Advertisement

MicroRNA-22 Is a Master Regulator of Bone Morphogenetic Protein-7/6 Homeostasis in the Kidney*

  • Jianyin Long
    Footnotes
    Affiliations
    Nephrology Section, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
    Search for articles by this author
  • Shawn S. Badal
    Footnotes
    Affiliations
    Nephrology Section, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

    Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas 77030
    Search for articles by this author
  • Yin Wang
    Affiliations
    Nephrology Section, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
    Search for articles by this author
  • Benny H.J. Chang
    Affiliations
    Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
    Search for articles by this author
  • Antony Rodriguez
    Affiliations
    Department of Surgery, Baylor College of Medicine, Houston, Texas 77030
    Search for articles by this author
  • Farhad R. Danesh
    Correspondence
    To whom correspondence should be addressed: Section of Nephrology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030. Tel.: 713-745-0858; Fax: 713-745-0854;
    Affiliations
    Nephrology Section, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

    Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas 77030

    Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants RO1DK091310 and RO1DK078900 (to F. R. D.).
    1 Both authors contributed equally to this work.
Open AccessPublished:October 25, 2013DOI:https://doi.org/10.1074/jbc.M113.498634
      Accumulating evidence suggests that microRNAs (miRNAs) contribute to a myriad of kidney diseases. However, the regulatory role of miRNAs on the key molecules implicated in kidney fibrosis remains poorly understood. Bone morphogenetic protein-7 (BMP-7) and its related BMP-6 have recently emerged as key regulators of kidney fibrosis. Using the established unilateral ureteral obstruction (UUO) model of kidney fibrosis as our experimental model, we examined the regulatory role of miRNAs on BMP-7/6 signaling. By analyzing the potential miRNAs that target BMP-7/6 in silica, we identified miR-22 as a potent miRNA targeting BMP-7/6. We found that expression levels of BMP-7/6 were significantly elevated in the kidneys of the miR-22 null mouse. Importantly, mice with targeted deletion of miR-22 exhibited attenuated renal fibrosis in the UUO model. Consistent with these in vivo observations, primary renal fibroblast isolated from miR-22-deficient UUO mice demonstrated a significant increase in BMP-7/6 expression and their downstream targets. This phenotype could be rescued when cells were transfected with miR-22 mimics. Interestingly, we found that miR-22 and BMP-7/6 are in a regulatory feedback circuit, whereby not only miR-22 inhibits BMP-7/6, but miR-22 by itself is induced by BMP-7/6. Finally, we identified two BMP-responsive elements in the proximal region of miR-22 promoter. These findings identify miR-22 as a critical miRNA that contributes to renal fibrosis on the basis of its pivotal role on BMP signaling cascade.

      Introduction

      Kidney fibrosis represents a failed wound healing process following a chronic and sustained injury and, regardless of the type of injury and etiology, is the common final outcome of many progressive chronic kidney diseases leading to the destruction and collapse of renal parenchyma and progressive loss of kidney function (
      • Boor P.
      • Ostendorf T.
      • Floege J.
      Renal fibrosis. Novel insights into mechanisms and therapeutic targets.
      ,
      • Farris A.B.
      • Colvin R.B.
      Renal interstitial fibrosis. Mechanisms and evaluation.
      ,
      • Liu Y.
      Cellular and molecular mechanisms of renal fibrosis.
      ). Activated fibroblasts, also known as myofibroblasts, are commonly regarded as the main effector cells responsible for the excess of extracellular matrix production, a key pathological feature of renal fibrosis. Although the origin of the activated fibroblasts pool in the kidney remains controversial, a growing body of evidence indicates that fibrogenic cues in the kidney evoke multiple intracellular signaling pathways that ultimately lead to phenotypic changes in the fibroblasts, resulting in their activation with characteristic expression of several smooth muscle cell markers (
      • Boor P.
      • Ostendorf T.
      • Floege J.
      Renal fibrosis. Novel insights into mechanisms and therapeutic targets.
      ,
      • Farris A.B.
      • Colvin R.B.
      Renal interstitial fibrosis. Mechanisms and evaluation.
      ,
      • Liu Y.
      Cellular and molecular mechanisms of renal fibrosis.
      ). Among the many fibrogenic factors that regulate renal fibrotic processes, TGF-β and connective tissue growth factor are regarded as key fibrogenic cytokines (
      • Boor P.
      • Ostendorf T.
      • Floege J.
      Renal fibrosis. Novel insights into mechanisms and therapeutic targets.
      ,
      • Farris A.B.
      • Colvin R.B.
      Renal interstitial fibrosis. Mechanisms and evaluation.
      ,
      • Liu Y.
      Cellular and molecular mechanisms of renal fibrosis.
      ,
      • Lan H.Y.
      Diverse roles of TGF-β/Smads in renal fibrosis and inflammation.
      ), whereas bone morphogenic protein-7 (BMP-7)
      The abbreviations used are: BMP
      bone morphogenetic protein
      miRNA
      microRNA
      BMPR1B
      bone morphogenetic protein receptor, type 1B
      αSMA
      smooth muscle α-actin
      UUO
      unilateral ureteral obstruction
      BRE
      BMP-responsive element
      RT-qPCR
      real time quantitative PCR.
      and BMP-6, have been known as natural antagonists of TGF-β signaling and anti-fibrogenic factors that have been shown to prevent renal fibrosis in several experimental models (
      • Morrissey J.
      • Hruska K.
      • Guo G.
      • Wang S.
      • Chen Q.
      • Klahr S.
      Bone morphogenetic protein-7 improves renal fibrosis and accelerates the return of renal function.
      ,
      • Zeisberg M.
      • Hanai J.
      • Sugimoto H.
