TGF b acts through PDGFR b to activate mTORC1 via the Akt/PRAS40 axis and causes glomerular mesangial cell hypertrophy and matrix protein expression

Interaction of transforming growth factor- b (TGF b )-induced canonical signaling with the noncanonical kinase cascades regulates glomerular hypertrophy and matrix protein deposition, which are early features of glomerulosclerosis. However, the specific target downstream of the TGF b receptor involved in the noncanonical signaling is unknown. Here, we show that TGF b increased the catalytic loop phosphorylation of platelet-derived growth factor receptor b (PDGFR b ), a receptor tyrosine kinase expressed abundantly in glomerular mesangial cells. TGF b increased phosphorylation of the PI 3-kinase – interacting Tyr-751 residue of PDGFR b , thus activating Akt. Inhibition of PDGFR b using a pharmacological inhibitor and siRNAs blocked TGF b -stimulated phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), an intrinsic inhibitory component of mTORC1, and prevented activation of mTORC1 in the absence of any effect on Smad 2/3 phosphorylation. Expression of constitutively active myristoylated Akt reversed the siPDGFR b -medi-ated inhibition of mTORC1 activity; however, co-expression of the phospho-deficient mutant of PRAS40 inhibited the effect of myristoylated Akt, suggesting a definitive role of PRAS40 phosphorylation in mTORC1 activation downstream of PDGFR b in mesangial cells. Additionally, we demonstrate that PDGFR b -ini-tiated phosphorylation of PRAS40 is required for TGF b -induced mesangial cell hypertrophy and fibronectin and collagen I ( a 2) production. Increased activating phosphorylation of PDGFR b is also associated with enhanced TGF b expression and mTORC1 activation in the kidney cortex and glomeruli of diabetic mice and rats, respectively. Thus, pursuing TGF b noncanonical signaling, we identified how TGF b receptor I achieves mTORC1 activation through PDGFR b -mediated Akt/PRAS40 phosphorylation to spur mesangial cell hypertrophy and matrix protein accumulation. These findings provide support for targeting PDGFR b in TGF b -driven renal fibrosis. were sepa-rated by SDS-PAGE. The proteins were transferred to PVDF membrane. For immunoblotting, the membrane containing the proteins was incubated The primary antibody dilutions used for immunoblotting were 1:1000. The membrane was washed, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:10,000). The membrane was then incubated with enhanced chemiluminescence reagent to develop a specific protein band, which was visualized by exposing the membrane to X-ray film (91, 96).

