Wenjun Ju

Abstract Foxp3, a winged-helix family transcription factor, serves as the master switch for CD4+ regulatory T cells (Treg). We identified a unique and evolutionarily conserved CpG-rich island of the Foxp3 nonintronic upstream enhancer and discovered that a specific site within it was unmethylated in natural Treg (nTreg) but heavily methylated in naive CD4+ T cells, activated CD4+ T cells, and peripheral TGFβ-induced Treg in which it was bound by DNMT1, DNMT3b, MeCP2, and MBD2. Demethylation of this CpG site using the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza) induced acetylation of histone 3, interaction with TIEG1 and Sp1, and resulted in strong and stable induction of Foxp3. Conversely, IL-6 resulted in methylation of this site and repression of Foxp3 expression. Aza plus TGFβ-induced Treg resembled nTreg, expressing similar receptors, cytokines, and stable suppressive activity. Strong Foxp3 expression and suppressor activity could be induced in a variety of T cells, including human CD4+CD25− T cells. Epigenetic regulation of Foxp3 can be predictably controlled with DNMT inhibitors to generate functional, stable, and specific Treg.

Chronic kidney disease (CKD) affects 8 to 16% people worldwide, with an increasing incidence and prevalence of end-stage kidney disease (ESKD). The effective management of CKD is confounded by the inability to identify patients at high risk of progression while in early stages of CKD. To address this challenge, a renal biopsy transcriptome-driven approach was applied to develop noninvasive prognostic biomarkers for CKD progression. Expression of intrarenal transcripts was correlated with the baseline estimated glomerular filtration rate (eGFR) in 261 patients. Proteins encoded by eGFR-associated transcripts were tested in urine for association with renal tissue injury and baseline eGFR. The ability to predict CKD progression, defined as the composite of ESKD or 40% reduction of baseline eGFR, was then determined in three independent CKD cohorts. A panel of intrarenal transcripts, including epidermal growth factor (EGF), a tubule-specific protein critical for cell differentiation and regeneration, predicted eGFR. The amount of EGF protein in urine (uEGF) showed significant correlation (P < 0.001) with intrarenal EGF mRNA, interstitial fibrosis/tubular atrophy, eGFR, and rate of eGFR loss. Prediction of the composite renal end point by age, gender, eGFR, and albuminuria was significantly (P < 0.001) improved by addition of uEGF, with an increase of the C-statistic from 0.75 to 0.87. Outcome predictions were replicated in two independent CKD cohorts. Our approach identified uEGF as an independent risk predictor of CKD progression. Addition of uEGF to standard clinical parameters improved the prediction of disease events in diverse CKD populations with a wide spectrum of causes and stages.

A prominent pathway of transforming growth factor (TGF)-β signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-β1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-β1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-β1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [3H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-β1. Lack of Smad2 or Smad3 expression did not affect TGF-β1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-β1. Moreover, TGF-β1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-β1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-β-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element)4-Lux reporter by TGF-β1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-β1 signaling. A prominent pathway of transforming growth factor (TGF)-β signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-β1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-β1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-β1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [3H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-β1. Lack of Smad2 or Smad3 expression did not affect