodies against FLAG (panel C) and Myc (panel D) or nonimmune IgG. The immunoprecipitates were immunoblotted with anti-Myc and anti-FLAG antibodies, respectively. Bottom panels show immunoblot analysis of each indicated proteins in the cell lysates. Representative of 3 to 4 independent experiments is shown. Quantification and significance of these data are shown in , A–D.

      TGFβ regulates association of DJ-1 with mTORC1 and mTORC2

      Our results demonstrate requirement of DJ-1 in TGFβ-stimulated activation of mTORC1 and mTORC2 (Figs. 2 and 3). Given that DJ-1 interacts with both mTORC1 and mTORC2 (Fig. 5), we next investigated the responsiveness to TGFβ. Proximal tubular epithelial cells were exposed to TGFβ. DJ-1 was immunoprecipitated from the cell lysates and immunoblotted with antibody against mLST8, which is a common subunit for both mTORC1 and mTORC2. Fig. 6A shows increased association of DJ-1 with mLST8 (Fig. S7A). Reciprocal mLST8 immunoprecipitation followed by DJ-1 immunoblotting confirmed increased association of these two proteins (Fig. 6B, Fig. S7B). These results demonstrate that DJ-1 may be incorporated in both mTOR complexes. Next, we confirmed specific association of DJ-1 with mTORC1 by coimmunoprecipitating raptor with DJ-1. The results showed incorporation of DJ-1 into mTORC1 (Fig. 6C, Fig. S7C and Fig. 6D, Fig. S7D). To examine the association of DJ-1 with mTORC2 complex, we used two specific subunits of it, rictor and mSin1. Reciprocal immunoprecipitation of DJ-1 and rictor, and mSin1 showed incorporation of DJ-1 into mTORC2 (Fig. 6, E–H and Fig. S7, E–H).
      Figure thumbnail gr6
      Figure 6TGFβ induces association of DJ-1 with mTORC1 and mTORC2. Human proximal tubular epithelial cells were incubated with 2 ng/ml TGFβ for 24 h. A, C, E, and G, the cell lysates were immunoprecipitated with DJ-1 antibody followed by immunoblotting with antibodies against mLST8 (panel A), raptor (panel C), rictor (panel E), mSin1 (panel G), and DJ-1 antibodies to detect the corresponding proteins. The bottom panels show immunoblot analysis of the indicated proteins in the cell lysates. B, D, F, and H, the cell lysates were immunoprecipitated with antibodies against mLST8 (panel B), raptor (panel D), rictor (panel F), and mSin1 (panel H) followed by immunoblotting with DJ-1 antibody and antibodies against mLST8, raptor, rictor, and mSin1 as indicated. Representative of 3 to 5 independent experiments is shown. Quantification and significance of these data are shown in , A–H. TGFβ, transforming growth factor β.

      DJ-1 regulates mTORC1 and mTORC2 kinase activities

      The observations above indicate complex formation between DJ-1 and mTORC1/mTORC2 in which mTOR is the common kinase subunit. Therefore, we examined association of DJ-1 with mTOR. DJ-1 immunoprecipitates were used for immunoblotting with mTOR antibody. TGFβ significantly increased association of mTOR with DJ-1 (Fig. 7A, Fig. S8A). Complementary experiment using mTOR immunoprecipitates for DJ-1 immunoblotting validated these results (Fig. 7B, Fig. S8B). Next, we determined the role of DJ-1 in both mTORC1 and mTORC2 kinase activities using immunecomplex kinase assays of mTOR immunoprecipitate in vitro. Proximal tubular epithelial cells were transfected with siRNAs against DJ-1 prior to incubation with TGFβ. To assay mTORC1 activity, mTOR immunoprecipitate was used in immunecomplex kinase assay with recombinant S6 kinase as substrate in the presence of ATP. Fig. 7C shows significantly increased S6 kinase phosphorylation at the Thr-389 site by TGFβ (Fig. S8C). Interestingly, DJ-1 siRNAs significantly inhibited the TGFβ-stimulated mTORC1 activity in the mTOR immunoprecipitate (Fig. 7C, Fig. S8C). Similarly, when we used recombinant Akt in mTOR immunecomplex kinase assay, siDJ-1 inhibited the TGFβ-induced phosphorylation of Akt at its mTORC2 hydrophobic motif site Ser-473 (Fig. 7D, Fig. S8D). These results demonstrate that DJ-1 is required for TGFβ-stimulated activities of both mTORC1 and mTORC2.
      Figure thumbnail gr7
      Figure 7TGFβ induces association of DJ-1 with mTOR to increase mTORC1 and mTORC2 activities. A and B, human proximal tubular epithelial cells were incubated with 2 ng/ml TGFβ for 24 h. A, the cell lysates were immunoprecipitated with DJ-1 antibody followed by immunoblotting with antibodies against mTOR and DJ-1. The bottom panels show actin immunoblot. B, the cell lysates were immunoprecipitated with mTOR antibody followed by immunoblotting with DJ-1 and mTOR antibodies, respectively. C and D, immunecomplex kinase assays for mTORC1 and mTORC2. Human proximal tubular epithelial cells were transfected with siRNAs against DJ-1 or scrambled RNA prior to incubation with 2 ng/ml TGFβ for 24 h. The cell lysates were immunoprecipitated with mTOR antibody. In panel (C), the immunoprecipitate was assayed for mTORC1 activity using 100 ng recombinant S6 kinase substrate in the presence of ATP in an immunecomplex kinase assay as described in the Experimental procedures. In panel (D), the mTOR immunoprecipitate was used to assay mTORC2 activity using 100 ng recombinant Akt as substrate. For S6 kinase and Akt blots in these panels, 20 ng recombinant proteins were run in parallel. Bottom panels show immunoblots of the indicated proteins in the cell lysates. Representative of four to five experiments is shown. Quantification and significance of these data are shown in , A–D. TGFβ, transforming growth factor β.

      DJ-1 regulates TGFβ-stimulated collagen I (α2) expression via PKCβII

      Using rapamycin, the role of mTOR especially mTORC1 in the expression of collagen I (α2) and in fibrotic renal diseases is established (
      • Das F.
      • Ghosh-Choudhury N.
      • Bera A.
      • Dey N.
      • Abboud H.E.
      • Kasinath B.S.
      • et al.
      Transforming growth factor beta integrates Smad 3 to mechanistic target of rapamycin complexes to arrest deptor abundance for glomerular mesangial cell hypertrophy.
      ,
      • Das F.
      • Ghosh-Choudhury N.
      • Mahimainathan L.
      • Venkatesan B.
      • Feliers D.
      • Riley D.J.
      • et al.
      Raptor-rictor axis in TGFbeta-induced protein synthesis.
      ,
      • Eid A.A.
      • Ford B.M.
      • Bhandary B.
      • Cavagliery R.
      • Block K.
      • Barnes J.L.
      • et al.
      Mammalian target of rapamycin regulates Nox4-mediated podocyte Depletion in diabetic renal injury.
      ,
      • Mori H.
      • Inoki K.
      • Masutani K.
      • Wakabayashi Y.
      • Komai K.
      • Nakagawa R.
      • et al.
      The mTOR pathway is highly activated in diabetic nephropathy and rapamycin has a strong therapeutic potential.
      ,
      • Lloberas N.
      • Cruzado J.M.
      • Franquesa M.
      • Herrero-Fresneda I.
      • Torras J.
      • Alperovich G.
      • et al.
      Mammalian target of rapamycin pathway blockade slows progression of diabetic kidney disease in rats.
      ,
      • Sakaguchi M.
      • Isono M.
      • Isshiki K.
      • Sugimoto T.
      • Koya D.
      • Kashiwagi A.
      Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice.
      ,
      • Sataranatarajan K.
      • Mariappan M.M.
      • Lee M.J.
      • Feliers D.
      • Choudhury G.G.
      • Barnes J.L.
      • et al.
      Regulation of elongation phase of mRNA translation in diabetic nephropathy: Amelioration by rapamycin.
      ,
      • Rozen-Zvi B.
      • Hayashida T.
      • Hubchak S.C.
      • Hanna C.
      • Platanias L.C.
      • Schnaper H.W.
      TGF-beta/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1alpha expression.
      ). However, it is known that prolonged rapamycin treatment can block mTORC2 (
      • Lamming D.W.
      • Ye L.
      • Katajisto P.
      • Goncalves M.D.
      • Saitoh M.
      • Stevens D.M.
      • et al.
      Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity.
      ,
      • Sarbassov D.D.
      • Ali S.M.
      • Sengupta S.
      • Sheen J.H.
      • Hsu P.P.
      • Bagley A.F.
      • et al.
      Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.
      ), suggesting that a role for mTORC2 in renal disease especially in fibrosis cannot be ruled out. Our results above show that DJ-1 controls the activities of both mTORC1 and mTORC2 (Figs. 2 and 4). Therefore, we examined whether DJ-1 regulates the fibrotic protein collagen I (α2) expression in proximal tubular epithelial cells. DJ-1 siRNAs were transfected prior to exposure of cells with TGFβ. Immunoblot analysis of the cell lysates showed that inhibition of DJ-1 expression blocked TGFβ-stimulated expression of collagen I (α2) (Fig. 8A, Fig. S9A). In contrast to these results, overexpression of DJ-1 was sufficient to increase collagen I (α2) expression similar to TGFβ (Fig. 8B, Fig. S9B). Next, we studied the role of PKCβII, which is phosphorylated and activated by TGFβ via mTORC2, to augment collagen I (α2) expression. Expression of a vector containing kinase-dead mutant of PKCβII (HA-tagged PKCβII K371R) in proximal tubular epithelial cells significantly inhibited the expression of collagen I (α2) in response to TGFβ (Fig. 8C, Fig. S9C). To complement this observation, we used siRNAs against PKCβII. Transfection of these siRNAs blocked the expression of PKCβII in proximal tubular epithelial cells resulting in the inhibition of expression of collagen I (α2) (Fig. 8D, Fig. S9D). However, TGFβ uses Smad 3 to regulate expression of genes (
      • Moustakas A.
      • Heldin C.H.
      The regulation of TGFbeta signal transduction.
      ). siRNAs against Smad 3 inhibited TGFβ-stimulated collagen I (α2) expression (Fig. 9A, Fig. S10A). To examine the existence of a cross talk between Smad 3 and PKCβII, we cotransfected Smad 3 and kinase-dead PKCβII. Expression of kinase-dead PKCβII significantly inhibited Smad 3-induced collagen I (α2) expression (Fig. 9B, Fig. S10B). Similarly, siRNAs against PKCβII blocked Smad-3-stimulated collagen I (α2) (Fig. 9C, Fig. S10C). These results demonstrate that both Smad 3 pathway and PKCβII contribute to the expression of DJ-1 by TGFβ.
      Figure thumbnail gr8
      Figure 8TGFβ-stimulated expression of collagen I (α2) is regulated by DJ-1 and PKCβII. Human proximal tubular epithelial cells were transfected with siRNAs against DJ-1 (panel A) and PKCβII (panel D) and with FLAG-tagged DJ-1 (panel B) and HA-tagged PKCβII K371R (panel C) prior to incubation with 2 ng/ml TGFβ for 24 h. The cell lysates were immunoblotted with antibodies to detect the indicated proteins. Representative of three to four independent experiments is shown. Quantification and significance of these data are shown in , A–D. TGFβ, transforming growth factor β.
      Figure thumbnail gr9
      Figure 9PKCβII cooperates with Smad 3 signaling for TGFβ-stimulated collagen I (α2) expression. A, human proximal tubular epithelial cells were transfected with siRNAs against Smad 3 prior to incubation with 2 ng/ml TGFβ for 24 h. B and C, cells were cotransfected with Smad 3 and PKCβII K371R (panel B) or siRNAs against PKCβII (panel C) prior to incubation with 2 ng/ml TGFβ for 24 h. The cell lysates were immunoblotted with antibodies to detect the indicated proteins. Representative of three independent experiments is shown. Quantification and significance of these data are shown in , A–C. TGFβ, transforming growth factor β.
      We have shown above the regulation of PKCβII by DJ-1 (Fig. 4, B, D, F and H). To examine whether TGFβ-stimulated DJ-1 integrates PKCβII with the expression of collagen I (α2), siRNAs against DJ-1 and a mutant of PKCβII (HA-tagged PKCβII CAT) conferring constitutive catalytic activity were cotransfected into proximal tubular epithelial cells. As shown in Fig. 10A, siDJ-1–induced inhibition of TGFβ-stimulated collagen I (α2) expression was reversed by the expression of catalytically active PKCβII (Fig. S11A). In fact, when a dominant negative PKCβII was expressed, collagen I (α2) expression was inhibited in response to both FLAG DJ-1 alone and FLAG DJ-1 plus TGFβ (Fig. 10B, Fig. S11B and Fig. 10C, Fig. S11C). Similarly, siRNAs against PKCβII blocked FLAG DJ-1– as well as both FLAG DJ-1– and TGFβ-mediated expression of collagen I (α2) (Fig. 10D, Fig. S11D and Fig. 10E, Fig. S11E). Collectively, our results demonstrate a role for PKCβII downstream of DJ-1 in the expression of collagen I (α2) induced by TGFβ.
      Figure thumbnail gr10
      Figure 10TGFβ-stimulated expression of collagen I (α2) is regulated by PKCβII downstream of DJ-1. Human proximal tubular epithelial cells were cotransfected with siDJ-1 plus HA-tagged PKCβII CAT (panel A), FLAG DJ-1 plus HA PKCβII K371R (panels B and C), and FLAG DJ-1 plus siPKCβII (panels D and E). The transfected cells were incubated with 2 ng/ml TGFβ for 24 h (panels A, C, and E). The cell lysates were immunoblotted with antibodies to detect the indicated proteins. Representative of three independent experiments is shown. Quantification and significance of these data are shown in , A–E. TGFβ, transforming growth factor β.