Interaction of transforming growth factor-b (TGFb)-induced canonical signaling with the noncanonical kinase cascades regulates glomerular hypertrophy and matrix protein deposition, which are early features of glomerulosclerosis. However, the specific target downstream of the TGFb receptor involved in the noncanonical signaling is unknown. Here, we show that TGFb increased the catalytic loop phosphorylation of plateletderived growth factor receptor b (PDGFRb), a receptor tyrosine kinase expressed abundantly in glomerular mesangial cells. TGFb increased phosphorylation of the PI 3-kinase-interacting Tyr-751 residue of PDGFRb, thus activating Akt. Inhibition of PDGFRb using a pharmacological inhibitor and siRNAs blocked TGFb-stimulated phosphorylation of proline-rich Akt substrate of 40 kDa (PRAS40), an intrinsic inhibitory component of mTORC1, and prevented activation of mTORC1 in the absence of any effect on Smad 2/3 phosphorylation. Expression of constitutively active myristoylated Akt reversed the siPDGFRb-mediated inhibition of mTORC1 activity; however, co-expression of the phospho-deficient mutant of PRAS40 inhibited the effect of myristoylated Akt, suggesting a definitive role of PRAS40 phosphorylation in mTORC1 activation downstream of PDGFRb in mesangial cells. Additionally, we demonstrate that PDGFRb-initiated phosphorylation of PRAS40 is required for TGFb-induced mesangial cell hypertrophy and fibronectin and collagen I (a2) production. Increased activating phosphorylation of PDGFRb is also associated with enhanced TGFb expression and mTORC1 activation in the kidney cortex and glomeruli of diabetic mice and rats, respectively. Thus, pursuing TGFb noncanonical signaling, we identified how TGFb receptor I achieves mTORC1 activation through PDGFRb-mediated Akt/PRAS40 phosphorylation to spur mesangial cell hypertrophy and matrix protein accumulation. These findings provide support for targeting PDGFRb in TGFb-driven renal fibrosis.
Chronic kidney disease affects 10% of the world's population, including 20 million Americans, and causes increased risk of cardiovascular diseases and loss of renal function, leading to end-stage renal disease with significant public health costs (1,2). Thus, understanding the progression of the disease process is of prime importance for its prevention and arrest. The pathologic correlates of progressive renal function impairment include renal structural changes with initial renal hypertrophy, especially glomerular hypertrophy, that leads to hyperfiltration and microalbuminuria, followed by accumulation of matrix proteins and a greater degree of proteinuria (3,4). A significant pathologic characteristic of chronic kidney disease is glomerulosclerosis. One-third of the cell population in the glomerulus is made up of mesangial cells, which have the capacity to communicate with the endothelial cells; they can also regulate glomerular filtration because of their contractile nature (5,6). Transforming growth factor-b (TGFb) contributes to glomerulosclerosis and albuminuria during progressive kidney injury, especially in diabetic kidney disease (4,7). Liver-specific TGFb transgenic mice with increased circulating levels of cytokine developed mesangial expansion with augmented glomerular immune deposits and matrix proteins (8). In the disease milieu or in vitro when mesangial cells are exposed to TGFb, they undergo hypertrophy and acquire a myofibroblast-like phenotype to synthesize larger amounts of matrix proteins (9,10). Also, anti-TGFb antibody decreases mesangial and fibrotic protein expression in rodent models of nephropathy in type 1 and type 2 diabetes where TGFb contributes to the pathology (11,12).
The dimeric active TGFb directly binds to TGFb receptor II, which is a constitutively active kinase. After TGFb binding, TGFbRI is recruited into the complex and undergoes phosphorylation by TGFbRII. Activated TGFbRI then binds and phosphorylates the receptor-specific Smad, Smad 2, and Smad 3, which form a complex with a common Smad, Smad 4, for translocation to the nucleus and regulation of gene transcription (13). Apart from this canonical signaling, TGFb also initiates other noncanonical signal transduction pathways involving mitogen-activated protein kinases, PI 3-kinase/Akt/mTORC1, and Rho GTPase, which contribute to the renal complications under disease conditions (13)(14)(15)(16)(17).
Among many hormones, growth factors, and cytokines, platelet-derived growth factor (PDGF) contributes to significant injury in different renal, especially glomerular, diseases (5). Four different isoforms of PDGF (A, B, C, and D) form a total of five homo-or heterodimers (18). These dimers bind to three This article contains supporting information. ‡ These authors contributed equally to this work. * For correspondence: Goutam Ghosh-Choudhury, choudhuryg@uthscsa. edu.
dimeric PDGF receptors (PDGFRs), aa, bb, and ab, with variable affinity (19). For example, whereas PDGF AA and CC show affinity for PDGFRa, CC also binds to PDGFRab. PDGF DD binds to PDGFRab at a low affinity, but it interacts with PDGFRbb with significantly higher affinity. On the other hand, PDGF AB and BB bind to both receptors. All these PDGF dimers and three dimeric receptors are expressed in mesangial cells (19). In glomerulonephritis, PDGF BB, CC, and DD play prominent roles and stimulate proliferation of mesangial cells, whereas PDGF AA does not (5,(20)(21)(22). The importance of PDGF BB in glomerular mesangial cell biology is established because mice deficient in either the B-chain or its receptor b lack these glomerular cells (23,24). In fibrotic disorders such as diabetic nephropathy where TGFb contributes to the pathology, expression of PDGF BB and PDGFRb is increased, especially in mesangial cells (25,26). Whether PDGFRb contributes to the signal transduction of TGFb in mesangial cell pathology through a cross-talk has not been investigated. In our studies, we show that TGFb activates PDGFRb to initiate Akt signaling to inactivate PRAS40, which results in the increased mTORC1 activity necessary for mesangial cell hypertrophy and expression of matrix proteins fibronectin and collagen I (a2).

Activation of TGFbRI stimulates PDGFRb autophosphorylation in mesangial cells
Previously, we demonstrated that TGFb increases the tyrosine phosphorylation of several proteins, including a protein at 190 kDa to induce noncanonical signaling in mesangial cells (15). To characterize the tyrosine kinase involved in the action of TGFb, we considered PDGFRb because it is a receptor tyrosine kinase, it is one of most abundant receptors expressed in mesangial cells, and it elicits pathology in glomerulosclerosis (5,19,27). Because activation of PDGFRb requires its phosphorylation at Tyr-857 in the activation loop, we examined this in glomerular mesangial cells. TGFb increased the phosphorylation of PDGFRb in a time-dependent manner (Fig. 1A). Because TGFb utilizes its type I serine/threonine kinase receptor for its signal transduction, we used a specific TGFbRI inhibitor, SB-431542 (SB), to test its involvement in PDGFRb phosphorylation. Similar to the effect on phosphorylation of Smad 2 and Smad 3, SB significantly inhibited TGFb-stimulated PDGFRb Tyr-857 phosphorylation (Fig. 1, B-D). PDGFRb undergoes autophosphorylation at this site (13). Therefore, we used a specific PDGFRb ATP competitive inhibitor, JNJ-10198409 (JNJ), in mesangial cells. JNJ blocked the autophosphorylation of PDGFRb in response to TGFb (Fig. 1E). Importantly, JNJ did not have any effect on phosphorylation of Smad 2 and Smad 3 in response to TGFb (Fig. 1, F and G). Similarly, siRNAs against PDGFRb to block its expression (Fig. 1H) showed no effect on TGFb-induced phosphorylation of Smad 2 and Smad 3 (Fig. 1, I and J). These data indicate that TGFb activates PDGFRb; however, PDGFRb does not control the canonical Smad 2/3 phosphorylation by TGFbRI.