TGF-β1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-β1. Moreover, TGF-β1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-β1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-β-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element)4-Lux reporter by TGF-β1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-β1 signaling. transforming growth factor activator protein 1 activin response element dermal fibroblast embryonic stem forkhead activin signal transducer knockout mouse embryo fibroblast Mad homology matrix metalloproteinase plasminogen activator inhibitor 1 Smad binding element TGF-β receptor type wild-type receptor-activated Smad multiplicity of infection Transforming growth factor (TGF)1-β is the prototypic member of the TGF-β superfamily and mediates a multiplicity of biological effects on different cell types. TGF-β regulates cellular proliferation, induces synthesis of extracellular matrix proteins such as fibronectin and plasminogen activator inhibitor-1 (PAI-1), modulates the immune response, and plays an important role in embryonic development and cellular differentiation (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors. Springer-Verlag, Berlin1990: 419-472Google Scholar).TGF-β evokes its biological effects by signaling through two different receptor serine/threonine kinases, TGF-β receptor type (TβR)-I and TβR-II, that form a tetrameric complex after binding of TGF-β to TβR-II. TβR-II activates TβR-I by phosphorylation of serine residues in the GS box. The anchor protein SARA (Smad anchor for receptor activation) recruits the cytoplasmic signal transducers Smad2 and Smad3, classified as so-called receptor-activated Smads (R-Smads), to the Tβ R-I kinase domain, resulting in their phosphorylation on serine residues in the C-terminal SSXS motif. Activated R-Smads heteroligomerize with the common partner (CO)-Smad4, and these complexes are transported into the nucleus, where they regulate gene expression. R-Smads and CO- Smads contain two highly conserved domains, the Mad homology (MH) 1 domain and the MH2 domain, which are connected by a linker region. Whereas their MH1 domains can interact with the DNA, the MH2 domains are endowed with transcriptional activation properties.Down-regulation of TGF-β signaling is effected, in part, by a feedback mechanism involving induction of expression of the inhibitory Smads, Smad6 and Smad7, which then prevent R-Smad activation (2Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar,3Piek E. Heldin C.H. ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (737) Google Scholar).Absence of Smad2 or Smad3 expression resulting from targeted deletion of the respective Smad genes in mice has revealed different developmental roles for Smad2 and Smad3. Homozygous loss of function mutations of the Smad2 gene by targeted deletion of the MH1 or MH2 domain resulted in embryonic lethality due to failure to establish an anterior-posterior axis, gastrulation, and mesoderm formation (4Nomura E. Nature. PubMed Scopus Google J. PubMed Scopus Google are by from the PubMed Scopus Google J. E. 1999; PubMed Scopus Google Smad2 embryos to embryonic but showed such as and as by and embryo J. E. 1999; PubMed Scopus Google mice of gene are and for that Smad3 is for embryonic Smad3 knockout mice are wild-type and M.B. 1999; PubMed Google Roberts A.B. J. 1999; PubMed Google expression of Smad3 from of as a of immune function in as revealed by in to and of can in to the of of cells to the effects of TGF-β as as to a response of to TGF-β M.B. 1999; PubMed Google Roberts A.B. J. 1999; PubMed Google mice with wild-type which is a of enhanced by and as as TGF-β by A.B. Roberts A.B. 1999; PubMed Scopus Google Homozygous Smad3 knockout mice by PubMed Scopus Google from and of a that was not in Smad3 mice derived by M.B. 1999; PubMed Google or Roberts A.B. J. 1999; PubMed Google investigate the relative importance of Smad2 and Smad3 in TGF-β1 we have mouse fibroblasts expression of the Smad2 or Smad3 gene J. E. 1999; PubMed Scopus Google Roberts A.B. J. 1999; PubMed Google to analysis of the function of