      • Mammoto T.
      • Charytan D.
      • Strutz F.
      • Kalluri R.
      BMP-7 counteracts TGF-β1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.
      ,
      • Dendooven A.
      • van Oostrom O.
      • van der Giezen D.M.
      • Leeuwis J.W.
      • Snijckers C.
      • Joles J.A.
      • Robertson E.J.
      • Verhaar M.C.
      • Nguyen T.Q.
      • Goldschmeding R.
      Loss of endogenous bone morphogenetic protein-6 aggravates renal fibrosis.
      ,
      • Jenkins R.H.
      • Fraser D.J.
      BMP-6 emerges as a potential major regulator of fibrosis in the kidney.
      ,
      • Weiskirchen R.
      • Meurer S.K.
      • Gressner O.A.
      • Herrmann J.
      • Borkham-Kamphorst E.
      • Gressner A.M.
      BMP-7 as antagonist of organ fibrosis.
      ,
      • Yan J.D.
      • Yang S.
      • Zhang J.
      • Zhu T.H.
      BMP6 reverses TGF-β1-induced changes in HK-2 cells. Implications for the treatment of renal fibrosis.
      ,
      • Boon M.R.
      • van der Horst G.
      • van der Pluijm G.
      • Tamsma J.T.
      • Smit J.W.
      • Rensen P.C.
      Bone morphogenetic protein 7. A broad-spectrum growth factor with multiple target therapeutic potency.
      ).
      BMPs are important signaling molecules that were first identified by their ability to induce bone and cartilage and subsequently were shown to be pleiotropic cytokines controlling a wide variety of biological processes (
      • Blitz I.L.
      • Cho K.W.
      Finding partners. How BMPs select their targets.
      ,
      • Bragdon B.
      • Moseychuk O.
      • Saldanha S.
      • King D.
      • Julian J.
      • Nohe A.
      Bone morphogenetic proteins. A critical review.
      ). Signaling by BMPs has been long implicated in kidney homeostasis and kidney injury repair (
      • Cain J.E.
      • Hartwig S.
      • Bertram J.F.
      • Rosenblum N.D.
      Bone morphogenetic protein signaling in the developing kidney. Present and future.
      ). In the BMP canonical signaling pathway, the constitutively active type II receptors, upon binding to BMP ligands, phosphorylate and thus activate their type I partners, which in turn phosphorylate their intracellular effectors, the receptor-regulated Smad proteins 1, 5, and 8 (Smad1/5/8) (
      • Blitz I.L.
      • Cho K.W.
      Finding partners. How BMPs select their targets.
      ,
      • Bragdon B.
      • Moseychuk O.
      • Saldanha S.
      • King D.
      • Julian J.
      • Nohe A.
      Bone morphogenetic proteins. A critical review.
      ,
      • Miyazono K.
      • Maeda S.
      • Imamura T.
      BMP receptor signaling. Transcriptional targets, regulation of signals, and signaling cross-talk.
      ). Phosphorylated Smads form complexes with Smad4 and translocate to the nucleus where they regulate expression of their target genes by binding to BMP-responsive elements (BRE) on the promoter region of their target genes (
      • Cain J.E.
      • Hartwig S.
      • Bertram J.F.
      • Rosenblum N.D.
      Bone morphogenetic protein signaling in the developing kidney. Present and future.
      ,
      • Miyazono K.
      • Maeda S.
      • Imamura T.
      BMP receptor signaling. Transcriptional targets, regulation of signals, and signaling cross-talk.
      ,
      • Feng X.H.
      • Derynck R.
      Specificity and versatility in TGF-β signaling through Smads.
      ,
      • Massagué J.
      TGFβ signalling in context.
      ). Although much is known on the BMP canonical pathway, the precise mechanism of its regulation remains inadequately described.
      MicroRNAs (miRNAs) comprise a broad class of small noncoding RNAs that negatively regulate gene expression by base-pairing to partial or perfect complementary sites in the 3′-UTRs of specific target mRNAs (
      • Ambros V.
      The functions of animal microRNAs.
      ,
      • Bartel D.P.
      MicroRNAs. Target recognition and regulatory functions.
      ). Recent studies from our group (
      • Long J.
      • Wang Y.
      • Wang W.
      • Chang B.H.
      • Danesh F.R.
      Identification of microRNA-93 as a novel regulator of vascular endothelial growth factor in hyperglycemic conditions.
      ,
      • Long J.
      • Wang Y.
      • Wang W.
      • Chang B.H.
      • Danesh F.R.
      MicroRNA-29c is a signature microRNA under high glucose conditions that targets Sprouty homolog 1, and its in vivo knockdown prevents progression of diabetic nephropathy.
      ) and others (
      • Dey N.
      • Das F.
      • Mariappan M.M.
      • Mandal C.C.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Choudhury G.G.
      MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes.
      ,
      • Wang Q.
      • Wang Y.
      • Minto A.W.
      • Wang J.
      • Shi Q.
      • Li X.
      • Quigg R.J.
      MicroRNA-377 is up-regulated and can lead to increased fibronectin production in diabetic nephropathy.
      ,
      • Kato M.
      • Zhang J.
      • Wang M.
      • Lanting L.
      • Yuan H.
      • Rossi J.J.
      • Natarajan R.
      MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors.
      ,
      • Wei Q.
      • Bhatt K.
      • He H.Z.
      • Mi Q.S.
      • Haase V.H.
      • Dong Z.
      Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury.
      ,
      • Chau B.N.
      • Xin C.
      • Hartner J.
      • Ren S.
      • Castano A.P.
      • Linn G.
      • Li J.
      • Tran P.T.
      • Kaimal V.
      • Huang X.
      • Chang A.N.