TGFb uses PDGFRb for Akt kinase activation
We and others demonstrated previously that TGFb regulates PI 3-kinase-dependent Akt activation (15,28,29). For activation of PI 3-kinase, the SH2 domain of the lipid kinase has to interact with the tyrosine phosphorylated proteins (30). In the case of activated PDGFRb, PI 3-kinase associates with the phosphorylated Tyr-751 (31). Therefore, we determined the phosphorylation of PDGFRb at this site in mesangial cells. TGFb increased the phosphorylation of PDGFRb at Tyr-751 in a time-dependent manner similar to its catalytic loop Tyr-857 phosphorylation ( Fig. 2A). Both TGFbRI and PDGFRb inhibitors SB and JNJ significantly blocked the phosphorylation of PDGFRb at Tyr-751 (Fig. 2, B and C). Activation of PI 3-kinase results in the phosphorylation of its downstream target Akt at the catalytic loop Thr-308 and hydrophobic motif Ser-473 sites (32). Therefore, to determine the activation of PI 3-kinase, we measured the phosphorylation of these Akt residues. TGFb increased the phosphorylation of both these residues in a time-dependent manner (Fig. 2D). JNJ significantly inhibited the TGFb-induced phosphorylation of Akt at both of these sites (Fig. 2E). Because phosphorylation of Akt increases its kinase activity, we determined the phosphorylation of one of its endogenous substrates, glycogen synthase kinase 3-b (GSK3b). JNJ significantly inhibited GSK3b phosphorylation by TGFb (Fig. 2F). To confirm this observation, we used siRNAs to down-regulate PDGFRb in mesangial cells. siRNAs against PDGFRb inhibited the TGFbinduced phosphorylation of Akt and its endogenous substrate GSK3b (Fig. 2, G and H).

PDGFRb regulates TGFb-induced mTORC1 activation
We and others demonstrated that TGFb regulates mTORC1 activity (14,16,33). The exclusive mTORC1 subunit PRAS40 acts as an inhibitor of mTORC1 activity (34). Similarly, the GTPase-activating protein tuberin inhibits mTORC1 by acting on the Rheb GTPase (35). When phosphorylated by Akt, both of these proteins undergo inactivation (36)(37)(38). We determined the effect of PDGFRb inhibition on the phosphorylation of these proteins. JNJ significantly inhibited the TGFb-stimulated phosphorylation of PRAS40 and tuberin (Fig. 3, A and B). Similarly, PDGFRb siRNAs blocked phosphorylation of PRAS40 and tuberin in response to TGFb (Fig. 3, C and D). Because phosphorylation/inactivation of PRAS40 and tuberin activate mTORC1, we determined activity of the latter by using phosphorylation of its substrates S6 kinase and 4EBP1. We also examined the phosphorylation of ribosomal protein S6 (rps6), which undergoes phosphorylation upon S6 kinase activation. TGFb increased the phosphorylation of S6 kinase and 4EBP1 at mTORC1 sites and rps6 at S6 kinase sites. Both JNJ and siPDGFRb markedly inhibited phosphorylation at these sites by TGFb (Fig. 3, E-J).
These results indicate that PDGFRb is involved in TGFbinduced Akt/mTORC1 activation but not in the regulation of ERK1/2 and STAT3.

TGFb regulates PDGF B expression
Our results above demonstrate that TGFb activates PDGFRb.
To determine the mechanism of activation of PDGFRb, we considered the expression of the PDGFRb ligand PDGF B, which forms BB homodimer to activate the receptor (41)(42)(43). TGFb time-dependently increased the expression of PDGF B in mesangial cells (Fig. 5A). These results indicate that a TGFb-induced increase in PDGF expression may regulate PDGFRb activation. We tested this hypothesis. We used siRNAs against PDGF B. siPDGF B inhibited TGFb-stimulated phosphorylation of PDGFRb (Fig. 5B). Also, TGFb-induced phosphorylation of Akt was inhibited by siRNAs against PDGF B (Fig. 5C). These data conclusively demonstrate that PDGF B regulates the activation of PDGFRb and the downstream activation of Akt/mTORC1 by TGFb. We tested this notion by using PDGF BB ligand directly in mesangial cells. PDGF BB increased the phosphorylation of PDGFRb in a time-de-pendent manner (Fig. 6A) similar to the kinetics of PDGF B expression by TGFb (Fig. 5A). Also, PDGF BB enhanced the phosphorylation of Akt (Fig. 6B), resulting in phosphorylation of PRAS40 and tuberin (Fig. 6, C and D), which led to the activation of mTORC1 as judged by phosphorylation of S6 kinase, rps6, and 4EBP1 ( Fig. 6, E-G). Furthermore, increased phosphorylation of PDGFRb was observed when PDGF BB ligand was added as compared with TGFb alone (Fig. S1A). No further increase in phosphorylation was found when both ligands were added (Fig. S1A). Importantly, this increased PDGFRb phosphorylation resulted in a similar increase in Akt phosphorylation leading to mTORC1 activation ( Fig. S1, B-G). Similar to the observations with TGFb, PDGFRb inhibitor JNJ blocked PDGF BB-stimulated phosphorylation of Akt, PRAS40, and tuberin ( Fig. S2, A-C). JNJ also blocked activation of mTORC1 in response to PDGF BB (Fig. S2, D-F).