Smad2 and Smad3 by these loss of function cell a to investigate the roles and relative importance of these R-Smads in TGF-β signaling and into the of TGF-β R-Smad function in to Our that expression of Smad2 or Smad3 in fibroblasts is important for TGF-β1-mediated growth inhibition as as for synthesis of whereas Smad2 and Smad3 to TGF-β1-induced activation of reporter that genes are selectively dependent on only of these two TGF-β receptor-activated Smads, such for the matrix metalloproteinase which is dependent on Smad2 but not Smad3 expression. indicate roles for Smad2 and Smad3 in TGF-β1-mediated signaling and into the of these specific signaling in have TGF-β signaling in mouse fibroblasts deficient in expression of Smad3 to the of loss of of these signaling on induction of target gene expression by TGF-β1. have target genes with or of that are by the loss of and genes that are selectively dependent on or the of these two R-Smad we have that TGF-β1-induced fibronectin synthesis in the of Smad2 or Smad3 whereas Smads have roles in the induction of protein and in the complex of TGF-β1-induced growth inhibition with of kinase and for the that TGF-β1-mediated induction of expression Smad3 and that induction of is selectively dependent on Moreover, to that for Smad3 and A.B. Roberts A.B. 1999; PubMed Scopus Google we that autoinduction of TGF-β1 in fibroblasts is strongly suppressed in the of Smad3. To that in are dependent on Smad2 and Smad3, we have assessed the activation of TGF-β-sensitive reporter genes in these these that Smad2 and Smad3 have and roles in TGF-β1 on the target gene and cellular of the embryonic of the Smad2 knockout mice (4Nomura E. Nature. PubMed Scopus Google J. PubMed Scopus Google and the in of S2KO embryonic we to of the role of Smad2 and Smad3 in TGF-β1 signaling fibroblasts that To the of we that expression of Smad3 is important for TGF-β1-mediated induction of Smad7, and in and that induction of expression of these genes by TGF-β1 can after of Smad3 in these whereas or of Smad2 or Smad3 expression in fibroblasts reporter gene induction dependent on Smads, was not to induction by TGF-β1 of gene to dependent on Smad2 or Smad3 not of the to by of Smads or signaling into cell have J. J. J. PubMed Scopus Google J. J. PubMed Scopus Google Our that of Smad expression is not to the Smad knockout fibroblasts to their loss of Smad expression in with with expression of genes important in signaling to complex of to that the role of different Smads by in in we that fibroblasts derived from mouse embryos expression of Smad2 or Smad3 a loss of function to investigate the different effects of these two R-Smads in TGF-β as is important for the of their roles in the different roles of Smad2 and Smad3 are in of where targeted deletion of Smad2 or Smad3 in embryonic lethality or (4Nomura E. Nature. PubMed Scopus Google J. PubMed Scopus Google PubMed Scopus Google J. E. 1999; PubMed Scopus Google M.B. 1999; PubMed Google Roberts A.B. J. 1999; PubMed Google PubMed Scopus Google of Smad2 or Smad3 have different effects A.B. Roberts A.B. 1999; PubMed Scopus Google are in where Smad2 has classified as a based on its in of J. PubMed Scopus Google J. PubMed Scopus Google but where for a role of Smad3 is PubMed Scopus Google 1997; Google Moreover, autoinduction of to protein signaling P. K. Sporn M.B. Roberts A.B. PubMed Google J. J. PubMed Scopus Google is dependent on Smad3 A.B. Roberts A.B. 1999; PubMed Scopus Google of Smad3 for in cells TGF-β1 by cells can by and in and in that R-Smad can by in have effects to its loss by E. Roberts A.B. J. PubMed Google of Smad2 and Smad3 have that or to their in to their Whereas the MH1 domain of Smad3 can interact with in the DNA, Smad2 an that in the MH1 domain of Smad3 and its binding to 1999; PubMed Scopus Google K. Miyazono K. J. 1999; PubMed Scopus Google with we that TGF-β1-induced activation of the which of derived from the mouse Heldin C.H. ten Dijke P. J. PubMed Scopus Google as in S2KO as in whereas in S3KO TGF-β1-induced reporter activation was