      • Li S.
      • Kalra A.
      • Grafals M.
      • Portilla D.
      • MacKenna D.A.
      • Orkin S.H.
      • Duffield J.S.
      MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways.
      ) suggest that miRNAs are involved in the pathogenesis and progression of a variety of kidney diseases and hold great therapeutic potential. However, limited information is available on the exact role of miRNAs on renal fibrosis in vivo.
      Herein, using the established unilateral ureteral obstruction (UUO) mouse model of renal fibrosis as our experimental model, we found that miR-22 deletion attenuates kidney fibrosis in vivo by targeting BMP-7/6 and BMP receptor type I receptor (BMPR1B). Our findings also suggest that there is a regulatory feedback circuit between miR-22 and BMP-7/6 signaling, whereby miR-22 targets BMP-7/6, but it is by itself induced by this pathway. Furthermore, we identified two BREs in the miR-22 promoter region, which are responsible for the transcriptional regulation of BMP-7/6 on miR-22 expression.

      DISCUSSION

      Emerging evidence suggests that BMP-7/6, members of the TGF-β superfamily of cysteine knot cytokines, play pivotal roles in kidney homeostasis mainly by antagonizing TGF-β-induced profibrogenic signaling that contribute to the accumulation of the extracellular matrix (
      • Morrissey J.
      • Hruska K.
      • Guo G.
      • Wang S.
      • Chen Q.
      • Klahr S.
      Bone morphogenetic protein-7 improves renal fibrosis and accelerates the return of renal function.
      ,
      • Zeisberg M.
      • Hanai J.
      • Sugimoto H.
      • Mammoto T.
      • Charytan D.
      • Strutz F.
      • Kalluri R.
      BMP-7 counteracts TGF-β1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.
      ,
      • Dendooven A.
      • van Oostrom O.
      • van der Giezen D.M.
      • Leeuwis J.W.
      • Snijckers C.
      • Joles J.A.
      • Robertson E.J.
      • Verhaar M.C.
      • Nguyen T.Q.
      • Goldschmeding R.
      Loss of endogenous bone morphogenetic protein-6 aggravates renal fibrosis.
      ,
      • Jenkins R.H.
      • Fraser D.J.
      BMP-6 emerges as a potential major regulator of fibrosis in the kidney.
      ,
      • Weiskirchen R.
      • Meurer S.K.
      • Gressner O.A.
      • Herrmann J.
      • Borkham-Kamphorst E.
      • Gressner A.M.
      BMP-7 as antagonist of organ fibrosis.
      ,
      • Yan J.D.
      • Yang S.
      • Zhang J.
      • Zhu T.H.
      BMP6 reverses TGF-β1-induced changes in HK-2 cells. Implications for the treatment of renal fibrosis.
      ,
      • Boon M.R.
      • van der Horst G.
      • van der Pluijm G.
      • Tamsma J.T.
      • Smit J.W.
      • Rensen P.C.
      Bone morphogenetic protein 7. A broad-spectrum growth factor with multiple target therapeutic potency.
      ). In this study, we uncover a critical role of miR-22 on BMP signaling and renal fibrosis by providing strong evidence that miR-22 directly targets BMP-7, BMP-6, and BMPR1B. In support of our conclusions, we found that targeted deletion of miR-22 significantly attenuated renal fibrosis in the well characterized UUO model of kidney fibrosis. Knockdown of miR-22 both genetically or pharmacologically, resulted in elevated BMP-7/6 protein levels and enhanced BMP-7/6 downstream targets. Importantly, using miR-22-deficient primary renal fibroblasts, we demonstrated that miR-22 mimic could partially rescue the profibrotic effect of TGF-β1. Finally, we made the novel observation that BMP-7/6 per se could induce miR-22 at the transcriptional level through two consensus BREs in the proximal promoter region of miR-22 gene. Our findings suggest that miR-22 is an immediate early target gene of BMP-7/6, requiring an intact BMP-7/6 signaling, because pretreatment with dorsomorphin, a selective small molecule inhibitor of BMP type 1 receptor, abolished the induction of miR-22 expression by BMP-6. Taken together, our data suggest that miR-22 serves as an important regulator of BMP homeostasis and kidney fibrosis in response to extracellular signals.
      Recent studies from our group (
      • Long J.
      • Wang Y.
      • Wang W.
      • Chang B.H.
      • Danesh F.R.
      Identification of microRNA-93 as a novel regulator of vascular endothelial growth factor in hyperglycemic conditions.
      ,
      • Long J.
      • Wang Y.
      • Wang W.
      • Chang B.H.
      • Danesh F.R.
      MicroRNA-29c is a signature microRNA under high glucose conditions that targets Sprouty homolog 1, and its in vivo knockdown prevents progression of diabetic nephropathy.
      ) and others (
      • Dey N.
      • Das F.
      • Mariappan M.M.
      • Mandal C.C.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Choudhury G.G.
      MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes.
      ,
      • Wang Q.
      • Wang Y.
      • Minto A.W.
      • Wang J.
      • Shi Q.
      • Li X.
      • Quigg R.J.
      MicroRNA-377 is up-regulated and can lead to increased fibronectin production in diabetic nephropathy.
      ,
      • Kato M.
      • Zhang J.
      • Wang M.
      • Lanting L.
      • Yuan H.
      • Rossi J.J.
      • Natarajan R.
      MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors.
      ,
      • Wei Q.
      • Bhatt K.
      • He H.Z.
      • Mi Q.S.
      • Haase V.H.
      • Dong Z.
      Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury.
      ,
      • Chau B.N.
      • Xin C.
      • Hartner J.
      • Ren S.
      • Castano A.P.
      • Linn G.
      • Li J.
      • Tran P.T.