PDGF regulates TGFb signaling
The above results show that TGFb utilizes PDGF ligand to activate PDGFRb. We examined whether there is a crosstalk between PDGF and TGFb. Incubation of mesangial cells with PDGF increased the expression of TGFb in a time-dependent manner (Fig. 7A). Inhibition of PDGFRb with JNJ blocked PDGF-stimulated TGFb expression (Fig. 7B). To test if PDGF signals through TGFb, we examined phosphorylation of Smads. PDGF increased phosphorylation of Smad 2 and Smad 3, and the TGFb receptor kinase inhibitor SB blocked these phosphorylations (Fig. 7, C and D). Interestingly, TGFb antibody inhibited PDGF-stimulated Smad 2 and Smad 3 phosphorylation (Fig. 7, E and F). These data demonstrate the presence of a positive feedback loop between TGFb and PDGF.

PRAS40 regulates TGFb-induced mTORC1 activity downstream of PDGFRb
Our results above show that TGFb-induced PDGFRb activation contributes to phosphorylation of Akt and PRAS40, resulting in mTORC1 activation ( Fig. 2 and Fig. 3). To determine whether Akt-mediated phosphorylation of PRAS40 regulates the activation of mTORC1 downstream of PDGFRb, we transfected mesangial cells with siRNAs against PDGFRb, along with constitutively active myristoylated Akt (myr-Akt) and the non-phosphorylatable mutant PRAS40 T246A. Expression of myr-Akt alone increased the phosphorylation of S6 kinase, rps6, and 4EBP1, similar to TGFb treatment (data not shown). However, constitutively active Akt kinase reversed the inhibition of phosphorylation of S6 kinase, rps6, and 4EBP1 by siPDGFRb in the presence of TGFb (Fig. 8, A-C; compare 4th lanes with 3rd lanes). Importantly, the phospho-deficient  mutant of PRAS40 abrogated the reversal of phosphorylation of these mTORC1 and S6 kinase substrates by constitutively active Akt (Fig. 8, A-C; compare 5th lanes with 4th lanes). These results conclusively demonstrate that Akt-mediated phosphorylationdependent inactivation of PRAS40 downstream of PDGFRb regulates TGFb-induced mTORC1 activation in mesangial cells.

Activation of Akt kinase by PDGFRb regulates TGFb-induced mesangial cell hypertrophy and matrix protein expression
We demonstrated that TGFb-stimulated Akt kinase regulates mesangial cell hypertrophy and expression of matrix proteins (14,44). Because PDGFRb contributes to the activation of Akt by TGFb, we investigated the involvement of this receptor tyrosine kinase in mesangial cell hypertrophy. JNJ significantly inhibited TGFb-induced protein synthesis and hypertrophy of mesangial cells (Fig. 9, A and B). Similarly, siRNAs against PDGFRb attenuated both these phenomena (Fig. 9, C and D). Because PRAS40 phosphorylation by Akt regulates its downstream signaling, we determined the involvement of Akt. Expression of constitutively active myr-Akt restored the siPDGFRb-mediated inhibition of mesangial cell protein synthesis and hypertrophy in the presence of TGFb (Fig. 9, E and F). Furthermore, expression of phospho-deficient PRAS40 T246A significantly prevented the myr-Akt-mediated reversal of siPDGFRb-induced inhibition of protein synthesis and hypertrophy (Fig. 9, E and F).
TGFb regulates renal fibrosis, including glomerulosclerosis, by increasing the synthesis and deposition of matrix proteins fibronectin and collagen (3). TGFb increased the expression of fibronectin and collagen I (a2) in mesangial cells (44)(45)(46). Both JNJ and siRNA against PDGFRb inhibited TGFb-stimulated expression of these matrix proteins (Fig. 10, A-D). We showed above that TGFb-induced expression of PDGF B regulates activation of PDGFRb and Akt kinase (Fig. 5). Therefore, we tested the effect of siPDGF B on expression of fibronectin and colla-gen I (a2). Inhibition of PDGF B blocked fibronectin and collagen I (a2) expression in response to TGFb (Fig. 10, E and F). To further evaluate the mechanism of matrix expression, we determined the role of Akt because it is activated downstream of TGFb by PDGFRb activation (Fig. 2D, Fig. 6B, and Fig. S1B). We used an Akt inhibitor, MK, which blocked Akt phosphorylation by TGFb (Fig. S3A). MK inhibited TGFb-induced expression of fibronectin and collagen I (a2) (Fig. S3, B and C). We showed above that siPDGFRb blocked TGFb-induced expression of fibronectin and collagen I (a2) (Fig. 10, C and D). Expression of myr-Akt reversed the siPDGFRb-mediated inhibition of TGFb-stimulated expression of both of these matrix proteins (Fig. 10, G and H; compare 4th lanes with 3rd lanes). Importantly, PRAS40 T246A phospho-deficient mutant inhibited this myr-Akt effect on fibronectin and collagen I (a2) expression (Fig. 10, G and H; compare 5th lanes with 4th lanes). Together, these data show a significant role of Akt downstream of PDGFRb in mesangial cell matrix protein expression. Furthermore, our results demonstrate a critical role for the Akt and its substrate PRAS40 in PDGFRb-mediated signaling by TGFb in mesangial cell pathology.