      DJ-1 regulates Hif1α via PKCβII to upregulate collagen I (α2) expression

      A role for Hif1α to drive renal fibrosis is established in CKD in which TGFβ is a major participant (
      • Liu J.
      • Wei Q.
      • Guo C.
      • Dong G.
      • Liu Y.
      • Tang C.
      • et al.
      Hypoxia, HIF, and associated signaling Networks in chronic kidney disease.
      ,
      • Kimura K.
      • Iwano M.
      • Higgins D.F.
      • Yamaguchi Y.
      • Nakatani K.
      • Harada K.
      • et al.
      Stable expression of HIF-1alpha in tubular epithelial cells promotes interstitial fibrosis.
      ). TGFβ increases expression of Hif1α to induce many fibrotic genes including collagen I (α2) (
      • Liu J.
      • Wei Q.
      • Guo C.
      • Dong G.
      • Liu Y.
      • Tang C.
      • et al.
      Hypoxia, HIF, and associated signaling Networks in chronic kidney disease.
      ,
      • Baumann B.
      • Hayashida T.
      • Liang X.
      • Schnaper H.W.
      Hypoxia-inducible factor-1alpha promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3.
      ). Although TGFβ-stimulated canonical Smad 3 regulates expression of collagen I (α2), recently, a role of Hif1α has been shown in normoxic renal cells (
      • Runyan C.E.
      • Schnaper H.W.
      • Poncelet A.C.
      The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1.
      ,
      • Poncelet A.C.
      • de Caestecker M.P.
      • Schnaper H.W.
      The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells.
      ,
      • Basu R.K.
      • Hubchak S.
      • Hayashida T.
      • Runyan C.E.
      • Schumacker P.T.
      • Schnaper H.W.
      Interdependence of HIF-1alpha and TGF-beta/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression.
      ). Furthermore, rapamycin inhibited TGFβ-stimulated Hif1α-mediated increase in collagen I (α2), suggesting involvement of mTORC1 (
      • Rozen-Zvi B.
      • Hayashida T.
      • Hubchak S.C.
      • Hanna C.
      • Platanias L.C.
      • Schnaper H.W.
      TGF-beta/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1alpha expression.
      ). We examined the role of mTORC2 in this process. Downregulation of rictor, which regulates mTORC2 activity (
      • Sarbassov D.D.
      • Ali S.M.
      • Kim D.H.
      • Guertin D.A.
      • Latek R.R.
      • Erdjument-Bromage H.
      • et al.
      Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton.
      ), by two independent shRNAs, significantly inhibited TGFβ-stimulated expression of Hif1α (Fig. 11A and B, Fig. S12, A and B). We have shown above that DJ-1 regulates mTORC2. Therefore, we examined whether DJ-1 regulates Hif1α expression in proximal tubular epithelial cells. siRNAs against DJ-1 significantly impaired TGFβ-induced expression of Hif1α (Fig. 11C, Fig. S12C). In addition, overexpression of DJ-1 enhanced the level of Hif1α similar to that induced by TGFβ (Fig. 11D, Fig. S12D). Since we have demonstrated activation of PKCβII downstream of mTORC2, we determined its role in Hif1α expression. Dominant negative PKCβII inhibited Hif1α expression in response to TGFβ (Fig. 11E, Fig. S12E). Similarly, siRNAs against PKCβII blocked the expression of Hif1α (Fig. 11F, Fig. S12F). Further, expression of constitutively active PKCβII increased Hif1α similar to TGFβ (Fig. 11G, Fig. S12G). These results demonstrate independent involvement of DJ-1 and PKCβII in the expression of Hif1α.
      Figure thumbnail gr11
      Figure 11Rictor as well as DJ-1 and PKCβII regulate TGFβ-stimulated Hif1α expression. Human proximal tubular epithelial cells were transfected with two independent shRNAs against rictor (panels A and B), siDJ-1 (panel C), FLAG DJ-1 (panel D), HA-tagged PKCβII K371R (panel E), siPKCβII (panel F), and HA-tagged PKCβII CAT (panel G). The transfected cells were incubated with 2 ng/ml TGFβ for 24 h. The cell lysates were immunoblotted with antibodies to detect the indicated proteins. Representative of three independent experiments is shown. Quantification and significance of these data are shown in , A–G. TGFβ, transforming growth factor β; Hif1α, hypoxia-inducible factor 1α.
      Since mTORC2 regulates PKCβII and expression of Hif1α, we examined the involvement of mTORC2 in the expression of collagen I (α2). Expression of two independent shRNAs against rictor blocked TGFβ-stimulated collagen I (α2) expression (Fig. 12, A and B, Fig. S13, A and B). Next, to probe further whether Hif1α has any connection with DJ-1 in expression of collagen I (α2), siRNAs against DJ-1 and a vector-expressing Hif1α were cotransfected into proximal tubular epithelial cells. Results showed overexpression of Hif1α reversed the siDJ-1–mediated inhibition of TGFβ-stimulated collagen I (α2) expression (Fig. 12C, Fig. S13C). To specifically examine the role of DJ-1, FLAG DJ-1 and siRNAs against Hif1α were cotransfected. DJ-1 was expressed alone and, in the presence of TGFβ, siHif1α inhibited the expression of collagen I (α2) (Fig. 12, D and E, Fig. S13, D and E). These results are consistent with the involvement of Hif1α in the expression of collagen I (α2) downstream of DJ-1. Given the role of PKCβII in the expression of Hif1α, and that these molecules are activated in renal fibrosis, it is important to investigate whether there is any link between them in the expression of collagen I (α2). For these studies, overexpression of Hif1α was utilized along with expression of dominant negative PKCβII. As seen above, kinase-dead PKCβII inhibited TGFβ-stimulated collagen I (α2) expression (Fig. 12F, Fig. S13F). However, coexpression of Hif1α prevented this inhibition (Fig. 12F, Fig. S13F). Similarly, expression of Hif1α reversed siPCKβII-mediated inhibition of TGFβ-stimulated collagen I (α2) expression (Fig. 12G, Fig. S13G). To complement these results, siRNAs against Hif1α were cotransfected with vector containing catalytically active PKCβII into proximal tubular epithelial cells. siHif1α inhibited the expression of collagen I (α2) in cells expressing constitutively active PKCβII alone as well as in cells expressing constitutively active PKCβII treated with TGFβ (Fig. 12, H and I, Fig. S13, H and I). Taken together, the above results strongly suggest that Hif1α downstream of TGFβ-DJ-1–mTORC2–PKCβII axis contributes to the expression of collagen I (α2).
      Figure thumbnail gr12
      Figure 12Rictor as well as Hif1α downstream of DJ-1–PKCβII axis regulates TGFβ-stimulated collagen I (α2) expression. Human proximal tubular epithelial cells were transfected with two independent shRNAs against rictor (panels A and B), with siDJ-1 plus Hif1α (panel C), FLAG DJ-1 plus siHif1α (panel D and E), PKCβII K371 R plus Hif1α (panel F), siPKCβII plus Hif1α (panel G), PKCβII CAT plus siHif1α (panel H), and PKCβII CAT plus siHif1α (panel I). The cell lysates were immunoblotted with antibodies to detect the indicated proteins. Representative of three to four independent experiments is shown. Quantification and significance of these data are shown in , A–I. TGFβ, transforming growth factor β; Hif1α, hypoxia-inducible factor 1α.
      We and others have shown that TGFβ-induced expression of collagen I (α2) is regulated by transcriptional mechanism via Hif1α in renal cells (
      • Das F.
      • Ghosh-Choudhury N.
      • Venkatesan B.
      • Kasinath B.S.
      • Ghosh Choudhury G.
      PDGF receptor-beta uses Akt/mTORC1 signaling node to promote high glucose-induced renal proximal tubular cell collagen I (alpha2) expression.
      ,
      • Liu J.
      • Wei Q.
      • Guo C.
      • Dong G.
      • Liu Y.
      • Tang C.
      • et al.
      Hypoxia, HIF, and associated signaling Networks in chronic kidney disease.
      ,
      • Baumann B.
      • Hayashida T.
      • Liang X.
      • Schnaper H.W.
      Hypoxia-inducible factor-1alpha promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3.
      ,
      • Basu R.K.
      • Hubchak S.
      • Hayashida T.
      • Runyan C.E.
      • Schumacker P.T.
      • Schnaper H.W.
      Interdependence of HIF-1alpha and TGF-beta/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression.
      ). To determine the role of TGFβ-stimulated DJ-1 in Hif1α-mediated transcription of collagen I (α2), we used luciferase reporter construct in proximal tubular epithelial cells. The cells were cotransfected with the reporter plasmid along with siDJ-1 and Hif1α. As shown in Fig. 13A, expression of Hif1α reversed the siDJ-1–mediated inhibition of TGFβ-stimulated transcription of collagen I (α2). Furthermore, siHif1α inhibited the collagen I (α2) promoter activity by FLAG-DJ-1 alone and in the presence of TGFβ (Fig. 13, B and C). Similar to the collagen I (α2) protein, expression of dominant negative PKCβII as well as siPKCβII inhibited the transcription of collagen I (α2) (Fig. 13, D and E). Coexpression of Hif1α prohibited this inhibition (Fig. 13, D and E). To confirm this observation, we used constitutively active PKCβII, which alone and with TGFβ, increased collagen I (α2) transcription (Fig. 13, F and G). Coexpression of siHif1α blocked this increase (Fig. 13, F and G). These results indicate that Hif1α downstream of DJ-1/PKCβII regulates collagen I (α2) expression in response to TGFβ in proximal tubular epithelial cells.
      Figure thumbnail gr13
      Figure 13Rictor and Hif1α following DJ-1/PKCβII regulates TGFβ-induced transcription of collagen I (α2). Proximal tubular epithelial cells were cotransfected with collagen I (α2) promoter-driven luciferase reporter plasmid along with siDJ-1 plus Hif1α (panel A), FLAG DJ-1 plus siHif1α (panels B and C), PKCβII K371 R plus Hif1α (panel D), siPKCβII plus Hif1α (panel E), and HA PKCβII CAT plus siHif1α (panels F and G). The transfected cells were incubated with 2 ng/ml TGFβ for 24 h. The cell lysates were assayed for luciferase activity as described in the Experimental procedures. Mean ± SD of six to eight measurements is shown. ∗p < 0.001 versus control (panels A–G); ∗∗p < 0.001 versus TGFβ (panels A, D, E); #p < 0.001 versus FLAG DJ-1 or PKCβII CAT (panels B and F); ∗∗∗p < 0.001 versus TGFβ plus siDJ-1 (panel A), versus TGFβ plus FLAG DJ-1 (panel C), versus TGFβ plus PKCβII K371R (panel D), versus TGFβ plus siPKCβII (panel E), and TGFβ plus PKCβII CAT (panel G). The bottom panels show expression of indicated proteins. TGFβ, transforming growth factor β; Hif1α, hypoxia-inducible factor 1α.