      • Kaimal V.
      • Huang X.
      • Chang A.N.
      • Li S.
      • Kalra A.
      • Grafals M.
      • Portilla D.
      • MacKenna D.A.
      • Orkin S.H.
      • Duffield J.S.
      MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways.
      ) have demonstrated that microRNAs play important roles in the development and progression of a variety of kidney diseases leading to kidney fibrosis as their final common pathway. Among miRNAs modulating kidney fibrosis, miR-21 has been recently reported by several groups to positively regulate renal fibrosis in UUO and ischemia reperfusion injury (
      • Dey N.
      • Das F.
      • Mariappan M.M.
      • Mandal C.C.
      • Ghosh-Choudhury N.
      • Kasinath B.S.
      • Choudhury G.G.
      MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes.
      ,
      • Chau B.N.
      • Xin C.
      • Hartner J.
      • Ren S.
      • Castano A.P.
      • Linn G.
      • Li J.
      • Tran P.T.
      • Kaimal V.
      • Huang X.
      • Chang A.N.
      • Li S.
      • Kalra A.
      • Grafals M.
      • Portilla D.
      • MacKenna D.A.
      • Orkin S.H.
      • Duffield J.S.
      MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways.
      ,
      • Ben-Dov I.Z.
      • Muthukumar T.
      • Morozov P.
      • Mueller F.B.
      • Tuschl T.
      • Suthanthiran M.
      MicroRNA sequence profiles of human kidney allografts with or without tubulointerstitial fibrosis.
      ,
      • Glowacki F.
      • Savary G.
      • Gnemmi V.
      • Buob D.
      • Van der Hauwaert C.
      • Lo-Guidice J.M.
      • Bouyé S.
      • Hazzan M.
      • Pottier N.
      • Perrais M.
      • Aubert S.
      • Cauffiez C.
      Increased circulating miR-21 levels are associated with kidney fibrosis.
      ,
      • Zarjou A.
      • Yang S.
      • Abraham E.
      • Agarwal A.
      • Liu G.
      Identification of a microRNA signature in renal fibrosis. Role of miR-21.
      ,
      • Zhong X.
      • Chung A.C.
      • Chen H.Y.
      • Meng X.M.
      • Lan H.Y.
      Smad3-mediated upregulation of miR-21 promotes renal fibrosis.
      ). Similarly, miR-200 has also been implicated in renal fibrosis (
      • Oba S.
      • Kumano S.
      • Suzuki E.
      • Nishimatsu H.
      • Takahashi M.
      • Takamori H.
      • Kasuya M.
      • Ogawa Y.
      • Sato K.
      • Kimura K.
      • Homma Y.
      • Hirata Y.
      • Fujita T.
      miR-200b precursor can ameliorate renal tubulointerstitial fibrosis.
      ,
      • Xiong M.
      • Jiang L.
      • Zhou Y.
      • Qiu W.
      • Fang L.
      • Tan R.
      • Wen P.
      • Yang J.
      The miR-200 family regulates TGF-β1-induced renal tubular epithelial to mesenchymal transition through Smad pathway by targeting ZEB1 and ZEB2 expression.
      ). The present study is the first to report the effect of miR-22 on BMP homeostasis and kidney fibrosis. However, we recognize that the effect of miR-22 on BMP-7/6 could be kidney- and tissue-specific. For instance, we have recently reported an association between miR-22 and cardiomyocyte hypertrophy and cardiac fibrosis (
      • Gurha P.
      • Abreu-Goodger C.
      • Wang T.
      • Ramirez M.O.
      • Drumond A.L.
      • van Dongen S.
      • Chen Y.
      • Bartonicek N.
      • Enright A.J.
      • Lee B.
      • Kelm Jr., R.J.
      • Reddy A.K.
      • Taffet G.E.
      • Bradley A.
      • Wehrens X.H.
      • Entman M.L.
      • Rodriguez A.
      Targeted deletion of microRNA-22 promotes stress-induced cardiac dilation and contractile dysfunction.
      ), where in contrast to the current findings, deletion of miR-22 was associated with enhanced cardiac fibrosis (
      • Gurha P.
      • Abreu-Goodger C.
      • Wang T.
      • Ramirez M.O.
      • Drumond A.L.
      • van Dongen S.
      • Chen Y.
      • Bartonicek N.
      • Enright A.J.
      • Lee B.
      • Kelm Jr., R.J.
      • Reddy A.K.
      • Taffet G.E.
      • Bradley A.
      • Wehrens X.H.
      • Entman M.L.
      • Rodriguez A.
      Targeted deletion of microRNA-22 promotes stress-induced cardiac dilation and contractile dysfunction.
      ,
      • Huang Z.P.
      • Chen J.
      • Seok H.Y.
      • Zhang Z.
      • Kataoka M.
      • Hu X.
      • Wang D.Z.
      MicroRNA-22 regulates cardiac hypertrophy and remodeling in response to stress.
      ). Our interpretation of these functional differences on the effect of miR-22 on tissue fibrosis is that the effect of miRNAs on their target mRNAs have been convincingly shown to be tissue- and temporal-dependent (
      • Sayed D.
      • Abdellatif M.
      MicroRNAs in development and disease.
      ,
      • van Rooij E.
      The art of microRNA research.
      ). Thus, miR-22 could preferentially target BMP-7/6 in the kidney, where BMP-7/6 are highly expressed, whereas other miR-22 targets (e.g., purine-rich element-binding protein B (
      • Gurha P.
      • Abreu-Goodger C.
      • Wang T.
      • Ramirez M.O.
      • Drumond A.L.
      • van Dongen S.
      • Chen Y.
      • Bartonicek N.
      • Enright A.J.