PDGFRb-stimulated mTORC1 regulates TGFb-induced mesangial cell hypertrophy and matrix protein expansion
We demonstrated previously that activation of mTORC1 by TGFb contributes to mesangial cell hypertrophy and matrix protein expression (14,46,47). Our results above show a role of PDGFRb in TGFb-stimulated mTORC1 activation in mesangial cells (Fig. 3). Therefore, we examined whether mTORC1 downstream of PDGFRb regulates mesangial cell hypertrophy. To test this, we used a vector expressing raptor, which is a constitutive component of mTORC1 and is essential for its kinase activity (48,49). Expression of raptor in mesangial cells increased mTORC1 activity as judged by phosphorylation of S6 kinase, rps6, and 4EBP1 (Fig. S4, A-C). Raptor reversed the siPDGFRb-mediated inhibition of protein synthesis and hypertrophy of mesangial cells in the presence of TGFb (Fig. 11, A and  B). Similarly, raptor restored the inhibition of fibronectin and collagen I (a2) by siPDGFRb in the TGFb-stimulated cells (Fig. 11, C and D). To confirm these observations, we used a mutant of mTOR, which delivers hyperactive mTORC1 (49,50). Expression of this mutant increased the phosphorylation of S6 kinase, rps6, and 4EBP1 in the absence of any effect on phosphorylation of Akt at Ser-473 in mesangial cells (Fig. S5, A-D), indicating that it acts as hyperactive mTORC1 and not mTORC2. Expression of this hyperactive mTORC1 reversed the siPDGFRb-mediated inhibition of protein synthesis, hypertrophy, and matrix proteins fibronectin and collagen I (a2) expression (Fig. S6, A-D). These results show a positive role of mTORC1 downstream of PDGFRb in the TGFb-induced mesangial cell pathology.
Phosphorylation of PDGFRb in the kidneys of type 1 diabetic OVE26 mice and streptozotocin-induced rats Diabetic nephropathy is a fibrotic disorder. In this disease, mesangial expansion proceeds to glomerular hypertrophy and accumulation of matrix proteins, which lead to glomerulosclerosis (51). Hyperglycemia produces multiple fibrotic growth factors, including TGFb, which contribute to the pathogenesis of diabetic nephropathy (4, 51). Our results described above show a role of PDGFRb in TGFb-induced mesangial cell hypertrophy and matrix protein expression. To investigate the in vivo relevance of our results, we used OVE26 type 1 diabetic mice. We demonstrated recently that the blood glucose levels in 3-month-old OVE26 mice are significantly increased and are associated with renal hypertrophy (52). Renal cortical lysates were used to examine the expression of TGFb. In diabetic renal cortical samples, a significant increase in TGFb was detected (Fig. 12, A and B). This increased TGFb correlated with an increase in phosphorylation of Smad 2 and Smad 3, two downstream TGFb receptor-specific Smads (Fig. 12, C-E). Furthermore, diabetic mice showed a significantly increased expression of PDGF B (Fig. 12, F and G). The level of phosphorylation of PDGFRb at Tyr-857, which is required for its tyrosine kinase activity, was significantly enhanced (Fig. 12, H and I). Also, the level of phosphorylation at Tyr-751 of PDGFRb was elevated in the diabetic renal cortex (Fig. 12, J and K). Our results above show that PDGFRb is necessary for TGFb-induced mTORC1 activation (Fig. 3). We determined the phosphorylation of S6 kinase, rps6, and 4EBP1 as readout of mTORC1 activation in renal cortical lysates. A significant increase in phosphorylation of S6 kinase, rps6, and 4EBP1 was observed in the diabetic mice, indicating activation of mTORC1 (Fig. 12, L-R). To confirm these observations in mice, we used a streptozotocin (STZ)-induced rat model of type 1 diabetes, which showed early pathologic changes of diabetic nephropathy (44). From kidney cortex, we prepared glomeruli, which contains the mesangial cells. Glomerular lysates were used to determine the expression of TGFb. In the diabetic glomeruli, significantly increased TGFb was detected, which correlated with enhanced Smad 2 and Smad 3 phosphorylation (Fig. S7, A-E). Furthermore, increased expression of PDGF B and phosphorylation of PDGFRb were observed in the diabetic glomeruli (Fig. S7, F-K). Additionally, diabetic rat glomeruli showed activation of mTORC1 as judged by phosphorylation of S6 kinase, rps6, and 4EBP1 (Fig. S7, L-R). These results show that activation of PDGFRb is associated with increased expression of TGFb in the diabetic kidney.