      Increased expression of DJ-1 in the renal cortex of diabetic rats

      Progressive diabetic kidney disease is characterized by renal fibrosis as a pathology (
      • Kato M.
      • Natarajan R.
      Diabetic nephropathy--emerging epigenetic mechanisms.
      ). Hyperglycemia induces renal production of TGFβ that contributes to the pathogenesis of diabetic nephropathy (
      • Kanwar Y.S.
      • Sun L.
      • Xie P.
      • Liu F.Y.
      • Chen S.
      A glimpse of various pathogenetic mechanisms of diabetic nephropathy.
      ,
      • Kato M.
      • Natarajan R.
      Diabetic nephropathy--emerging epigenetic mechanisms.
      ,
      • Sharma K.
      • Ziyadeh F.N.
      • Alzahabi B.
      • McGowan T.A.
      • Kapoor S.
      • Kurnik B.R.
      • et al.
      Increased renal production of transforming growth factor-beta1 in patients with type II diabetes.
      ,
      • Ziyadeh F.N.
      • Hoffman B.B.
      • Han D.C.
      • Iglesias-De La Cruz M.C.
      • Hong S.W.
      • Isono M.
      • et al.
      Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice.
      ). Rodent models of diabetes are a useful tool to study renal fibrosis in diabetic nephropathy (
      • Miric G.
      • Dallemagne C.
      • Endre Z.
      • Margolin S.
      • Taylor S.M.
      • Brown L.
      Reversal of cardiac and renal fibrosis by pirfenidone and spironolactone in streptozotocin-diabetic rats.
      ,
      • Deelman L.
      • Sharma K.
      Mechanisms of kidney fibrosis and the role of antifibrotic therapies.
      ,
      • Sharma K.
      • McCue P.
      • Dunn S.R.
      Diabetic kidney disease in the db/db mouse.
      ,
      • Kitada M.
      • Ogura Y.
      • Koya D.
      Rodent models of diabetic nephropathy: Their utility and limitations.
      ). Previously, we and others reported that mTOR is activated in the kidneys of streptozotocin-induced diabetes in rat and in mice models of diabetes and, in human kidney (
      • Maity S.
      • Das F.
      • Kasinath B.S.
      • Ghosh-Choudhury N.
      • Ghosh Choudhury G.
      TGFbeta acts through PDGFRbeta to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression.
      • Mori H.
      • Inoki K.
      • Masutani K.
      • Wakabayashi Y.
      • Komai K.
      • Nakagawa R.
      • et al.
      The mTOR pathway is highly activated in diabetic nephropathy and rapamycin has a strong therapeutic potential.
      ,
      • Lloberas N.
      • Cruzado J.M.
      • Franquesa M.
      • Herrero-Fresneda I.
      • Torras J.
      • Alperovich G.
      • et al.
      Mammalian target of rapamycin pathway blockade slows progression of diabetic kidney disease in rats.
      ,
      • Sakaguchi M.
      • Isono M.
      • Isshiki K.
      • Sugimoto T.
      • Koya D.
      • Kashiwagi A.
      Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice.
      ,
      • Sataranatarajan K.
      • Mariappan M.M.
      • Lee M.J.
      • Feliers D.
      • Choudhury G.G.
      • Barnes J.L.
      • et al.
      Regulation of elongation phase of mRNA translation in diabetic nephropathy: Amelioration by rapamycin.
      ,
      • Dey N.
      • Ghosh-Choudhury N.
      • Das F.
      • Li X.
      • Venkatesan B.
      • Barnes J.L.
      • et al.
      PRAS40 acts as a nodal regulator of high glucose-induced TORC1 activation in glomerular mesangial cell hypertrophy.
      ,
      • Dey N.
      • Das F.
      • Mariappan M.M.
      • Mandal C.C.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • et al.
      MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes.
      ,
      • Das F.
      • Maity S.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Ghosh Choudhury G.
      Deacetylation of S6 kinase promotes high glucose-induced glomerular mesangial cell hypertrophy and matrix protein accumulation.
      ,
      • Inoki K.
      • Mori H.
      • Wang J.
      • Suzuki T.
      • Hong S.
      • Yoshida S.
      • et al.
      mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice.
      ). Administration of rapamycin ameliorates the pathologies of diabetic nephropathy including renal fibrosis, suggesting a significant role of mTOR in this process (
      • Eid A.A.
      • Ford B.M.
      • Bhandary B.
      • Cavagliery R.
      • Block K.
      • Barnes J.L.
      • et al.
      Mammalian target of rapamycin regulates Nox4-mediated podocyte Depletion in diabetic renal injury.
      ,
      • Lloberas N.
      • Cruzado J.M.
      • Franquesa M.
      • Herrero-Fresneda I.
      • Torras J.
      • Alperovich G.
      • et al.
      Mammalian target of rapamycin pathway blockade slows progression of diabetic kidney disease in rats.
      ,
      • Sakaguchi M.
      • Isono M.
      • Isshiki K.
      • Sugimoto T.
      • Koya D.
      • Kashiwagi A.
      Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice.
      ,
      • Sataranatarajan K.
      • Mariappan M.M.
      • Lee M.J.
      • Feliers D.
      • Choudhury G.G.
      • Barnes J.L.
      • et al.
      Regulation of elongation phase of mRNA translation in diabetic nephropathy: Amelioration by rapamycin.
      ,
      • Inoki K.
      • Mori H.
      • Wang J.
      • Suzuki T.
      • Hong S.
      • Yoshida S.
      • et al.
      mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice.
      ). Our results above show a conclusive role of DJ-1 in the activation of mTOR and in the expression of proximal tubular cell collagen I (α2) expression. To investigate the in vivo relevance of our observations, we used streptozotocin-induced type 1 diabetic rats, which showed early changes of diabetic nephropathy including expression of fibrotic markers (
      • Mahimainathan L.
      • Das F.
      • Venkatesan B.
      • Choudhury G.G.
      Mesangial cell hypertrophy by high glucose is mediated by downregulation of the tumor suppressor PTEN.
      ). Renal cortical lysates were used to determine the expression of TGFβ. Results showed significant increase in the expression of this fibrotic cytokine in the diabetic rats (Fig. 14, A and B). This increase in TGFβ correlated with enhanced expression of DJ-1 (Fig. 14, C and D). In proximal tubular epithelial cells, increased expression of DJ-1 by TGFβ was associated with the activation of mTORC1 and mTORC2. Consistently, the phosphorylation of S6 kinase at Thr-389 and Akt at Ser-473 as measures of mTORC1 and mTORC2 activation, respectively, was significantly elevated in the diabetic cortex (Fig. 14, E–H). In fact, we detected increased phosphorylation of rps6 and GSK3β, two substrates of S6 kinase and Akt, respectively, indicating activation of these kinases (Fig. 14, I–L). We have shown that DJ-1 promoted the hydrophobic motif site phosphorylation and activation of PKCβII in proximal tubular epithelial cells. We examined these phenomena. The results shown in Fig. 14, M–P demonstrate elevated phosphorylation of PKCβII at its hydrophobic motif site, resulting in phosphorylation of its substrate MARCKS. Our results above show that DJ-1–regulated PKCβII controls the expression of Hif1α, which increases the expression of collagen I (α2) in response to TGFβ. We determined the expression of Hif1α. A significant increase in the expression of Hif1α was observed in the renal cortex of diabetic animals (Fig. 14, Q and R). This increase was associated with elevated levels of collagen I (α2) (Fig. 14, S and T). These results indicate a possible role of DJ-1 in regulation of mTOR and PKCβII for Hif1α-dependent collagen I (α2) expression in the pathology of diabetic kidney injury.
      Figure thumbnail gr14
      Figure 14Increased expression of TGFβ is associated with enhanced expression of DJ-1, Hif1α, and collagen I (α2) along with phosphorylation/activation of mTORC1 (as judged by phosphorylation of S6 kinase/rps6) and mTORC2 (as judged by phosphorylation of Akt, GSK3β, PKCβII and MARCKS) in diabetic rats. Renal cortical lysates were immunoblotted with antibodies to detect the indicated proteins. C, control animal; D, diabetic animal. Scatter graphs show quantification of top immunoblot. Mean ± SD of four animals per group is shown. p values are indicated. TGFβ, transforming growth factor β; Hif1α, hypoxia-inducible factor 1α.