      • Lee B.
      • Kelm Jr., R.J.
      • Reddy A.K.
      • Taffet G.E.
      • Bradley A.
      • Wehrens X.H.
      • Entman M.L.
      • Rodriguez A.
      Targeted deletion of microRNA-22 promotes stress-induced cardiac dilation and contractile dysfunction.
      ) or HDAC4 (
      • Huang Z.P.
      • Chen J.
      • Seok H.Y.
      • Zhang Z.
      • Kataoka M.
      • Hu X.
      • Wang D.Z.
      MicroRNA-22 regulates cardiac hypertrophy and remodeling in response to stress.
      )) could be preferentially targeted by miR-22 in the heart. Finally, it is a canonical feature of miRNAs to exert their effects on target genes in a pleiotropic manner. Therefore, several other important targets, in addition to BMP-7/6 and BMPR1B, may potentially mediate the effect of miR-22 on renal fibrosis.
      Our findings also suggest that miR-22 forms a negative feedback circuit with BMP-7/6 signaling (Fig. 5E). This finding is of considerable interest because accumulating evidence suggests that not only miRNAs can regulate their target genes, but also these target genes may in turn interact with and regulate these specific miRNAs positively or negatively, either at the transcriptional or post-transcriptional level (
      • Mendell J.T.
      • Olson E.N.
      MicroRNAs in stress signaling and human disease.
      ,
      • Davis B.N.
      • Hilyard A.C.
      • Lagna G.
      • Hata A.
      SMAD proteins control DROSHA-mediated microRNA maturation.
      ). In our current study, we established such a relationship between miR-22 and BMP-7/6, whereby we identified BMP-7/6 and BMPR1B as direct targets of miR-22. On the other hand, we provided strong evidence that BMP-7/6 are also positive regulators of miR-22 levels mainly via their direct transcriptional regulation on miR-22 expression. These findings further extend the observations of others and validate the regulatory effects of miRNAs as fine-tuning elements (
      • Iliopoulos D.
      • Malizos K.N.
      • Oikonomou P.
      • Tsezou A.
      Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks.
      ), critical in the homeostasis of genes in response to a particular stimulus.
      Much attention has recently been paid to canonical BMP signaling, which is known to be largely mediated through activation of Smad1/5/8, whereas TGF-β signaling is mainly mediated by activation of Smad2/3. Both signaling pathways share Smad4 as a common assembly point into a heteromeric signaling complex that translocates into the nucleus to regulate target gene expression in coordination with several other transcription factors (
      • Blitz I.L.
      • Cho K.W.
      Finding partners. How BMPs select their targets.
      ,
      • Bragdon B.
      • Moseychuk O.
      • Saldanha S.
      • King D.
      • Julian J.
      • Nohe A.
      Bone morphogenetic proteins. A critical review.
      ,
      • Cain J.E.
      • Hartwig S.
      • Bertram J.F.
      • Rosenblum N.D.
      Bone morphogenetic protein signaling in the developing kidney. Present and future.
      ,
      • Miyazono K.
      • Maeda S.
      • Imamura T.
      BMP receptor signaling. Transcriptional targets, regulation of signals, and signaling cross-talk.
      ,
      • Feng X.H.
      • Derynck R.
      Specificity and versatility in TGF-β signaling through Smads.
      ,
      • Massagué J.
      TGFβ signalling in context.
      ). Cross-talk between TGF-β and BMP signaling is a well known feature of the TGF-β superfamily. BMP-7/6 is a potent antagonistic factor of TGF-β signaling (
      • Zeisberg M.
      • Hanai J.
      • Sugimoto H.
      • Mammoto T.
      • Charytan D.
      • Strutz F.
      • Kalluri R.
      BMP-7 counteracts TGF-β1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.
      ), and repression of BMP-7/6 expression leads to increased activation of TGF-β signaling, a key pathogenic factor within renal fibrosis (Fig. 4B). Within our model system, knock-out of miR-22 led to increased BMP-7/6 mRNA and protein levels, while it concomitantly caused blunting of the TGF-β1 induced profibrogenic genes (Fig. 4, A and B). These findings suggest that miR-22 plays a critical role in the cross-talk between TGF-β and BMP-7/6 signaling pathways.
      Interestingly, miR-22 itself is reduced under wild type UUO conditions, most likely as a consequence of reduced expression of its key regulators BMP-7/6, and an indirect effect via up-regulation of its negative regulator, TGF-β1 (Fig. 5E). The renoprotective effect of complete knock-out of miR-22 highlights the importance of sustained miR-22 expression to modulate levels of BMP-7/6 and BMPR1B under normal and pathogenic conditions.
      A surprising finding of this study was that we identified two conserved BREs within the proximal promoter region of the miR-22 host gene. A reporter gene expression assay indicated that these elements are necessary to enhance miR-22 promoter activity in response to BMP-7/6. These BREs presumably elicit their activation of miR-22 via Smad transcription interactions, likely Smad1/5/8, whose activity is directly related to BMP-mediated signaling. Further studies are needed to determine the role of different Smad complexes on BREs.
      In summary, our results provide a significant advance in our current understanding of BMP signaling and regulation in three distinct, yet related aspects: first, our in vitro and in vivo experiments indicate that BMP-7/6 and BMPR1B are direct targets of miR-22; second, our results demonstrate that the targeted deletion of miR-22 significantly attenuates renal fibrosis in an established model of kidney fibrosis; and third, our results reveal the existence of a local regulatory circuit, wherein miR-22 inhibits BMP-7/6 expression, which in turn acts to enhance miR-22 expression in a rapid, time-dependent manner. In this regard, we also characterized the regulatory mechanism of BMP-7/6 on miR-22 by identifying two BREs in the proximal region of miR-22 promoter. Taken together, our findings uncover a miR-22-BMP regulatory relationship in the kidney and suggest that BMP homeostasis is balanced through interactions between miR-22 and TGF-β activation.