is in with that Smad2, in to Smad3, not in to and that of Smad2 only weakly to TGF-β1-induced activation of the whereas of Smad3 enhanced reporter in the of TGF-β Heldin C.H. ten Dijke P. J. PubMed Scopus Google a Smad3 is in activation of gene whereas Smad2 not have a role in its induction by TGF-β K. J. PubMed Scopus Google J. J. 1999; PubMed Scopus Google to the binding of Smad2 and Smad3. of Smad2 that to K. Miyazono K. J. 1999; PubMed Scopus Google K. K. Miyazono K. K. PubMed Scopus Google and for loss of Smad3 in activation of in of Smad2 and Smad3 on the target element are by that Smad2 and Smad3 have for different transcription and to TGF-β signaling. of Smad3 activation of the or TGF-β target gene that are dependent on Smad2, and Nature. PubMed Scopus Google E. Nature. 1997; PubMed Scopus Google inhibition has to from Smad3 and for binding to J. J. 1999; PubMed Scopus Google or of Smad3 and for in the gene E. PubMed Scopus Google Our loss of function the of on Smad2 as as its by Smad3. we activation of the reporter in in with enhanced activation of reporter in cells as as in cells in which a form of Smad3 is with the function of the protein Roberts A.B. J. 1999; PubMed Google to activation of genes and TGF-β-sensitive reporter genes that are by signaling of cell growth signaling to the cell that complex is to the genes that are in of inhibition of cellular to findings in or has that the effects of TGF-β are in and M.B. 1999; PubMed Google M.B. J. 1999; PubMed Scopus Google Whereas M.B. 1999; PubMed Google did not in the induction of in expression of and and in S2KO and S3KO fibroblasts with with suppressed and activation of by TGF-β have in M.B. J. 1999; PubMed Scopus Google the and and the growth of fibroblasts is that activation of genes by TGF-β1 is dependent on of the two TGF-β Smad2 or Smad3, that the in of mice are not a of and of gene expression of these two R-Smads development but roles for Smad2 and Smad3 in of target gene which for in with signal analysis of TGF-β target gene expression in Smad2 Smad3 cell has the to into their respective roles in of genes that are of importance for and development as as in Transforming growth factor (TGF)1-β is the prototypic member of the TGF-β superfamily and mediates a multiplicity of biological effects on different cell types. TGF-β regulates cellular proliferation, induces synthesis of extracellular matrix proteins such as fibronectin and plasminogen activator inhibitor-1 (PAI-1), modulates the immune response, and plays an important role in embryonic development and cellular differentiation (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors. Springer-Verlag, Berlin1990: 419-472Google TGF-β evokes its biological effects by signaling through two different receptor serine/threonine kinases, TGF-β receptor type (TβR)-I and TβR-II, that form a tetrameric complex after binding of TGF-β to TβR-II. TβR-II activates TβR-I by phosphorylation of serine residues in the GS box. The anchor protein SARA (Smad anchor for receptor activation) recruits the cytoplasmic signal transducers Smad2 and Smad3, classified as so-called receptor-activated Smads (R-Smads), to the Tβ R-I kinase domain, resulting in their phosphorylation on serine residues in the C-terminal SSXS motif. Activated R-Smads heteroligomerize with the common partner (CO)-Smad4, and these complexes are transported into the nucleus, where they regulate gene expression. R-Smads and CO- Smads contain two highly conserved domains, the Mad homology (MH) 1 domain and the MH2 domain, which are connected by a linker region. Whereas their MH1 domains can interact with the DNA, the MH2 domains are endowed with transcriptional activation of TGF-β signaling is effected, in part, by a feedback mechanism involving induction of expression of the inhibitory Smads, Smad6 and Smad7, which then prevent R-Smad activation (2Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar,3Piek E. Heldin C.H. ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (737) Google of Smad2 or Smad3 expression resulting from targeted deletion of the respective Smad genes in mice has revealed different developmental roles for Smad2 and Smad3. Homozygous loss of function mutations of the Smad2 gene by targeted deletion of the MH1 or MH2 domain resulted in embryonic lethality due to failure to establish an anterior-posterior axis, gastrulation, and mesoderm formation (4Nomura E. Nature. PubMed Scopus Google J. PubMed Scopus Google are by from the PubMed Scopus Google J. E. 1999; PubMed Scopus Google Smad2 embryos to embryonic but showed such as and as by and embryo J. E. 1999; PubMed Scopus Google mice of gene are and for that Smad3 is for embryonic Smad3 knockout mice are wild-type and M.B. 1999; PubMed Google Roberts A.B. J. 1999; PubMed Google expression of Smad3 from of as a of immune function in as revealed by in to and of can in to the of of cells to the effects of TGF-β as as to a response of to TGF-β M.B. 1999; PubMed Google Roberts A.B. J. 1999; PubMed Google mice with wild-type which is a of enhanced by and as as TGF-β by A.B. Roberts A.B. 1999; PubMed Scopus Google Homozygous Smad3 knockout mice by PubMed Scopus Google from and of a that was not in Smad3 mice derived by M.B. 1999; PubMed Google or Roberts A.B. J. 1999; PubMed Google To investigate the relative importance of Smad2 and Smad3 in TGF-β1 we have mouse fibroblasts expression of the Smad2 or Smad3 gene J. E. 1999; PubMed Scopus Google Roberts A.B. J. 1999; PubMed Google to analysis of the function of Smad2 and Smad3 by these loss of function cell a to investigate the roles and relative importance of these R-Smads in TGF-β signaling and into the of TGF-β R-Smad function in to Our that expression of Smad2 or Smad3 in fibroblasts is important for TGF-β1-mediated growth inhibition as as for synthesis of whereas Smad2 and Smad3 to TGF-β1-induced activation of reporter that genes are selectively dependent on only of these two TGF-β receptor-activated Smads, such for the matrix metalloproteinase which is dependent on Smad2 but not Smad3 expression. indicate roles for Smad2 and Smad3 in TGF-β1-mediated signaling and into the of these specific signaling in have TGF-β signaling in mouse fibroblasts deficient in expression of Smad3 to the of loss of of these signaling on induction of target gene expression by TGF-β1. have target genes with or of that are by the loss of and genes that are selectively dependent on or the of these two R-Smad we have that TGF-β1-induced fibronectin synthesis in the of Smad2 or Smad3 whereas Smads have roles in the induction of protein and in the complex of TGF-β1-induced growth inhibition with of kinase and for the that TGF-β1-mediated induction of expression Smad3 and that induction of is selectively dependent on Moreover, to that for Smad3 and A.B. Roberts A.B. 1999; PubMed Scopus Google we that autoinduction of TGF-β1 in fibroblasts is strongly suppressed in the of Smad3. To that in are dependent on Smad2 and Smad3, we have assessed the activation of TGF-β-sensitive reporter genes in these these that Smad2 and Smad3 have and roles in TGF-β1 on the target gene and cellular of the embryonic of the Smad2 knockout mice (4Nomura E. Nature. PubMed Scopus Google J. PubMed Scopus Google and the in of S2KO embryonic we to of the role of Smad2 and Smad3 in TGF-β1 signaling fibroblasts that To the of we that expression of Smad3 is important for TGF-β1-mediated induction of Smad7, and in and that induction of expression of these genes by TGF-β1 can after of Smad3 in these whereas or of Smad2 or Smad3 expression in fibroblasts reporter gene induction dependent on Smads, was not to induction by TGF-β1 of gene to dependent on Smad2 or Smad3 not of the to by of Smads or signaling into cell have J. J. J. PubMed Scopus Google J. J. PubMed Scopus Google Our that of Smad expression is not to the Smad knockout fibroblasts to their loss of Smad expression in with with expression of genes important in signaling to complex of to that the role of different Smads by in in we that fibroblasts derived from mouse embryos expression of Smad2 or Smad3 a loss