      Discussion

      TGFβ plays significant role in CKD (
      • Sharma K.
      • Ziyadeh F.N.
      • Alzahabi B.
      • McGowan T.A.
      • Kapoor S.
      • Kurnik B.R.
      • et al.
      Increased renal production of transforming growth factor-beta1 in patients with type II diabetes.
      ,
      • Yoshioka K.
      • Takemura T.
      • Murakami K.
      • Okada M.
      • Hino S.
      • Miyamoto H.
      • et al.
      Transforming growth factor-beta protein and mRNA in glomeruli in normal and diseased human kidneys.
      ). Increased expression of TGFβ in a rat model of glomerulonephritis promoted glomerulosclerosis (
      • Okuda S.
      • Languino L.R.
      • Ruoslahti E.
      • Border W.A.
      Elevated expression of transforming growth factor-beta and proteoglycan production in experimental glomerulonephritis. Possible role in expansion of the mesangial extracellular matrix.
      ). Importantly, a TGFβ antibody ameliorated the fibrosis in this model (
      • Border W.A.
      • Okuda S.
      • Languino L.R.
      • Sporn M.B.
      • Ruoslahti E.
      Suppression of experimental glomerulonephritis by antiserum against transforming growth factor beta 1.
      ). Also, significant ameliorative effect of a TGFβ monoclonal antibody was observed in the models of adriamycin- and podocyte ablation–induced nephropathy in mice (
      • Liang X.
      • Schnaper H.W.
      • Matsusaka T.
      • Pastan I.
      • Ledbetter S.
      • Hayashida T.
      Anti-TGF-beta antibody, 1D11, ameliorates glomerular fibrosis in mouse models after the onset of proteinuria.
      ). Renal fibrosis elicited by diabetic nephropathy shows hyperexpression of TGFβ, which contributes to the pathology (
      • Kanwar Y.S.
      • Sun L.
      • Xie P.
      • Liu F.Y.
      • Chen S.
      A glimpse of various pathogenetic mechanisms of diabetic nephropathy.
      ,
      • Meng X.M.
      • Nikolic-Paterson D.J.
      • Lan H.Y.
      TGF-Beta: The master regulator of fibrosis.
      ). In fact, renal hypertrophy and matrix protein expression, two features of diabetic nephropathy in mice models of type 1 and type 2 diabetes, were prevented by TGFβ neutralizing antibody (
      • Ziyadeh F.N.
      • Hoffman B.B.
      • Han D.C.
      • Iglesias-De La Cruz M.C.
      • Hong S.W.
      • Isono M.
      • et al.
      Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice.
      ,
      • Sharma K.
      • Jin Y.
      • Guo J.
      • Ziyadeh F.N.
      Neutralization of TGF-beta by anti-TGF-beta antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice.
      ). Generation of whole body TGFβ hypomorphic and overexpression mice models with type 1 diabetes showed decreased and increased albuminuria, respectively (
      • Hathaway C.K.
      • Gasim A.M.
      • Grant R.
      • Chang A.S.
      • Kim H.S.
      • Madden V.J.
      • et al.
      Low TGFbeta1 expression prevents and high expression exacerbates diabetic nephropathy in mice.
      ). In fact, increased expression of TGFβ in type 1 diabetic mice showed significantly reduced proximal tubular expression of megalin, which endocytose albumin (
      • Hathaway C.K.
      • Gasim A.M.
      • Grant R.
      • Chang A.S.
      • Kim H.S.
      • Madden V.J.
      • et al.
      Low TGFbeta1 expression prevents and high expression exacerbates diabetic nephropathy in mice.
      ). When TGFβ expression was induced specifically in the proximal tubules of the same diabetic mouse model, albuminuria and fibrosis were affected significantly (
      • Hathaway C.K.
      • Gasim A.M.
      • Grant R.
      • Chang A.S.
      • Kim H.S.
      • Madden V.J.
      • et al.
      Low TGFbeta1 expression prevents and high expression exacerbates diabetic nephropathy in mice.
      ). Furthermore, in a more recent study where a soluble TGFβRII was administered to a model of renal fibrosis via a viral vector-mediated gene therapy, the kidney pathology was significantly ameliorated (
      • Davidsohn N.
      • Pezzone M.
      • Vernet A.
      • Graveline A.
      • Oliver D.
      • Slomovic S.
      • et al.
      A single combination gene therapy treats multiple age-related diseases.
      ). These results conclusively demonstrate a role of TGFβ in renal fibrosis. Thus, blocking the action of TGFβ may be beneficial for renal fibrosis. However, because TGFβ plays an important role in many other biological responses including immune homeostasis, potential adverse effects present a major challenge for employing anti-TGFβ therapy (
      • Meng X.M.
      • Nikolic-Paterson D.J.
      • Lan H.Y.
      TGF-Beta: The master regulator of fibrosis.
      ). Thus, it is important to delineate intricacies of TGFβ signaling to identify alternative therapeutic strategies for inhibiting renal fibrosis.
      In the present article, we identified the familial early-onset Parkinson’s disease protein DJ-1 as a target of TGFβ-induced canonical Smad 3 signaling leading to its increased expression in the renal proximal tubular epithelial cells. Similar to the role of oxidative stress in Parkinson’s disease, a role of reactive oxygen species in the pathogenesis of renal fibrosis has been established (
      • Kanwar Y.S.
      • Sun L.
      • Xie P.
      • Liu F.Y.
      • Chen S.
      A glimpse of various pathogenetic mechanisms of diabetic nephropathy.
      ,
      • Eid A.A.
      • Ford B.M.
      • Bhandary B.
      • Cavagliery R.
      • Block K.
      • Barnes J.L.
      • et al.
      Mammalian target of rapamycin regulates Nox4-mediated podocyte Depletion in diabetic renal injury.
      ,
      • Su H.
      • Wan C.
      • Song A.
      • Qiu Y.
      • Xiong W.
      • Zhang C.
      Oxidative stress and renal fibrosis: mechanisms and therapies.
      ,
      • Das F.
      • Ghosh-Choudhury N.
      • Dey N.
      • Bera A.
      • Mariappan M.M.
      • Kasinath B.S.
      • et al.
      High glucose forces a positive feedback loop connecting Akt kinase and FoxO1 transcription factor to activate mTORC1 kinase for mesangial cell hypertrophy and matrix protein expression.
      ). In fact, TGFβ has been shown to increase reactive oxygen species due to increased expression of NADPH oxidases especially Nox4 that contribute to the expression of fibrotic markers (
      • Maity S.
      • Das F.
      • Kasinath B.S.
      • Ghosh-Choudhury N.
      • Ghosh Choudhury G.
      TGFbeta acts through PDGFRbeta to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression.
      ,
      • Das F.
      • Ghosh-Choudhury N.
      • Venkatesan B.
      • Li X.
      • Mahimainathan L.
      • Choudhury G.G.
      Akt kinase targets association of CBP with SMAD 3 to regulate TGFbeta-induced expression of plasminogen activator inhibitor-1.
      ,
      • Bondi C.D.
      • Manickam N.
      • Lee D.Y.
      • Block K.
      • Gorin Y.
      • Abboud H.E.
      • et al.
      NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts.
      ,
      • Ramundo V.
      • Giribaldi G.
      • Aldieri E.
      Transforming growth factor-beta and oxidative stress in cancer: a Crosstalk in driving tumor Transformation.
      ). Along with quenching reactive oxygen species in neuronal cells in Parkinson’s disease, DJ-1 has been shown to regulate proliferation of cancer cells including breast, lung, thyroid, pancreas, and prostate among many (
      • Jin W.
      Novel Insights into PARK7 (DJ-1), a potential anti-cancer therapeutic target, and implications for cancer progression.
      ,
      • Ariga H.
      Common mechanisms of onset of cancer and neurodegenerative diseases.
      ). However, it was suggested that DJ-1 function is reciprocally regulated in neurodegenerative disorder and cancer (
      • Mencke P.
      • Hanss Z.
      • Boussaad I.
      • Sugier P.E.
      • Elbaz A.
      • Kruger R.
      Bidirectional relation between Parkinson's disease and glioblastoma Multiforme.
      ). Molecular analysis showed that the PI 3 kinase–Akt signaling is downregulated in the Parkinson’s disease, causing apoptosis of neurons while these enzymes act as the drivers of cancer cell proliferation and metastasis including glioblastoma (
      • Manning B.D.
      • Toker A.
      AKT/PKB signaling: navigating the Network.
      ,
      • Timmons S.
      • Coakley M.F.
      • Moloney A.M.
      • C O.N.
      Akt signal transduction dysfunction in Parkinson's disease.
      ,
      • Langhans J.
      • Schneele L.
      • Trenkler N.
      • von Bandemer H.
      • Nonnenmacher L.
      • Karpel-Massler G.
      • et al.
      The effects of PI3K-mediated signalling on glioblastoma cell behaviour.
      ). Previously, in a model of eye development in Drosophila, DJ-1 was placed upstream of Akt kinase (
      • Kim R.H.
      • Peters M.
      • Jang Y.
      • Shi W.
      • Pintilie M.
      • Fletcher G.C.
      • et al.
      DJ-1, a novel regulator of the tumor suppressor PTEN.
      ). Also, DJ-1 was shown to regulate Akt activation in cancer cells (
      • Kim R.H.
      • Peters M.
      • Jang Y.
      • Shi W.
      • Pintilie M.
      • Fletcher G.C.
      • et al.
      DJ-1, a novel regulator of the tumor suppressor PTEN.
      ,
      • Ariga H.
      Common mechanisms of onset of cancer and neurodegenerative diseases.
      ). Recently, we and others have established a role of TGFβ-stimulated PI 3 kinase–Akt noncanonical signaling in increased synthesis of matrix protein in renal cells (
      • Ghosh Choudhury G.
      • Abboud H.E.
      Tyrosine phosphorylation-dependent PI 3 kinase/Akt signal transduction regulates TGFbeta-induced fibronectin expression in mesangial cells.
      ,
      • Runyan C.E.
      • Schnaper H.W.
      • Poncelet A.C.
      The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1.
      ). In the present study, we demonstrate a mechanism of TGFβ-stimulated Akt phosphorylation and its activation via increased DJ-1 in proximal tubular epithelial cells. In fact, we show that DJ-1 regulates the mTORC2 activity, which phosphorylates the hydrophobic motif site of Akt.
      mTOR exists in three different complexes of which complexes 1 and 2 have sizes of 1.0 and 1.3 mDa respectively and contain common and distinct multiproteins which are not present in the mTORC3 (
      • Saxton R.A.
      • Sabatini D.M.
      mTOR signaling in growth, metabolism, and disease.
      ,
      • Fantus D.
      • Rogers N.M.
      • Grahammer F.
      • Huber T.B.
      • Thomson A.W.
      Roles of mTOR complexes in the kidney: Implications for renal disease and transplantation.
      ,
      • Harwood F.C.
      • Klein Geltink R.I.
      • O'Hara B.P.
      • Cardone M.
      • Janke L.
      • Finkelstein D.
      • et al.
      ETV7 is an essential component of a rapamycin-insensitive mTOR complex in cancer.
      ). While both mTORC1 and mTORC2 manifest rapamycin sensitivity, mTORC3 is resistant (
      • Yang H.
      • Rudge D.G.
      • Koos J.D.
      • Vaidialingam B.
      • Yang H.J.
      • Pavletich N.P.
      mTOR kinase structure, mechanism and regulation.
      ). mTORC1 and mTORC2 phosphorylate distinct proteins and enzymes while mTORC3 uses substrates which can be phosphorylated by both mTORC1 and C2 (
      • Saxton R.A.
      • Sabatini D.M.
      mTOR signaling in growth, metabolism, and disease.
      • Harwood F.C.
      • Klein Geltink R.I.
      • O'Hara B.P.
      • Cardone M.
      • Janke L.
      • Finkelstein D.
      • et al.
      ETV7 is an essential component of a rapamycin-insensitive mTOR complex in cancer.
      ). Importantly, Akt is phosphorylated by the mTORC2 for its full activation (
      • Sarbassov D.D.
      • Guertin D.A.
      • Ali S.M.
      • Sabatini D.M.
      Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.
      ). Our observation that DJ-1 regulates Akt hydrophobic motif site phosphorylation demonstrates DJ-1 regulation of mTORC2, which is known to regulate mTORC1 via Akt (
      • Saxton R.A.
      • Sabatini D.M.
      mTOR signaling in growth, metabolism, and disease.
      ). In fact, we found that DJ-1 contributes to the activation of mTORC1. Interestingly, we demonstrated that TGFβ increased phosphorylation of S6 kinase at Thr-389 (measure of mTORC1) and Akt at Ser-473 (measure of mTORC2) along with Thr-308, similar to that increased by activation of a receptor tyrosine kinase platelet-derived growth factor (PDGF) receptor by its ligand (Fig. S14, A–D). Similarly, the both ligands significantly stimulated phosphorylation of PKCβII at Ser-660 (Fig. S15, A and B).