      Acknowledgments

      We are grateful to Dr. Ralph C. Nicholas at Dartmouth Medical School for luciferase reporter construct 3.1-luc. We thank the Sequencing Core at Baylor College of Medicine.

      REFERENCES

        • Boor P.
        • Ostendorf T.
        • Floege J.
        Renal fibrosis. Novel insights into mechanisms and therapeutic targets.
        Nat. Rev. Nephrol. 2010; 6: 643-656
        • Farris A.B.
        • Colvin R.B.
        Renal interstitial fibrosis. Mechanisms and evaluation.
        Curr. Opin. Nephrol. Hypertens. 2012; 21: 289-300
        • Liu Y.
        Cellular and molecular mechanisms of renal fibrosis.
        Nat. Rev. Nephrol. 2011; 7: 684-696
        • Lan H.Y.
        Diverse roles of TGF-β/Smads in renal fibrosis and inflammation.
        Int. J. Biol. Sci. 2011; 7: 1056-1067
        • Morrissey J.
        • Hruska K.
        • Guo G.
        • Wang S.
        • Chen Q.
        • Klahr S.
        Bone morphogenetic protein-7 improves renal fibrosis and accelerates the return of renal function.
        J. Am. Soc. Nephrol. 2002; 13: S14-S21
        • Zeisberg M.
        • Hanai J.
        • Sugimoto H.
        • Mammoto T.
        • Charytan D.
        • Strutz F.
        • Kalluri R.
        BMP-7 counteracts TGF-β1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury.
        Nat. Med. 2003; 9: 964-968
        • Dendooven A.
        • van Oostrom O.
        • van der Giezen D.M.
        • Leeuwis J.W.
        • Snijckers C.
        • Joles J.A.
        • Robertson E.J.
        • Verhaar M.C.
        • Nguyen T.Q.
        • Goldschmeding R.
        Loss of endogenous bone morphogenetic protein-6 aggravates renal fibrosis.
        Am. J. Pathol. 2011; 178: 1069-1079
        • Jenkins R.H.
        • Fraser D.J.
        BMP-6 emerges as a potential major regulator of fibrosis in the kidney.
        Am. J. Pathol. 2011; 178: 964-965
        • Weiskirchen R.
        • Meurer S.K.
        • Gressner O.A.
        • Herrmann J.
        • Borkham-Kamphorst E.
        • Gressner A.M.
        BMP-7 as antagonist of organ fibrosis.
        Front. Biosci. 2009; 14: 4992-5012
        • Yan J.D.
        • Yang S.
        • Zhang J.
        • Zhu T.H.
        BMP6 reverses TGF-β1-induced changes in HK-2 cells. Implications for the treatment of renal fibrosis.
        Acta Pharmacol. Sin. 2009; 30: 994-1000
        • Boon M.R.
        • van der Horst G.
        • van der Pluijm G.
        • Tamsma J.T.
        • Smit J.W.
        • Rensen P.C.
        Bone morphogenetic protein 7. A broad-spectrum growth factor with multiple target therapeutic potency.
        Cytokine Growth Factor Rev. 2011; 22: 221-229
        • Blitz I.L.
        • Cho K.W.
        Finding partners. How BMPs select their targets.
        Dev. Dyn. 2009; 238: 1321-1331
        • Bragdon B.
        • Moseychuk O.
        • Saldanha S.
        • King D.
        • Julian J.
        • Nohe A.
        Bone morphogenetic proteins. A critical review.
        Cell Signal. 2011; 23: 609-620
        • Cain J.E.
        • Hartwig S.
        • Bertram J.F.
        • Rosenblum N.D.
        Bone morphogenetic protein signaling in the developing kidney. Present and future.
        Differentiation. 2008; 76: 831-842
        • Miyazono K.
        • Maeda S.
        • Imamura T.
        BMP receptor signaling. Transcriptional targets, regulation of signals, and signaling cross-talk.
        Cytokine Growth Factor Rev. 2005; 16: 251-263
        • Feng X.H.
        • Derynck R.
        Specificity and versatility in TGF-β signaling through Smads.
        Annu. Rev. Cell Dev. Biol. 2005; 21: 659-693
        • Massagué J.
        TGFβ signalling in context.
        Nat. Rev. Mol. Cell Biol. 2012; 13: 616-630
        • Ambros V.
        The functions of animal microRNAs.
        Nature. 2004; 431: 350-355
        • Bartel D.P.
        MicroRNAs. Target recognition and regulatory functions.
        Cell. 2009; 136: 215-233
        • Long J.
        • Wang Y.
        • Wang W.
        • Chang B.H.
        • Danesh F.R.
        Identification of microRNA-93 as a novel regulator of vascular endothelial growth factor in hyperglycemic conditions.
        J. Biol. Chem. 2010; 285: 23457-23465
        • Long J.
        • Wang Y.
        • Wang W.
        • Chang B.H.
        • Danesh F.R.
        MicroRNA-29c is a signature microRNA under high glucose conditions that targets Sprouty homolog 1, and its in vivo knockdown prevents progression of diabetic nephropathy.
        J. Biol. Chem. 2011; 286: 11837-11848
        • Dey N.
        • Das F.
        • Mariappan M.M.
        • Mandal C.C.
        • Ghosh-Choudhury N.
        • Kasinath B.S.
        • Choudhury G.G.
        MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes.
        J. Biol. Chem. 2011; 286: 25586-25603
        • Wang Q.
        • Wang Y.
        • Minto A.W.
        • Wang J.
