Jörg Heyer

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

The expression of the murine interleukin (IL)-2 receptor alpha chain/CD25 is strongly induced at the transcriptional level after T cell activation. We show here that nuclear factor of activated T cell (NF-AT) factors are involved in the control of CD25 promoter induction in T cells. NF-ATp and NF-ATc bind to two sites around positions -585 and -650 located upstream of the proximal CD25 promoter. Immediately 3' from these NF-AT motifs, nonconsensus sites are located for the binding of AP-1-like factors. Mutations of sites that suppress NF-AT binding impair the induction and strong NF-ATp-mediated transactivation of the CD25 promoter in T cells. In T lymphocytes from NF-ATp-deficient mice, the expression of CD25 is severely impaired, leading to a delayed IL-2 receptor expression after T cell receptor (TCR)/CD3 stimulation. Our data indicate an important role for NF-AT in the faithful expression of high affinity IL-2 receptors and a close link between the TCR-mediated induction of IL-2 and IL-2 receptor alpha chain promoters, both of which are regulated by NF-AT factors.

NF-ATp and NF-ATc are the most prominent nuclear NF-AT transcription factors in peripheral T lymphocytes. After T cell activation both factors bind to and control the promoters and enhancers of numerous lymphokine and receptor ligand genes. In order to define a specific role for NF-ATp in vivo we have inactivated the NF-ATp gene by gene targeting in mice. We show that NF-ATp deficiency leads to the accumulation of peripheral T cells with a "preactivated" phenotype, enhanced immune responses of T cells after secondary stimulation in vitro and severe defects in the proper termination of antigen responses, as shown by a reduced deletion of superantigen-reactive CD4+ T cells. These alterations in the function of the immune system are correlated with drastic changes in the morphology of lymphoid organs. Approximately 25 % of NF-ATp-deficient mice older than 6 months develop large germinal centers in the spleen and peripheral lymph nodes. In addition, they exhibit a pronounced retardation in the involution of the thymus. The thymus of these NF-ATp-deficient mice exhibits large cortical areas typical for newborn mice and a massive infiltration of IgM+/ IgD+ B lymphocytes. Contrary to the T lymphocytes from IL-2-deficient mice which develop a phenotype similar to the NF-ATp-/- mice, NF-ATp-/- T cells do not show obvious defects in Fas-mediated apoptosis. This might indicate defects in other types of programmed cell death which are controlled by the activity of NF-ATp.

Smad2 is a member of the intracellular mediators that transduce signals from TGF-beta receptors and activin receptors. Targeted inactivation of Smad2 in mice leads to early lethality before gastrulation. It was shown previously that TGF-betaRII deficiency in vivo leads to defects in B cell homeostasis, Ag responsiveness, and IgA class switch recombination of B cells. To investigate the importance of Smad2-mediated signaling in B lymphocytes, we generated a B cell-specific inactivation of Smad2 in mice (bSmad2(-/-)). bSmad2(-/-) mice had normal B cell numbers in the spleen but showed a reduced population of marginal zone B cells. In contrast, B cells in Peyer's patches and peritoneal B-1a cells of bSmad2(-/-) mice were increased in numbers. bSmad2(-/-) mice showed a reduced number of surface-IgA(+) B cells and of IgA-secreting cells in Peyer's patches, decreased levels of IgA in serum, and, after immunization with a T cell-dependent Ag, a reduced IgA response. Class switch recombination to IgA was impaired in Smad2-deficient B cells, when stimulated in vitro with LPS in the presence of TGF-beta. The growth-inhibitory effects of TGF-beta in LPS-stimulated B cells were not affected in Smad2-deficient B cells. In summary, our data indicate a crucial role of Smad2 in mediating signals for the TGF-beta-directed class switch to IgA and the induction of IgA responses in vivo. Other B cell functions like growth-inhibitory signaling, which are known to be regulated by signals via the TGF-betaR, are not affected in Smad2-deficient B cells.