Discussion
We report activation of PDGFRb by TGFb via expression of PDGF, which also contributes to TGFb signaling in glomerular mesangial cells (Fig. 13). We show that TGFb-induced Akt kinase activation requires PDGFRb tyrosine kinase activity. Furthermore, we provide novel evidence that Akt-mediated phosphorylation of PRAS40 by TGFb is mediated by PDGFRb and is required for mesangial cell hypertrophy and matrix protein expression. Finally, we show activation of PDGFRb and mTORC1 in kidneys of diabetic mice that are undergoing matrix expansion.
A role of TGFb in human chronic kidney disease was established decades ago (53,54). Also, in the animal models of renal fibrosis such as diabetic nephropathy, increased expression of TGFb promotes the pathology (4,17,55). Similarly, in a rat model of glomerulonephritis, increased expression of TGFb promotes fibrosis (56). Administration of anti-TGFb antibody ameliorates the disease progression (57). Similarly, a murine monoclonal TGFb antibody reduced matrix expansion and disease-associated histological changes in adriamycin-and podocyte ablation-induced nephropathies in mice (58). Furthermore, in diabetic nephropathy associated with STZ-induced type 1 diabetes in mice and with type 2 diabetic db/db mice, neutralization of TGFb with anti-TGFb antibody ameliorates the glomerular hypertrophy and matrix protein expansion (11,12). A hypomorphic mouse with TGFb expression at 10% the level of that in WT showed significant reduction in glomerular filtration rate and albuminuria. In fact, 3-fold overexpression of TGFb in mice causes glomerulosclerosis and albuminuria (59). More recently, administration of a soluble TGFb receptor II by a gene therapy protocol to mice with renal fibrosis, where TGFb contributes to the pathology, showed significant beneficial effects (60). These results conclusively demonstrate a significant pathologic role of TGFb in renal diseases, suggesting that blocking of TGFb may be beneficial in renal fibrosis. However, the lack of progress is due to the concerns that inhibition of TGFb may enhance the risk of autoimmune disease (17). Because TGFb has multiple roles in maintaining various homeostatic processes, direct targeting of this cytokine may elicit multi-organ side effects. Thus, an alternative therapeutic strategy to in-hibit pathologic actions of TGFb in glomerulosclerosis needs to be identified.
The role of receptor tyrosine kinases in renal disease is established (61). Both positive and negative regulatory roles of epidermal growth factor receptor (EGFR) in renal pathology have been reported. For example, in ischemic kidney injury, activation of EGFR ameliorates the disease progression (62). Furthermore, in an EGFR mutant mouse with significantly reduced tyrosine kinase activity, tubular damage was more severe with increased apoptosis after nephrotoxic injury than in the WT mice, demonstrating a protective role of EGFR (63). On the other hand, a pathologic role of this receptor in TGFb-mediated renal fibrosis was reported (17). EGFR is abundantly expressed in the renal proximal tubular epithelial cells and interstitial fibroblasts (62). Inhibition of EGFR and proximal tubule-specific deletion of this receptor tyrosine kinase ameliorate TGFb-mediated renal fibrosis (64,65). Also, involvement of Src tyrosine kinase has been reported in angiotensin IIinduced kidney fibrosis (66). Thus, a role of tyrosine kinases in renal fibroblasts and tubular epithelial cells is established for induction of fibrosis. In the present study, we found that TGFb activates the PDGFRb in the renal mesangial cells. These results are in contrast to the observations of a recent report in which TGFb did not have any effect on PDGFRb phosphorylation in fibroblasts, suggesting cell-specificity for this interaction (67). In fact, we showed that TGFb increased the expression of PDGF, which binds to the PDGFRb to induce autophosphorylation and its activation. Addition of PDGF directly to mesangial cells also increased phosphorylation/activation of PDGFRb.