      Beneficial and significant deleterious effects of different PKC isoforms have been reported in renal cells (
      • Meier M.
      • Menne J.
      • Haller H.
      Targeting the protein kinase C family in the diabetic kidney: Lessons from analysis of mutant mice.
      ).Although PKCβ contributes to significant pathologies during the progression of renal fibrosis, other isoforms such as PKCε has been shown to be protective in other organ fibrosis (
      • Klein G.
      • Schaefer A.
      • Hilfiker-Kleiner D.
      • Oppermann D.
      • Shukla P.
      • Quint A.
      • et al.
      Increased collagen deposition and diastolic dysfunction but preserved myocardial hypertrophy after pressure overload in mice lacking PKCepsilon.
      ). Deletion of PKCε induced renal fibrosis in mice. Furthermore, induction of diabetes in this model aggravated the pathology (
      • Meier M.
      • Menne J.
      • Park J.K.
      • Holtz M.
      • Gueler F.
      • Kirsch T.
      • et al.
      Deletion of protein kinase C-epsilon signaling pathway induces glomerulosclerosis and tubulointerstitial fibrosis in vivo.
      ). TGFβ has been shown to activate PKCε in proximal tubular epithelial cells possibly as a protective mechanism, where hyperactivation of this kinase inhibited TGFβ-induced Smad 3 phosphorylation and increased Smad 7 expression to block fibrotic marker expression (
      • Wang L.Y.
      • Diao Z.L.
      • Zheng J.F.
      • Wu Y.R.
      • Zhang Q.D.
      • Liu W.H.
      Apelin attenuates TGF-beta1-induced epithelial to mesenchymal transition via activation of PKC-epsilon in human renal tubular epithelial cells.
      ). In contrast, in the renal cortex, which is predominantly constituted by proximal tubular epithelial cells, increased expression and apical translocation of PKCα is observed in diabetic mice. Although in this model, increased TGFβ expression is obvious, its expression did not correlate with PKCα activation (
      • Yao L.J.
      • Wang J.Q.
      • Zhao H.
      • Liu J.S.
      • Deng A.G.
      Effect of telmisartan on expression of protein kinase C-alpha in kidneys of diabetic mice.
      ). Similarly, TGFβ expression was not affected in the kidneys of diabetic PKCα KO mice (
      • Menne J.
      • Park J.K.
      • Boehne M.
      • Elger M.
      • Lindschau C.
      • Kirsch T.
      • et al.
      Diminished loss of proteoglycans and lack of albuminuria in protein kinase C-alpha-deficient diabetic mice.
      ). In the proximal tubular epithelial cells, urinary protein–induced epithelial-mesenchymal transdifferentiation and fibronectin expression were mediated by PKCα and PKCβI and not by PKCβII (
      • Tang R.
      • Yang C.
      • Tao J.L.
      • You Y.K.
      • An N.
      • Li S.M.
      • et al.
      Epithelial-mesenchymal transdifferentiation of renal tubular epithelial cells induced by urinary proteins requires the activation of PKC-alpha and betaI isozymes.
      ). In contrast to these results, overexpression of PKCβII in proximal tubular epithelial cells increased TGFβ and fibronectin expression (
      • Slattery C.
      • Ryan M.P.
      • McMorrow T.
      Protein kinase C beta overexpression induces fibrotic effects in human proximal tubular epithelial cells.
      ). Furthermore, in a rat model of ureteric obstruction, which predominantly involves TGFβ, inhibition of PKCβ ameliorated the pathology (
      • Juan Y.S.
      • Chuang S.M.
      • Long C.Y.
      • Lin R.J.
      • Liu K.M.
      • Wu W.J.
      • et al.
      Protein kinase C inhibitor prevents renal apoptotic and fibrotic changes in response to partial ureteric obstruction.
      ). It should be noted that phosphorylation-dependent activation of PKCβII has not been investigated in these studies related to kidney diseases. However, the major pathologic effects of PKCβ are mediated through TGFβ, suggesting that this cytokine is a downstream target of the kinase (
      • Ohshiro Y.
      • Ma R.C.
      • Yasuda Y.
      • Hiraoka-Yamamoto J.
      • Clermont A.C.
      • Isshiki K.
      • et al.
      Reduction of diabetes-induced oxidative stress, fibrotic cytokine expression, and renal dysfunction in protein kinase Cbeta-null mice.
      ,
      • Meier M.
      • Menne J.
      • Park J.K.
      • Holtz M.
      • Gueler F.
      • Kirsch T.
      • et al.
      Deletion of protein kinase C-epsilon signaling pathway induces glomerulosclerosis and tubulointerstitial fibrosis in vivo.
      ,
      • Slattery C.
      • Ryan M.P.
      • McMorrow T.
      Protein kinase C beta overexpression induces fibrotic effects in human proximal tubular epithelial cells.
      ,
      • Takeda M.
      • Babazono T.
      • Nitta K.
      • Iwamoto Y.
      High glucose stimulates hyaluronan production by renal interstitial fibroblasts through the protein kinase C and transforming growth factor-beta cascade.
      ,
      • Park S.H.
      • Choi H.J.
      • Lee J.H.
      • Woo C.H.
      • Kim J.H.
      • Han H.J.
      High glucose inhibits renal proximal tubule cell proliferation and involves PKC, oxidative stress, and TGF-beta 1.
      ,
      • Chen S.
      • Cohen M.P.
      • Lautenslager G.T.
      • Shearman C.W.
      • Ziyadeh F.N.
      Glycated albumin stimulates TGF-beta 1 production and protein kinase C activity in glomerular endothelial cells.
      ,
      • Koya D.
      • Jirousek M.R.
      • Lin Y.W.
      • Ishii H.
      • Kuboki K.
      • King G.L.
      Characterization of protein kinase C beta isoform activation on the gene expression of transforming growth factor-beta, extracellular matrix components, and prostanoids in the glomeruli of diabetic rats.
      ). In contrast to these observations, we demonstrate that PKCβII is part of the TGFβ noncanonical signaling. We find that TGFβ increases the hydrophobic motif site phosphorylation of PKCβII, resulting in its activation and phosphorylation of its substrate. In fact, TGFβ increased the PKCβII hydrophobic motif phosphorylation to the same extent as by activation of PDGF receptor in response to PDGF (Fig. 15, A and B).
      Importantly, we show that TGFβ-stimulated DJ-1 regulates these phenomena. Thus, with our findings that TGFβ-stimulated DJ-1 regulates phosphorylation at the hydrophobic motif sites of Akt and PKCβII and phosphorylation of S6 kinase, we conclude that DJ-1 controls the mTORC2 and mTORC1 activities in proximal tubular epithelial cells. The mechanism by which DJ-1 activates the mTOR complexes is not known. DJ-1 is a versatile protein and is able to bind or form complex with multiple signaling proteins such as ErbB3, androgen receptor, Raf, SIRT1, and more to either activate or inhibit their functions (
      • Jin W.
      Novel Insights into PARK7 (DJ-1), a potential anti-cancer therapeutic target, and implications for cancer progression.
      ,
      • Tillman J.E.
      • Yuan J.
      • Gu G.
      • Fazli L.
      • Ghosh R.
      • Flynt A.S.
      • et al.
      DJ-1 binds androgen receptor directly and mediates its activity in hormonally treated prostate cancer cells.
      ,
      • Takahashi-Niki K.
      • Kato-Ose I.
      • Murata H.
      • Maita H.
      • Iguchi-Ariga S.M.M.
      • Ariga H.
      Epidermal growth factor-dependent activation of the extracellular signal-regulated kinase pathway by DJ-1 protein through its direct binding to c-Raf protein.
      ,
      • Takahashi-Niki K.
      • Ganaha Y.
      • Niki T.
      • Nakagawa S.
      • Kato-Ose I.
      • Iguchi-Ariga S.M.M.
      • et al.
      DJ-1 activates SIRT1 through its direct binding to SIRT1.
      ,
      • Zhang S.
      • Mukherjee S.
      • Fan X.
      • Salameh A.
      • Mujoo K.
      • Huang Z.
      • et al.
      Novel association of DJ-1 with HER3 potentiates HER3 activation and signaling in cancer.
      ). Interestingly, we identified that TGFβ stimulated complex formation between DJ-1 and raptor and mLST8 as well as with rictor and mSin1. Furthermore, we demonstrate association of DJ-1 with mTOR. Importantly, we show that DJ-1 regulates the kinase activities of both mTORC1 and mTORC2. These data provide a mechanism on how DJ-1 may regulate both mTORC1 and mTORC2 activities.
      A role of TGFβ and PKCβ has been established in renal fibrosis (
      • Ohshiro Y.
      • Ma R.C.
      • Yasuda Y.
      • Hiraoka-Yamamoto J.
      • Clermont A.C.
      • Isshiki K.
      • et al.
      Reduction of diabetes-induced oxidative stress, fibrotic cytokine expression, and renal dysfunction in protein kinase Cbeta-null mice.
      ,
      • Ziyadeh F.N.
      • Hoffman B.B.
      • Han D.C.
      • Iglesias-De La Cruz M.C.
      • Hong S.W.
      • Isono M.
      • et al.
      Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice.
      ,
      • Juan Y.S.
      • Chuang S.M.
      • Long C.Y.
      • Lin R.J.
      • Liu K.M.
      • Wu W.J.
      • et al.
      Protein kinase C inhibitor prevents renal apoptotic and fibrotic changes in response to partial ureteric obstruction.
      ,
      • Gu Y.Y.
      • Liu X.S.
      • Huang X.R.
      • Yu X.Q.
      • Lan H.Y.
      Diverse role of TGF-beta in kidney disease.
      ). TGFβ significantly contributes to the expression of the matrix proteins including collagen I (α2) during the progression of renal fibrosis (
      • Gu Y.Y.
      • Liu X.S.
      • Huang X.R.
      • Yu X.Q.
      • Lan H.Y.
      Diverse role of TGF-beta in kidney disease.
      ,
      • Meng X.M.
      • Huang X.R.
      • Xiao J.
      • Chen H.Y.
      • Zhong X.
      • Chung A.C.
      • et al.
      Diverse roles of TGF-beta receptor II in renal fibrosis and inflammation in vivo and in vitro.
      ). Previously, a cross talk between TGFβ receptor-specific Smad 3 and mTOR has been implicated in the expression of collagen I (α2) in response to TGFβ (
      • Runyan C.E.
      • Schnaper H.W.
      • Poncelet A.C.
      The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1.
      ,
      • Meng X.M.
      • Huang X.R.
      • Xiao J.
      • Chung A.C.
      • Qin W.
      • Chen H.Y.
      • et al.
      Disruption of Smad4 impairs TGF-beta/Smad3 and Smad7 transcriptional regulation during renal inflammation and fibrosis in vivo and in vitro.
      ). In a mouse model using unilateral ureteral obstruction (UUO) of kidney, the interstitial fibrosis is mainly mediated by significant expression of TGFβ due to activation of mTOR in the macrophages and myofibroblasts but not in the proximal tubular cells (
      • Chen G.
      • Chen H.
      • Wang C.
      • Peng Y.
      • Sun L.
      • Liu H.
      • et al.
      Rapamycin ameliorates kidney fibrosis by inhibiting the activation of mTOR signaling in interstitial macrophages and myofibroblasts.
      ,
      • De Miguel C.
      • Kraus A.C.
      • Saludes M.A.
      • Konkalmatt P.
      • Ruiz Dominguez A.
      • Asico L.D.
      • et al.
      ND-13, a DJ-1-derived Peptide, attenuates the renal expression of fibrotic and inflammatory markers associated with unilateral ureter obstruction.
      ). A role of DJ-1 in preventing renal damage in UUO model has been shown using a DJ-1 KO mouse. Interestingly, the DJ-1 KO mice showed decreased TGFβ levels compared to WT UUO model. However, there was no difference in the increase in expression of collagen I (α1) (
      • De Miguel C.
      • Kraus A.C.
      • Saludes M.A.
      • Konkalmatt P.
      • Ruiz Dominguez A.
      • Asico L.D.
      • et al.
      ND-13, a DJ-1-derived Peptide, attenuates the renal expression of fibrotic and inflammatory markers associated with unilateral ureter obstruction.
      ). In contrast to these results, our results show that DJ-1 is downstream of TGFβ in which its upregulated expression contributes to the expression of collagen I (α2) in the proximal tubular epithelial cells, suggesting a possible role in tubular fibrosis. These results are opposite to that observed in cardiac fibrosis induced by ischemia reperfusion injury where DJ-1 protects the organ from fibrosis (
      • Shimizu Y.
      • Nicholson C.K.
      • Polavarapu R.
      • Pantner Y.