        • Shi Q.
        • Li X.
        • Quigg R.J.
        MicroRNA-377 is up-regulated and can lead to increased fibronectin production in diabetic nephropathy.
        FASEB J. 2008; 22: 4126-4135
        • Kato M.
        • Zhang J.
        • Wang M.
        • Lanting L.
        • Yuan H.
        • Rossi J.J.
        • Natarajan R.
        MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-β-induced collagen expression via inhibition of E-box repressors.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 3432-3437
        • Wei Q.
        • Bhatt K.
        • He H.Z.
        • Mi Q.S.
        • Haase V.H.
        • Dong Z.
        Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury.
        J. Am. Soc. Nephrol. 2010; 21: 756-761
        • Chau B.N.
        • Xin C.
        • Hartner J.
        • Ren S.
        • Castano A.P.
        • Linn G.
        • Li J.
        • Tran P.T.
        • Kaimal V.
        • Huang X.
        • Chang A.N.
        • Li S.
        • Kalra A.
        • Grafals M.
        • Portilla D.
        • MacKenna D.A.
        • Orkin S.H.
        • Duffield J.S.
        MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways.
        Sci. Transl. Med. 2012; 4: 121ra118
        • Grimwood L.
        • Masterson R.
        Propagation and culture of renal fibroblasts.
        in: Hewitson T.D. Becker G.J. Methods in Molecular Biology: Kidney Research. Humana Press, Totowa, NJ2009: 25-37
        • Long J.
        • Matsuura I.
        • He D.
        • Wang G.
        • Shuai K.
        • Liu F.
        Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 9791-9796
        • Livak K.J.
        • Schmittgen T.D.
        Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔC(T)) method.
        Methods. 2001; 25: 402-408
        • Jørgensen S.
        • Baker A.
        • Møller S.
        • Nielsen B.S.
        Robust one-day in situ hybridization protocol for detection of microRNAs in paraffin samples using LNA probes.
        Methods. 2010; 52: 375-381
        • Várallyay E.
        • Burgyán J.
        • Havelda Z.
        MicroRNA detection by Northern blotting using locked nucleic acid probes.
        Nat. Protoc. 2008; 3: 190-196
        • Rehmsmeier M.
        • Steffen P.
        • Hochsmann M.
        • Giegerich R.
        Fast and effective prediction of microRNA/target duplexes.
        RNA. 2004; 10: 1507-1517
        • Du M.
        • Roy K.M.
        • Zhong L.
        • Shen Z.
        • Meyers H.E.
        • Nichols R.C.
        VEGF gene expression is regulated post-transcriptionally in macrophages.
        FEBS J. 2006; 273: 732-745
        • Bar N.
        • Dikstein R.
        miR-22 forms a regulatory loop in PTEN/AKT pathway and modulates signaling kinetics.
        PLoS ONE. 2010; 5: e10859
        • Loots G.G.
        • Ovcharenko I.
        rVISTA 2.0. Evolutionary analysis of transcription factor binding sites.
        Nucleic Acids Res. 2004; 32: W217-W221
        • Yu P.B.
        • Hong C.C.
        • Sachidanandan C.
        • Babitt J.L.
        • Deng D.Y.
        • Hoyng S.A.
        • Lin H.Y.
        • Bloch K.D.
        • Peterson R.T.
        Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.
        Nat. Chem. Biol. 2008; 4: 33-41
        • Lal A.
        • Thomas M.P.
        • Altschuler G.
        • Navarro F.
        • O'Day E.
        • Li X.L.
        • Concepcion C.
        • Han Y.C.
        • Thiery J.
        • Rajani D.K.
        • Deutsch A.
        • Hofmann O.
        • Ventura A.
        • Hide W.
        • Lieberman J.
        Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling.
        PLoS Genet. 2011; 7: e1002363
        • Orom U.A.
        • Lund A.H.
        Isolation of microRNA targets using biotinylated synthetic microRNAs.
        Methods. 2007; 43: 162-165
        • Gurha P.
        • Abreu-Goodger C.
        • Wang T.
        • Ramirez M.O.
        • Drumond A.L.
        • van Dongen S.
        • Chen Y.
        • Bartonicek N.
        • Enright A.J.
        • Lee B.
        • Kelm Jr., R.J.
        • Reddy A.K.
        • Taffet G.E.
        • Bradley A.
        • Wehrens X.H.
        • Entman M.L.
        • Rodriguez A.
        Targeted deletion of microRNA-22 promotes stress-induced cardiac dilation and contractile dysfunction.
        Circulation. 2012; 125: 2751-2761
        • Satoh M.
        • Kashihara N.
        • Yamasaki Y.
        • Maruyama K.
        • Okamoto K.
        • Maeshima Y.
        • Sugiyama H.
        • Sugaya T.
        • Murakami K.
        • Makino H.
        Renal interstitial fibrosis is reduced in angiotensin II type 1a receptor-deficient mice.
        J. Am. Soc. Nephrol. 2001; 12: 317-325
        • Wang W.
        • Wang Y.
        • Long J.
        • Wang J.
        • Haudek S.B.
        • Overbeek P.
        • Chang B.H.
        • Schumacker P.T.
        • Danesh F.R.
        Mitochondrial fission triggered by hyperglycemia is mediated by ROCK1 activation in podocytes and endothelial cells.
        Cell Metab. 2012; 15: 186-200
        • Klahr S.
        • Morrissey J.
        Obstructive nephropathy and renal fibrosis.
        Am. J. Physiol. Renal Physiol. 2002; 283: F861-F875
        • Alvarez-Diaz S.
        • Valle N.
        • Ferrer-Mayorga G.
        • Lombardia L.
        • Herrera M.
        • Dominguez O.