Our data in renal mesangial cells suggest that PDGFRb may serve as the initiator of TGFb noncanonical signaling. We and others previously reported that both Smad 3 and PI 3-kinase/ Akt signaling regulate mesangial cell pathology in response to TGFb (15,29,45,68). PI 3-kinase activation requires its association with tyrosine phosphorylated proteins (69). In the case of PDGFRb, the specific residue was identified as Tyr-751 (31). We showed that TGFb increased the tyrosine phosphorylation of this residue, which was sensitive to the inhibition of PDGFRb tyrosine kinase activity, demonstrating autophosphorylation by PDGFRb. These results indicate that TGFb may activate PI 3-kinase via this direct interaction mechanism with PDGFRb. In fact, we found that TGFb-stimulated phosphorylation of Akt, a downstream target of active PI 3-kinase, depended upon PDGFRb and its tyrosine kinase activity. This observation was similar to that obtained with direct addition of PDGF to mesangial cells. Interestingly, we found that PDGF increased the expression of TGFb to induce Smad 2/3 phosphorylation. These results identify a positive feedback loop between TGFb and PDGF signaling in mesangial cells (Fig. 13) Recently, we and others demonstrated that mTORC1 controls renal cell pathology in rodent models of fibrosis where TGFb plays important role (70)(71)(72)(73)(74)(75)(76). Three protein subunits of mTORC1, raptor, deptor, and PRAS40, regulate the activity of this kinase complex (48). Both nutrients and growth factors use independent mechanisms for mTORC1 activation. Amino acids promote formation of GTP-bound Rag proteins via Ragulator to recruit mTORC1 to GTP-bound Rheb for its activation (77). During growth factor receptor tyrosine kinase stimulation of cells, activated Akt kinase phosphorylates the raptor binding protein, PRAS40, which under basal state inhibits the substrate recruitment to mTORC1. Akt-mediated phosphorylation of PRAS40 results in its inactivation to increase the mTORC1 activity (34). For example, inactivation of PRAS40 in HeLa cells increased mTORC1 activity, leading to inhibition of their apoptosis. Interestingly, rapamycin did not reverse this anti-apoptotic effect, indicating that the effect of PRAS40 does not involve inhibition of mTORC1 (78). In 293 cells, PRAS40 inactivation showed inhibition of mTORC1 activity (79). These results are similar to those observed in Drosophila eyes where the hypomorphic allele of lobe, the fly ortholog of PRAS40, resulted in inhibition of mTORC1 and reduced eye size (80,81). We reported previously that TGFb stimulates phosphorylation of PRAS40 in mesangial cells (46). Now we show that PDGFRb tyrosine kinase is required for this phosphorylation of PRAS40 by TGFb, similar to PDGF addition. In contrast to the results in 293 cells and in Drosophila described above, phosphorylation of PRAS40 by PDGFRb-mediated activated Akt kinase enhances mTORC1 activity in mesangial cells by both TGFb and PDGF.
Induction of mesangial cell hypertrophy and matrix protein accumulation correlate with increased expression of TGFb in rodent models of fibrosis (17,51,82). Also, renal cells derived from TGFb null mice show impaired hypertrophic response and fibronectin expression (83). Similarly, glomerular hypertrophy and mesangial matrix expansion are reduced in TGFb receptor II heterozygous mice models of diabetes (84). We demonstrated previously that TGFb regulates mesangial cell hypertrophy and fibronectin expression (14,46,47). A role of Akt kinase was initially identified in cell-size control in the fruit fly. Overexpression of Akt in the imaginal disc of Drosophila increases the cell size (85). Similarly, transgenic mice expressing constitutively active Akt in the heart show cardiomyocyte hypertrophy (86). Also, Akt kinase activity is required for TGFb-induced mesangial cell hypertrophy (44). We showed that expression of both fibronectin and collagen I (a2) by TGFb was meditated by Akt kinase. However, the specific substrate that regulates this TGFb response is not clear. In mouse hearts, Akt-mediated phosphorylation of PRAS40 increases mTORC1 activity to induce pathological hypertrophy and fibrosis (87). We reported the requirement of mTORC1 in TGFb-induced mesangial cell hypertrophy and matrix protein expression (14,46,47). Now we demonstrate that Akt-phosphorylated PRAS40 is required for mesangial cell hypertrophy and fibronectin and collagen I (a2) expression in response to TGFb. In fact, we provide evidence that PDGFRb tyrosine kinase downstream of TGFbRI acts as a mediator of these phenomena. To our knowledge, this is the first demonstration of the involvement of PDGFRb tyrosine kinase in TGFb-stimulated Akt/PRAS40/mTORC1 signaling that contributes to mesangial cell hypertrophy and matrix protein expansion. Furthermore, our results demonstrate an in vivo relevance showing association of increased TGFb expression with activation of PDGFRb and mTORC1 in the renal cortex and glomeruli of diabetic mice and rats, respectively. Thus, in TGFb-regulated fibrotic renal diseases such as diabetic nephropathy, attacking PDGFRb may provide an attractive therapeutic option that bypasses the detrimental effects of directly targeting TGFb.