      • Husain A.
      • Naqvi N.
      • et al.
      Role of DJ-1 in modulating glycative stress in Heart Failure.
      ). However, our results are in line with the pathologic role of DJ-1 in liver fibrosis (
      • Yu Y.
      • Sun X.
      • Gu J.
      • Yu C.
      • Wen Y.
      • Gao Y.
      • et al.
      Deficiency of DJ-1 ameliorates liver fibrosis through inhibition of hepatic ROS production and inflammation.
      ). Interestingly, we not only show a role of DJ-1 in regulation of collagen I (α2) expression but it regulates PKCβII to increase the expression of the matrix protein. These results for the first time provide a mechanism for expression of this matrix protein involving the upstream and downstream targets of mTOR in response to TGFβ.
      TGFβ uses the canonical Smad 3 signal transduction for expression of fibrogenic genes (
      • Lan H.Y.
      Diverse roles of TGF-beta/Smads in renal fibrosis and inflammation.
      ). We demonstrate the presence of a cross talk between Smad 3 and PKCβII in the regulation of collagen I (α2). Hif1α, which is mainly stabilized by hypoxia, has been shown to be upregulated along with TGFβ and promotes epithelial to mesenchymal transdifferentiation during the progression of tubulointerstitial fibrosis (
      • Liu M.
      • Ning X.
      • Li R.
      • Yang Z.
      • Yang X.
      • Sun S.
      • et al.
      Signalling pathways involved in hypoxia-induced renal fibrosis.
      ). However, a beneficial role of myeloid-derived Hif1α has been reported in the UUO and remnant kidney models of renal fibrosis (
      • Kobayashi H.
      • Gilbert V.
      • Liu Q.
      • Kapitsinou P.P.
      • Unger T.L.
      • Rha J.
      • et al.
      Myeloid cell-derived hypoxia-inducible factor attenuates inflammation in unilateral ureteral obstruction-induced kidney injury.
      ,
      • Song Y.R.
      • You S.J.
      • Lee Y.M.
      • Chin H.J.
      • Chae D.W.
      • Oh Y.K.
      • et al.
      Activation of hypoxia-inducible factor attenuates renal injury in rat remnant kidney.
      ). Furthermore, stable expression of Hif1α in a model of subtotal nephrectomy increased renal fibrosis (
      • Kimura K.
      • Iwano M.
      • Higgins D.F.
      • Yamaguchi Y.
      • Nakatani K.
      • Harada K.
      • et al.
      Stable expression of HIF-1alpha in tubular epithelial cells promotes interstitial fibrosis.
      ). However, expression of Hif1α is significantly increased in the tubules of patients with diabetic nephropathy (
      • Higgins D.F.
      • Kimura K.
      • Bernhardt W.M.
      • Shrimanker N.
      • Akai Y.
      • Hohenstein B.
      • et al.
      Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition.
      ). In conjunction with these studies, inhibition of Hif1α in a model of diabetic nephropathy ameliorated tubular injury (
      • Takiyama Y.
      • Harumi T.
      • Watanabe J.
      • Fujita Y.
      • Honjo J.
      • Shimizu N.
      • et al.
      Tubular injury in a rat model of type 2 diabetes is prevented by metformin: A possible role of HIF-1alpha expression and oxygen metabolism.
      ). Similarly, inhibition of Hif1α by YC1 ameliorated progression of renal fibrosis in the UUO model (
      • Kimura K.
      • Iwano M.
      • Higgins D.F.
      • Yamaguchi Y.
      • Nakatani K.
      • Harada K.
      • et al.
      Stable expression of HIF-1alpha in tubular epithelial cells promotes interstitial fibrosis.
      ). Also, proximal tubular ablation of Hif1α blocked renal fibrosis including inhibition of expression of matrix modification enzyme PAI-1 and collagen deposition (
      • Higgins D.F.
      • Kimura K.
      • Bernhardt W.M.
      • Shrimanker N.
      • Akai Y.
      • Hohenstein B.
      • et al.
      Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition.
      ). Interestingly, we and others have shown hypoxia-independent activation of Hif1α in renal cells including proximal tubular epithelial cells (
      • Das F.
      • Ghosh-Choudhury N.
      • Venkatesan B.
      • Kasinath B.S.
      • Ghosh Choudhury G.
      PDGF receptor-beta uses Akt/mTORC1 signaling node to promote high glucose-induced renal proximal tubular cell collagen I (alpha2) expression.
      ,
      • Basu R.K.
      • Hubchak S.
      • Hayashida T.
      • Runyan C.E.
      • Schumacker P.T.
      • Schnaper H.W.
      Interdependence of HIF-1alpha and TGF-beta/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression.
      ). In fact, an interaction between TGFβ-stimulated Smad 3 and Hif1α has been reported to regulate the expression of collagen I (α2) (
      • Rozen-Zvi B.
      • Hayashida T.
      • Hubchak S.C.
      • Hanna C.
      • Platanias L.C.
      • Schnaper H.W.
      TGF-beta/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1alpha expression.
      ,
      • Baumann B.
      • Hayashida T.
      • Liang X.
      • Schnaper H.W.
      Hypoxia-inducible factor-1alpha promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3.
      ,
      • Basu R.K.
      • Hubchak S.
      • Hayashida T.
      • Runyan C.E.
      • Schumacker P.T.
      • Schnaper H.W.
      Interdependence of HIF-1alpha and TGF-beta/Smad3 signaling in normoxic and hypoxic renal epithelial cell collagen expression.
      ). Normoxic Hif1α level is regulated by PI 3 kinase/Akt and mTOR (
      • Rozen-Zvi B.
      • Hayashida T.
      • Hubchak S.C.
      • Hanna C.
      • Platanias L.C.
      • Schnaper H.W.
      TGF-beta/Smad3 activates mammalian target of rapamycin complex-1 to promote collagen production by increasing HIF-1alpha expression.
      ,
      • Runyan C.E.
      • Schnaper H.W.
      • Poncelet A.C.
      The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1.
      ,
      • Brugarolas J.B.
      • Vazquez F.
      • Reddy A.
      • Sellers W.R.
      • Kaelin Jr., W.G.
      TSC2 regulates VEGF through mTOR-dependent and -independent pathways.
      ). Also, these enzymes downstream of TGFβ controls collagen I (α2) expression in renal cells (
      • Dey N.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Choudhury G.G.
      TGFbeta-stimulated microRNA-21 utilizes PTEN to orchestrate AKT/mTORC1 signaling for mesangial cell hypertrophy and matrix expansion.
      ,
      • Runyan C.E.
      • Schnaper H.W.
      • Poncelet A.C.
      The phosphatidylinositol 3-kinase/Akt pathway enhances Smad3-stimulated mesangial cell collagen I expression in response to transforming growth factor-beta1.
      ). However, the intricacies of this signal transduction that regulates Hif1α-mediated collagen I (α2) expression has not been clarified. Upstream of mTORC2, we identified DJ-1 to regulate mTOR activity. In fact, our data for the first time show that DJ-1 controls the TGFβ-stimulated Hif1α, which increases the expression of collagen I (α2). Furthermore, expression of collagen I (α2) protein was dependent upon PKCβII downstream of DJ-1. Similarly, we observed that DJ-1–regulated PKCβII increased the transcription of collagen I (α2) by Hif1α in response to TGFβ. Together, our data provide the first evidence for a role of DJ-1 to regulate mTORC2-dependent activation of PKCβII to induce Hif1α that increases proximal tubular collagen I (α2) expression. In fact, in a rat model of diabetes which exhibits features of diabetic nephropathy, we demonstrate expression of DJ-1 concomitant with the activation of mTOR–PKCβII axis and increased Hif1α in association with increase in collagen I (α2).
      Due to sequence homology and structural similarities among the catalytic domains of various PKC isozymes, it has been difficult to develop selective inhibitors for specific isotypes. However, ruboxistaurin, a PKCβ-specific inhibitor was shown to ameliorate renal pathologies in preclinical models of diabetic nephropathy (
      • Ishii H.
      • Jirousek M.R.
      • Koya D.
      • Takagi C.
      • Xia P.
      • Clermont A.
      • et al.
      Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor.
      ,
      • Koya D.
      • Haneda M.
      • Nakagawa H.
      • Isshiki K.
      • Sato H.
      • Maeda S.
      • et al.
      Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes.
      ). This compound was evaluated in an underpowered human study with 123 patients to test its efficacy to delay diabetic nephropathy. The albumin-creatinine ratio decreased in the ruboxistaurin group without any effect on glomerular filtration rate; however, no statistical significance was observed when the data were analyzed for between-group differences (
      • Tuttle K.R.
      • Bakris G.L.
      • Toto R.D.
      • McGill J.B.
      • Hu K.
      • Anderson P.W.
      The effect of ruboxistaurin on nephropathy in type 2 diabetes.
      ,
      • He Z.
      • King G.L.
      Can protein kinase C beta-selective inhibitor, ruboxistaurin, stop vascular complications in diabetic patients?.
      ). In a large study with patients with diabetic retinopathy, ruboxistaurin did not show any difference in kidney outcomes (
      • Tuttle K.R.
      • McGill J.B.
      • Haney D.J.
      • Lin T.E.
      • Anderson P.W.
      • Pkc-Drs P.-D.
      • et al.
      Kidney outcomes in long-term studies of ruboxistaurin for diabetic eye disease.
      ). Targeting mTOR for intervention, we and others have shown beneficial effects of rapamycin in rodent models of diabetic nephropathy (
      • Eid A.A.
      • Ford B.M.
      • Bhandary B.
      • Cavagliery R.
      • Block K.
      • Barnes J.L.
      • et al.
      Mammalian target of rapamycin regulates Nox4-mediated podocyte Depletion in diabetic renal injury.
      ,
      • Mori H.
      • Inoki K.
      • Masutani K.
      • Wakabayashi Y.
      • Komai K.
      • Nakagawa R.
      • et al.
      The mTOR pathway is highly activated in diabetic nephropathy and rapamycin has a strong therapeutic potential.
      ,
      • Sakaguchi M.
      • Isono M.
      • Isshiki K.
      • Sugimoto T.
      • Koya D.
      • Kashiwagi A.
      Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice.
      ,
      • Sataranatarajan K.
      • Mariappan M.M.
      • Lee M.J.
      • Feliers D.
      • Choudhury G.G.
      • Barnes J.L.
      • et al.
      Regulation of elongation phase of mRNA translation in diabetic nephropathy: Amelioration by rapamycin.
      ,
      • Inoki K.
      • Mori H.
      • Wang J.
      • Suzuki T.
      • Hong S.
      • Yoshida S.
      • et al.
      mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice.
      ). However, inhibition of mTOR by chronic treatment with rapamycin shows significant adverse effects including insulin resistance and glucose intolerance (
      • Cunningham J.T.
      • Rodgers J.T.
      • Arlow D.H.
      • Vazquez F.
      • Mootha V.K.
      • Puigserver P.
      mTOR controls mitochondrial oxidative function through a YY1-PGC-1alpha transcriptional complex.
      ,
      • Houde V.P.
      • Brule S.
      • Festuccia W.T.
      • Blanchard P.G.
      • Bellmann K.
      • Deshaies Y.
      • et al.
      Chronic rapamycin treatment causes glucose intolerance and hyperlipidemia by upregulating hepatic gluconeogenesis and impairing lipid deposition in adipose tissue.
      ,
      • Johnston O.
      • Rose C.L.
      • Webster A.C.
      • Gill J.S.
      Sirolimus is associated with new-onset diabetes in kidney transplant recipients.
      ). Similar adverse effects may be observed in humans (
      • Johnston O.
      • Rose C.L.
      • Webster A.C.
      • Gill J.S.
      Sirolimus is associated with new-onset diabetes in kidney transplant recipients.
      ,
      • Teutonico A.
      • Schena P.F.
      • Di Paolo S.
      Glucose metabolism in renal transplant recipients: Effect of calcineurin inhibitor withdrawal and conversion to sirolimus.
      ). In this report, we identify a linear signaling pathway in which DJ-1 regulates mTOR, PKCβII, and Hif1α in TGFβ-stimulated fibrotic collagen I (α2) expression. We also demonstrate expression of DJ-1 in the kidneys of mice with diabetic nephropathy concomitant with the activation of PKCβII and expression of Hif1α. Thus, our results show importance of DJ-1 to be considered as an alternative therapeutic target to block the pathologic effects of mTOR and PKCβII in states of TGFβ-mediated renal fibrosis including diabetic nephropathy.