        • Segura M.F.
        • Bonilla F.
        • Hernando E.
        • Munoz A.
        MicroRNA-22 is induced by vitamin D and contributes to its antiproliferative, antimigratory and gene regulatory effects in colon cancer cells.
        Hum. Mol. Genet. 2012; 21: 2157-2165
        • Wang W.L.
        • Chatterjee N.
        • Chittur S.V.
        • Welsh J.
        • Tenniswood M.P.
        Effects of 1α,25 dihydroxyvitamin D3 and testosterone on miRNA and mRNA expression in LNCaP cells.
        Mol. Cancer. 2011; 10: 58
        • Delic D.
        • Grosser C.
        • Dkhil M.
        • Al-Quraishy S.
        • Wunderlich F.
        Testosterone-induced upregulation of miRNAs in the female mouse liver.
        Steroids. 2010; 75: 998-1004
        • Jian P.
        • Li Z.W.
        • Fang T.Y.
        • Jian W.
        • Zhuan Z.
        • Mei L.X.
        • Yan W.S.
        • Jian N.
        Retinoic acid induces HL-60 cell differentiation via the upregulation of miR-663.
        J. Hematol. Oncol. 2011; 4: 20
        • López-Rovira T.
        • Chalaux E.
        • Massagué J.
        • Rosa J.L.
        • Ventura F.
        Direct binding of Smad1 and Smad4 to two distinct motifs mediates bone morphogenetic protein-specific transcriptional activation of Id1 gene.
        J. Biol. Chem. 2002; 277: 3176-3185
        • Nakahiro T.
        • Kurooka H.
        • Mori K.
        • Sano K.
        • Yokota Y.
        Identification of BMP-responsive elements in the mouse Id2 gene.
        Biochem. Biophys. Res. Commun. 2010; 399: 416-421
        • Korchynskyi O.
        • ten Dijke P.
        Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter.
        J. Biol. Chem. 2002; 277: 4883-4891
        • Katagiri T.
        • Imada M.
        • Yanai T.
        • Suda T.
        • Takahashi N.
        • Kamijo R.
        Identification of a BMP-responsive element in Id1, the gene for inhibition of myogenesis.
        Genes Cells. 2002; 7: 949-960
        • Shepherd T.G.
        • Thériault B.L.
        • Nachtigal M.W.
        Autocrine BMP4 signalling regulates ID3 proto-oncogene expression in human ovarian cancer cells.
        Gene. 2008; 414: 95-105
        • Ben-Dov I.Z.
        • Muthukumar T.
        • Morozov P.
        • Mueller F.B.
        • Tuschl T.
        • Suthanthiran M.
        MicroRNA sequence profiles of human kidney allografts with or without tubulointerstitial fibrosis.
        Transplantation. 2012; 94: 1086-1094
        • Glowacki F.
        • Savary G.
        • Gnemmi V.
        • Buob D.
        • Van der Hauwaert C.
        • Lo-Guidice J.M.
        • Bouyé S.
        • Hazzan M.
        • Pottier N.
        • Perrais M.
        • Aubert S.
        • Cauffiez C.
        Increased circulating miR-21 levels are associated with kidney fibrosis.
        PLoS ONE. 2013; 8: e58014
        • Zarjou A.
        • Yang S.
        • Abraham E.
        • Agarwal A.
        • Liu G.
        Identification of a microRNA signature in renal fibrosis. Role of miR-21.
        Am. J. Physiol. Renal Physiol. 2011; 301: F793-F801
        • Zhong X.
        • Chung A.C.
        • Chen H.Y.
        • Meng X.M.
        • Lan H.Y.
        Smad3-mediated upregulation of miR-21 promotes renal fibrosis.
        J. Am. Soc. Nephrol. 2011; 22: 1668-1681
        • Oba S.
        • Kumano S.
        • Suzuki E.
        • Nishimatsu H.
        • Takahashi M.
        • Takamori H.
        • Kasuya M.
        • Ogawa Y.
        • Sato K.
        • Kimura K.
        • Homma Y.
        • Hirata Y.
        • Fujita T.
        miR-200b precursor can ameliorate renal tubulointerstitial fibrosis.
        PLoS ONE. 2010; 5: e13614
        • Xiong M.
        • Jiang L.
        • Zhou Y.
        • Qiu W.
        • Fang L.
        • Tan R.
        • Wen P.
        • Yang J.
        The miR-200 family regulates TGF-β1-induced renal tubular epithelial to mesenchymal transition through Smad pathway by targeting ZEB1 and ZEB2 expression.
        Am. J. Physiol. Renal. Physiol. 2012; 302: F369-F379
        • Huang Z.P.
        • Chen J.
        • Seok H.Y.
        • Zhang Z.
        • Kataoka M.
        • Hu X.
        • Wang D.Z.
        MicroRNA-22 regulates cardiac hypertrophy and remodeling in response to stress.
        Circ. Res. 2013; 112: 1234-1243
        • Sayed D.
        • Abdellatif M.
        MicroRNAs in development and disease.
        Physiol. Rev. 2011; 91: 827-887
        • van Rooij E.
        The art of microRNA research.
        Circ. Res. 2011; 108: 219-234
        • Mendell J.T.
        • Olson E.N.
        MicroRNAs in stress signaling and human disease.
        Cell. 2012; 148: 1172-1187
        • Davis B.N.
        • Hilyard A.C.
        • Lagna G.
        • Hata A.
        SMAD proteins control DROSHA-mediated microRNA maturation.
        Nature. 2008; 454: 56-61
        • Iliopoulos D.
        • Malizos K.N.
        • Oikonomou P.
        • Tsezou A.
        Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks.
        PLoS ONE. 2008; 3: e3740