Cell culture
Human mesangial cells were originally prepared by Abboud and co-workers (90) from glomeruli isolated by differential sieving. The resuspended glomeruli were digested with collagenase. Mesangial cells were cultured from outgrowths of collagenase-treated glomeruli and characterized (90). The frozen cells were thawed and grown in DMEM in the presence of 10% FBS as described previously (47,(91)(92)(93). The cells were used between passages 9 and 12. Before performing experiments, the cells were grown in complete medium until they reached confluency. The cell monolayer was then starved in serum-free medium for 24 h prior to incubation with TGFb (2 ng/ml) for indicated periods of time.

Animals
FVB mice overexpressing calmodulin transgene in the b-cell of pancreas, called OVE26 mice, were used and purchased from The Jackson Laboratory. At 3 days of age the OVE26 mice are hyperglycemic (94). At 2 months of age, these mice develop pathologies of diabetic nephropathy, including significant renal hypertrophy, glomerular hypertrophy, and albuminuria (52, 95). The control FVB and diabetic OVE26 mice had free access to food and water. At 3 months of age, the animals were sacrificed, the kidneys were removed, and renal cortical tissues were isolated and frozen as described previously (52).
To induce diabetes in rats (Sprague Dawley; 200-250 g), 55 mg/kg of body weight STZ in sodium citrate buffer (pH 4.5) was injected by tail vein. The blood glucose was measured after 24 h. The animals had free access to water and food. At 5 days after STZ injection, the rats were euthanized and the kidneys were removed to isolate cortical sections. Glomeruli were prepared from the kidney cortexes by a differential sieving technique as described (90). Both mice and rats were kept at the University of Texas Health Science Center animal facility. The University of Texas Health Science Center Institutional Animal Care and Use Committee approved both protocols.

Preparation of cell, cortical, and glomerular lysates
At the end of the incubation period, the mesangial cell monolayer was washed twice with PBS. The monolayer was then harvested in radioimmune precipitation assay buffer (RIPA; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na 3 VO 4 , 1% Nonidet P-40, 1 mM PMSF, and 0.1% protease inhibitor mixture). The renal cortexes from control and diabetic mice and the glomeruli from the control and STZinduced diabetic rats were harvested in the same RIPA buffer. The cells, cortexes, and glomeruli were lysed in RIPA at 4°C for 30 min. After the incubation, the cell, cortical, and glomerular extracts were centrifuged at 12,000 3 g for 30 min at 4°C. The cleared supernatant was collected as cell, cortical, and glomerular lysates. Protein concentration was determined.

Immunoblotting
Cell lysates containing equal amounts of protein were separated by SDS-PAGE. The proteins were transferred to PVDF membrane. For immunoblotting, the membrane containing the proteins was incubated with indicated primary antibody at 4°C. The primary antibody dilutions used for immunoblotting were 1:1000. The membrane was washed, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:10,000). The membrane was then incubated with enhanced chemiluminescence reagent to develop a specific protein band, which was visualized by exposing the membrane to X-ray film (91,96).

Transfection
The semiconfluent mesangial cell monolayer was washed with PBS. Opti-MEM was added. The expression vector or siR-NAs against PDGFRb were transfected in Opti-MEM with FuGENE HD as described previously (91). After 24 h, the cells were starved as described above prior to adding TGFb.

Measurement of protein synthesis
Protein synthesis was determined as described previously (14,89,91). Briefly, during the last four hours of incubation with TGFb, the cells were incubated with 1 mCi of [ 35 S]methionine as described before (44). At the end of incubation, the cells were lysed in RIPA as described above and the protein was estimated. Equal amount of protein was spotted onto 3MM filter paper and washed in boiling 10% trichloroacetic acid containing 0.1 gm/l methionine for 1 min. The filters were then dried and the radioactivity was counted using scintillation fluid.

Measurement of hypertrophy
The cells were trypsinized in the medium at the end of the incubation and counted using a hemocytometer (91,96). The cells were then gently centrifuged at 1,000 3 g for 5 min at 4°C. The cell pellet was washed with PBS and resuspended in RIPA buffer to lyse, as described above. Protein concentration was measured in the cell lysate and the ratio of total protein to cell number was determined. The increase in the ratio was considered as cell hypertrophy (44,89).

Statistics
Prism GraphPad software was used to determine the significance of the data. Analysis of variance followed by Tukey's multiple comparison test was used. A p-value of ,0.05 was considered significant (97).

Data availability
All data are contained within the article and in the supporting information. Conflict of interest-The authors declare that they do not have any conflicts of interest with the contents of the article.