      Experimental procedures

      Reagents

      Materials for cell culture including OPTIMEM medium for transfection, RNA spin mini isolation kit, cDNA synthesizing SuperScript VILO master mix, and PowerUp SYBR Green master mix were purchased from Thermo Fisher. TGFβ and PDGF BB were obtained from R & D Systems. The transfection reagent FuGENE HD and luciferase assay kit were acquired from Promega. NP-40, protease inhibitor cocktail, PMSF, Na3VO4, FLAG antibody, and GAPDH primers were obtained from Sigma. TGFβ1 was purchased from R & D Systems. SB 431542 was obtained from CalBiochem. DJ-1, Myc, collagen I (α2), and actin antibodies were acquired from Santa Cruz. Antibodies against phospho-Smad 3 (Ser-423/425), Smad 3, phospho-Akt (Ser-473), p-Akt (Thr-308), Akt, phospho-GSK3β (Ser-9), GSK3β, phospho-S6 kinase (Thr-389), S6 kinase, phospho-rps6 (Ser-240/244), rps6, phospho-PKCβII (Ser-660), PKCβII, phospho-MARCKS (Ser-152/156), raptor, rictor, mTOR, mLST8, and mSin1 were purchased from Cell Signaling. MARCKS antibody was obtained from ProteinTech. HA antibody was obtained from Covance. Pool of three siRNAs against DJ-1 and PKCβII were obtained from Santa Cruz. Recombinant S6 kinase and Akt were obtained from Novus Biological. DJ-1 primers to detect its mRNA were purchased from Qiagen. The FLAG-tagged DJ-1 expression vector was a kind gift from Dr H. Ariga (Hokkaido University). HA-tagged Hif1α plasmid was provided by Dr A. Kung (Dana-Farber Cancer Institute). HA-tagged constitutively active PKCβII CAT and dominant negative PKCβII K371R expression plasmids were purchased from Addgene. The collagen I (α2) promoter–luciferase reporter plasmid has been described previously (
      • Das F.
      • Maity S.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Ghosh Choudhury G.
      Deacetylation of S6 kinase promotes high glucose-induced glomerular mesangial cell hypertrophy and matrix protein accumulation.
      ).