of function to investigate the different effects of these two R-Smads in TGF-β as is important for the of their roles in the different roles of Smad2 and Smad3 are in of where targeted deletion of Smad2 or Smad3 in embryonic lethality or (4Nomura E. Nature. PubMed Scopus Google J. PubMed Scopus Google PubMed Scopus Google J. E. 1999; PubMed Scopus Google M.B. 1999; PubMed Google Roberts A.B. J. 1999; PubMed Google PubMed Scopus Google of Smad2 or Smad3 have different effects A.B. Roberts A.B. 1999; PubMed Scopus Google are in where Smad2 has classified as a based on its in of J. PubMed Scopus Google J. PubMed Scopus Google but where for a role of Smad3 is PubMed Scopus Google 1997; Google Moreover, autoinduction of to protein signaling P. K. Sporn M.B. Roberts A.B. PubMed Google J. J. PubMed Scopus Google is dependent on Smad3 A.B. Roberts A.B. 1999; PubMed Scopus Google of Smad3 for in cells TGF-β1 by cells can by and in and in that R-Smad can by in have effects to its loss by E. Roberts A.B. J. PubMed Google of Smad2 and Smad3 have that or to their in to their Whereas the MH1 domain of Smad3 can interact with in the DNA, Smad2 an that in the MH1 domain of Smad3 and its binding to 1999; PubMed Scopus Google K. Miyazono K. J. 1999; PubMed Scopus Google with we that TGF-β1-induced activation of the which of derived from the mouse Heldin C.H. ten Dijke P. J. PubMed Scopus Google as in S2KO as in whereas in S3KO TGF-β1-induced reporter activation was is in with that Smad2, in to Smad3, not in to and that of Smad2 only weakly to TGF-β1-induced activation of the whereas of Smad3 enhanced reporter in the of TGF-β Heldin C.H. ten Dijke P. J. PubMed Scopus Google a Smad3 is in activation of gene whereas Smad2 not have a role in its induction by TGF-β K. J. PubMed Scopus Google J. J. 1999; PubMed Scopus Google to the binding of Smad2 and Smad3. of Smad2 that to K. Miyazono K. J. 1999; PubMed Scopus Google K. K. Miyazono K. K. PubMed Scopus Google and for loss of Smad3 in activation of in of Smad2 and Smad3 on the target element are by that Smad2 and Smad3 have for different transcription and to TGF-β signaling. of Smad3 activation of the or TGF-β target gene that are dependent on Smad2, and Nature. PubMed Scopus Google E. Nature. 1997; PubMed Scopus Google inhibition has to from Smad3 and for binding to J. J. 1999; PubMed Scopus Google or of Smad3 and for in the gene E. PubMed Scopus Google Our loss of function the of on Smad2 as as its by Smad3. we activation of the reporter in in with enhanced activation of reporter in cells as as in cells in which a form of Smad3 is with the function of the protein Roberts A.B. J. 1999; PubMed Google to activation of genes and TGF-β-sensitive reporter genes that are by signaling of cell growth signaling to the cell that complex is to the genes that are in of inhibition of cellular to findings in or has that the effects of TGF-β are in and M.B. 1999; PubMed Google M.B. J. 1999; PubMed Scopus Google Whereas M.B. 1999; PubMed Google did not in the induction of in expression of and and in S2KO and S3KO fibroblasts with with suppressed and activation of by TGF-β have in M.B. J. 1999; PubMed Scopus Google the and and the growth of fibroblasts is that activation of genes by TGF-β1 is dependent on of the two TGF-β Smad2 or Smad3, that the in of mice are not a of and of gene expression of these two R-Smads development but roles for Smad2 and Smad3 in of target gene which for in with signal analysis of TGF-β target gene expression in Smad2 Smad3 cell has the to into their respective roles in of genes that are of importance for and development as as in have TGF-β signaling in mouse fibroblasts deficient in expression of Smad3 to the of loss of of these signaling on induction of target gene expression by TGF-β1. have target genes with or of that are by the loss of and genes that are selectively dependent on or the of these two R-Smad we have that TGF-β1-induced fibronectin synthesis in the of Smad2 or Smad3 whereas Smads have roles in the induction of protein and in the complex of TGF-β1-induced growth inhibition with of kinase and for the that TGF-β1-mediated induction of expression Smad3 and that induction of is selectively dependent on Moreover, to that for Smad3 and A.B. Roberts A.B. 1999; PubMed Scopus Google we that autoinduction of TGF-β1 in fibroblasts is strongly suppressed in the of Smad3. To that in are dependent on Smad2 and Smad3, we have assessed the activation of TGF-β-sensitive reporter genes in these these that Smad2 and Smad3 have and roles in TGF-β1 on the target gene and cellular of the embryonic of the Smad2 knockout mice (4Nomura E. Nature. PubMed Scopus Google J. PubMed Scopus Google and the in of S2KO embryonic we to of the role of Smad2 and Smad3 in TGF-β1 signaling fibroblasts that To the of we that expression of Smad3 is important for TGF-β1-mediated induction of Smad7, and in and that induction of expression of these genes by TGF-β1 can after of Smad3 in these whereas or of Smad2 or Smad3 expression in fibroblasts reporter gene induction dependent on Smads, was not to induction by TGF-β1 of gene to dependent on Smad2 or Smad3 not of the to by of Smads or signaling into cell have J. J. J. PubMed Scopus Google J. J. PubMed Scopus Google Our that of Smad expression is not to the Smad knockout fibroblasts to their loss of Smad expression in with with expression of genes important in signaling to complex of to that the role of different Smads by in in we that fibroblasts derived from mouse embryos expression of Smad2 or Smad3 a loss of function to investigate the different effects of these two R-Smads in TGF-β as is important for the of their roles in the different roles of Smad2 and Smad3 are in of where targeted deletion of Smad2 or Smad3 in embryonic lethality or (4Nomura E. Nature. PubMed Scopus Google J. PubMed Scopus Google PubMed Scopus Google J. E. 1999; PubMed Scopus Google M.B. 1999; PubMed Google Roberts A.B. J. 1999; PubMed Google PubMed Scopus Google of Smad2 or Smad3 have different effects A.B. Roberts A.B. 1999; PubMed Scopus Google are in where Smad2 has classified as a based on its in of J. PubMed Scopus Google J. PubMed Scopus Google but where for a role of Smad3 is PubMed Scopus Google 1997; Google Moreover, autoinduction of to protein signaling P. K. Sporn M.B. Roberts A.B. PubMed Google J. J. PubMed Scopus Google is dependent on Smad3 A.B. Roberts A.B. 1999; PubMed Scopus Google of Smad3 for in cells TGF-β1 by cells can by and in and in that R-Smad can by in have effects to its loss by E. Roberts A.B. J. PubMed Google A of Smad2 and Smad3 have that or to their in to their Whereas the MH1 domain of Smad3 can interact with in the DNA, Smad2 an that in the MH1 domain of Smad3 and its binding to 1999; PubMed Scopus Google K. Miyazono K. J. 1999; PubMed Scopus Google with we that TGF-β1-induced activation of the which of derived from the mouse Heldin C.H. ten Dijke P. J. PubMed Scopus Google as in S2KO as in whereas in S3KO TGF-β1-induced reporter activation was is in with that Smad2, in to Smad3, not in to and that of Smad2 only weakly to TGF-β1-induced activation of the whereas of Smad3 enhanced reporter in the of TGF-β Heldin C.H. ten Dijke P. J. PubMed Scopus Google a Smad3 is in activation of gene whereas Smad2 not have a role in its induction by TGF-β K. J. PubMed Scopus Google J. J. 1999; PubMed Scopus Google to the binding of Smad2 and Smad3. of Smad2 that to K. Miyazono K. J. 1999; PubMed Scopus Google K. K. Miyazono K. K. PubMed Scopus Google and for loss of Smad3 in activation of in of Smad2 and Smad3 on the target element are by that Smad2 and Smad3 have for different transcription and to TGF-β signaling. of Smad3 activation of the or TGF-β target gene that are dependent on Smad2, and Nature. PubMed Scopus Google E. Nature. 