      Cell culture

      The HK-2 proximal tubular epithelial cells were purchased from ATCC. These cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium with 10% bovine serum albumin (
      • Das F.
      • Ghosh-Choudhury N.
      • Venkatesan B.
      • Kasinath B.S.
      • Ghosh Choudhury G.
      PDGF receptor-beta uses Akt/mTORC1 signaling node to promote high glucose-induced renal proximal tubular cell collagen I (alpha2) expression.
      ,
      • Dey N.
      • Das F.
      • Ghosh-Choudhury N.
      • Mandal C.C.
      • Parekh D.J.
      • Block K.
      • et al.
      microRNA-21 governs TORC1 activation in renal cancer cell proliferation and invasion.
      ). The murine proximal tubular epithelial cells, originally obtained from Dr Eric Neilson at Northwestern University, were grown in DMEM with 5 mM glucose and 7% fetal bovine serum as described previously (
      • Bera A.
      • Das F.
      • Ghosh-Choudhury N.
      • Mariappan M.M.
      • Kasinath B.S.
      • Ghosh Choudhury G.
      Reciprocal regulation of miR-214 and PTEN by high glucose regulates renal glomerular mesangial and proximal tubular epithelial cell hypertrophy and matrix expansion.
      ). For experiments, the cells were starved in serum-free medium for 24 h prior to incubation with 2 ng/ml TGFβ for indicated periods of time. For TGFβ receptor I inhibitor, the serum-starved cells were treated with 5 μM SB for 1 hour prior to TGFβ addition.

      Animals

      Sprague-Dawley rats (200–250 gm) were used for the study. Streptozotocin in sodium citrate buffer (pH 4.5) at a dose of 55 mg/kg body weight was injected through tail vein of the animal. At 24 h postinjection, the blood glucose levels were monitored (
      • Dey N.
      • Ghosh-Choudhury N.
      • Das F.
      • Li X.
      • Venkatesan B.
      • Barnes J.L.
      • et al.
      PRAS40 acts as a nodal regulator of high glucose-induced TORC1 activation in glomerular mesangial cell hypertrophy.
      ). The rats were housed in the animal facility at UT Health San Antonio. They had free access to food and water. The rats were euthanized after 5 days of streptozotocin injection. The kidneys were removed and renal cortical sections were isolated (
      • Dey N.
      • Ghosh-Choudhury N.
      • Das F.
      • Li X.
      • Venkatesan B.
      • Barnes J.L.
      • et al.
      PRAS40 acts as a nodal regulator of high glucose-induced TORC1 activation in glomerular mesangial cell hypertrophy.
      ). The cortical preparation was stored in an ultralow freezer at -70 °C. The animal protocol was approved by the UT Health San Antonio Animal Care and Use Committee.

      Cell lysis and preparation of renal cortical lysates

      At the end of TGFβ incubation, the cell monolayer was washed twice with PBS. RIPA buffer (20 mM Tris–HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 1 mM Na3VO4, and 0.1% protease inhibitor cocktail) was added to the cells and incubated at 4 °C for 30 min to permit cell lysis. Lysed cell debris was scraped off and collected in centrifuge tubes. Similarly, the frozen renal cortex was thawed on ice and lysed in RIPA buffer. The lysed cell debris and cortical extracts were spun at 10,000 x g for 30 min at 4 °C. The cleared supernatant was collected in a fresh tube. The protein concentration was determined in this supernatant.

      Immunoblotting

      Equal amounts of cell or renal cortical lysates were mixed with SDS-PAGE sample buffer, boiled for 5 min, and separated by electrophoresis. The separated proteins were transferred to PVDF membrane using an electroblotting apparatus. To perform immunoblotting, the membrane containing the separated proteins was incubated with the indicated primary antibody at 4 °C. The dilution of antibody used was 1:1000. After the incubation, the membrane was washed and further incubated with horseradish peroxidase–conjugated secondary antibody (1:10,000). The membrane was treated with enhanced chemiluminescence reagent. Subsequently, the membrane was exposed to X-ray film in a dark room to visualize the specific protein band recognized by the primary antibody (
      • Das F.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Choudhury G.G.
      Tyrosines-740/751 of PDGFRbeta contribute to the activation of Akt/Hif1alpha/TGFbeta nexus to drive high glucose-induced glomerular mesangial cell hypertrophy.
      ).

      Immunoprecipitation

      After incubation with TGFβ, the immunoprecipitation (IP) buffer (40 mM Hepes, pH 7.5, 0.3% CHAPS, 1 mM EDTA, 120 mM NaCl, 10 mM pyrophosphate, 50 mM NaF, 1.5 mM Na3VO4, 10 mM glycerophosphate, and 0.1% EDTA-free protease cocktail) was added to the PBS-washed cell monolayer at 4 °C for 30 min. The cell extracts were collected and centrifuged as described above. The cleared supernatant was transferred to a fresh tube and protein concentration was determined. Equal amounts of proteins were incubated with indicated antibody at 4 °C for 30 min (
      • Das F.
      • Maity S.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Ghosh Choudhury G.
      Deacetylation of S6 kinase promotes high glucose-induced glomerular mesangial cell hypertrophy and matrix protein accumulation.
      ). This protein-antibody mixture was then incubated overnight with protein G-agarose on rotating device at 4 °C. The mixture was centrifuged briefly to collect the immunebeads. The beads were then washed three times with IP buffer. Finally, the beads were suspended in the SDS polyacrylamide gel sample buffer. The boiled protein sample was separated by SDS-PAGE. Subsequently, the separated proteins were transferred to PVDF membrane and immunoblotted as described above.

      RNA preparation and real-time RT-PCR

      Total RNAs were isolated from proximal tubular epithelial cells using RNA spin mini isolation kit as described by the vendor’s protocol. Five hundred nanogram of RNA was used to prepare first strand cDNAs using Superscript VILO master mix. The cDNA was amplified in a 96-well plate using primers for DJ-1 and GAPDH in a 7500 real time PCR machine (Applied Biosystems). The conditions for PCR were 95 °C for 10 min followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, respectively. The relative mRNA levels were normalized to the reference GAPDH in the samples. Data analysis was carried out by the comparative ΔΔCt method (
      • Das F.
      • Maity S.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Ghosh Choudhury G.
      Deacetylation of S6 kinase promotes high glucose-induced glomerular mesangial cell hypertrophy and matrix protein accumulation.
      ).

      Immunecomplex kinase assays for mTORC1 and mTORC2

      The cells were extracted in IP buffer and centrifuged as described above. The cleared cell lysates were immunoprecipitated with mTOR antibody. After washing the immunebeads with IP buffer, the beads were washed twice with immunecomplex kinase assay buffer (25 mM Hepes, pH 7.4, 100 mM potassium acetate, and 1 mM MgCl2). The immunecomplexes were resuspended in 20 μl immunecomplex kinase assay buffer, which contains 100 ng of recombinant S6 kinase (for mTORC1 substrate) or recombinant Akt (for mTORC2 substrate). The reaction was started with 500 μM ATP at 37 °C and incubated for 30 min. The kinase assay was terminated by adding 4X SDS sample buffer. The reaction mixture was then separated by PAGE and immunoblotted with p-S6 kinase (Thr-389) and p-Akt (Ser-473) antibodies to detect mTORC1 and mTORC2 activities, respectively. For controls, one fifth of the recombinant S6 kinase and Akt were separated by PAGE and immunoblotted with S6 kinase and Akt antibodies, respectively.

      Transfection

      The cell monolayer was washed with PBS once inside the cell culture hood. OPTIMEM was added to the monolayer. The expression plasmids, vector, siRNAs, or scramble RNA were mixed with OPTIMEM and FuGENE HD in a tube and incubated at room temperature for 5 min. Subsequently, the mixture was added to the cells. The cells were then incubated at 37 °C in a humidified cell culture incubator for 6 h. Complete medium was added after this incubation period. Twenty-four hours postincubation, the cells were serum-starved for 24 h before addition of TGFβ as described above (
      • Maity S.
      • Das F.
      • Kasinath B.S.
      • Ghosh-Choudhury N.
      • Ghosh Choudhury G.
      TGFbeta acts through PDGFRbeta to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression.
      ,
      • Das F.
      • Maity S.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Ghosh Choudhury G.
      Deacetylation of S6 kinase promotes high glucose-induced glomerular mesangial cell hypertrophy and matrix protein accumulation.
      ).

      Luciferase activity

      Proximal tubular epithelial cells were cotransfected with collagen I (α2) promoter–luciferase reporter plasmid, siRNAs against DJ-1, Hif1α, FLAG DJ-1, siHif1α, PKCβII K371R, PKCβII CAT, vector, or scrambled RNA as described in the figure legends. The transfected cells were starved for 24 h prior to incubation with 2 ng/ml TGFβ for 24 h. The cell lysates were assayed for luciferase activity using a kit as described previously (
      • Bera A.
      • Ghosh-Choudhury N.
      • Dey N.
      • Das F.
      • Kasinath B.S.
      • Abboud H.E.
      • et al.
      NFkappaB-mediated cyclin D1 expression by microRNA-21 influences renal cancer cell proliferation.
      ).

      Statistics

      The data were expressed as mean ± SD. The significance of the data was determined by using GraphPad Prism using analysis of variance or paired t test. A p-value of < 0.05 was considered significant. The significance of all the immunoblotting experiments has been included in the Supplementary Figures.

      Data availability

      All data are contained within the article.

      Supporting information

      This article contains supporting information.

      Author contributions

      F. D. and S. M. data curation; F. D. and G. G. C. formal analysis; G. G. C. conceptualization; G. G. C. supervision; G. G. C. funding acquisition; G. G. C. writing–original draft; G. G. C. project administration; N. G.-C. and G. G. C. writing–review and editing; B. S. K. formal analysis.

      Funding and additional information

      This work was supported by the Department of Veterans Affairs Biomedical Laboratory Research and Development Service Merit Review Award 2I01 BX000926 to G. G. C. G. G. C. is a recipient of Research Career Scientist Award IK6 BX005795-01 from the Department of Veterans Affairs Biomedical Laboratory Research and Development Service.

      Conflict of interest

      The authors do not have any conflicts of interest with the content of the article.

      Supplementary data

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