1997; PubMed Scopus Google inhibition has to from Smad3 and for binding to J. J. 1999; PubMed Scopus Google or of Smad3 and for in the gene E. PubMed Scopus Google Our loss of function the of on Smad2 as as its by Smad3. we activation of the reporter in in with enhanced activation of reporter in cells as as in cells in which a form of Smad3 is with the function of the protein Roberts A.B. J. 1999; PubMed Google to activation of genes and TGF-β-sensitive reporter genes that are by signaling of cell growth signaling to the cell that complex is to the genes that are in of inhibition of cellular to findings in or has that the effects of TGF-β are in and M.B. 1999; PubMed Google M.B. J. 1999; PubMed Scopus Google Whereas M.B. 1999; PubMed Google did not in the induction of in expression of and and in S2KO and S3KO fibroblasts with with suppressed and activation of by TGF-β have in M.B. J. 1999; PubMed Scopus Google the and and the growth of fibroblasts is The that activation of genes by TGF-β1 is dependent on of the two TGF-β Smad2 or Smad3, that the in of mice are not a of and of gene expression of these two R-Smads development but roles for Smad2 and Smad3 in of target gene which for in with signal analysis of TGF-β target gene expression in Smad2 Smad3 cell has the to into their respective roles in of genes that are of importance for and development as as in J. for Smad2 J. for Smad3 and reporter P. ten Dijke for reporter for and reporter for and K. Miyazono for Smad3 and and K. and for of the dermal

Smad2 and Smad3 interact and mediate TGF-beta signaling. Although Smad3 promotes fibrosis, the role of Smad2 in fibrogenesis is largely unknown. In this study, conditional deletion of Smad2 from the kidney tubular epithelial cells markedly enhanced fibrosis in response to unilateral ureteral obstruction. In vitro, Smad2 knockdown in tubular epithelial cells increased expression of collagen I, collagen III, and TIMP-1 and decreased expression of the matrix-degrading enzyme MMP-2 in response to TGF-beta1 compared with similarly treated wild-type cells. We obtained similar results in Smad2-knockout fibroblasts. Mechanistically, Smad2 deletion promoted fibrosis through enhanced TGF-beta/Smad3 signaling, evidenced by greater Smad3 phosphorylation, nuclear translocation, promoter activity, and binding of Smad3 to a collagen promoter (COL1A2). Moreover, deletion of Smad2 increased autoinduction of TGF-beta1. Conversely, overexpression of Smad2 attenuated TGF-beta1-induced Smad3 phosphorylation and collagen I matrix expression in tubular epithelial cells. In conclusion, in contrast to Smad3, Smad2 protects against TGF-beta-mediated fibrosis by counteracting TGF-beta/Smad3 signaling.

Cell-lineage-specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage-specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage-specific expression, even in lineages not separable by experimental microdissection. Our machine-learning-based approach leverages high-throughput data from tissue homogenates in a novel iterative statistical framework. We applied this method to chronic kidney disease and identified transcripts specific to podocytes, key cells in the glomerular filter responsible for hereditary and most acquired glomerular kidney disease. In a systematic evaluation of our predictions by immunohistochemistry, our in silico approach was significantly more accurate (65% accuracy in human) than predictions based on direct measurement of in vivo fluorescence-tagged murine podocytes (23%). Our method identified genes implicated as causal in hereditary glomerular disease and involved in molecular pathways of acquired and chronic renal diseases. Furthermore, based on expression analysis of human kidney disease biopsies, we demonstrated that expression of the podocyte genes identified by our approach is significantly related to the degree of renal impairment in patients. Our approach is broadly applicable to define lineage specificity in both cell physiology and human disease contexts. We provide a user-friendly website that enables researchers to apply this method to any cell-lineage or tissue of interest. Identified cell-lineage-specific transcripts are expected to play essential tissue-specific roles in organogenesis and disease and can provide starting points for the development of organ-specific diagnostics and therapies.