Hypoxia-activated Smad3-specific Dephosphorylation by PP2A

Abstract

The transforming growth factor-β (TGF-β) maintains epithelial homeostasis and suppresses early tumor formation, but paradoxically at later stages of tumor progression, TGF-β promotes malignancy. TGF-β activates phosphorylation of Smad2 and -3 effectors. Smad2 and -3 are known to have different functions, but differential regulation of their phosphorylation has not been described. Here we show that upon hypoxia, the TGF-β-induced phosphorylation of Smad3 was inhibited, although Smad2 remained phosphorylated. The inhibition of Smad3 phosphorylation was not due to TGF-β receptor inactivation. We show that Smad3 was dephosphorylated by PP2A (protein phosphatase 2A) specifically under hypoxic conditions. The hypoxic Smad3 dephosphorylation required intact expression of the essential scaffold component PR65 of PP2A. PP2A physically interacted with Smad3 that occurred only in hypoxia. Accordingly, Smad3-associated PP2A activity was found under hypoxic conditions. Hypoxia attenuated the nuclear accumulation of TGF-β-induced Smad3 but did not affect Smad2. Moreover, the influence of TGF-β on a set of Smad3-activated genes was attenuated by hypoxia, and this was reversed by chemical PP2A inhibition. Our data demonstrate the existence of a Smad3-specific phosphatase and identify a novel role for PP2A. Moreover, our data implicate a novel mechanism by which hypoxia regulates growth factor responses. The transforming growth factor-β (TGF-β) maintains epithelial homeostasis and suppresses early tumor formation, but paradoxically at later stages of tumor progression, TGF-β promotes malignancy. TGF-β activates phosphorylation of Smad2 and -3 effectors. Smad2 and -3 are known to have different functions, but differential regulation of their phosphorylation has not been described. Here we show that upon hypoxia, the TGF-β-induced phosphorylation of Smad3 was inhibited, although Smad2 remained phosphorylated. The inhibition of Smad3 phosphorylation was not due to TGF-β receptor inactivation. We show that Smad3 was dephosphorylated by PP2A (protein phosphatase 2A) specifically under hypoxic conditions. The hypoxic Smad3 dephosphorylation required intact expression of the essential scaffold component PR65 of PP2A. PP2A physically interacted with Smad3 that occurred only in hypoxia. Accordingly, Smad3-associated PP2A activity was found under hypoxic conditions. Hypoxia attenuated the nuclear accumulation of TGF-β-induced Smad3 but did not affect Smad2. Moreover, the influence of TGF-β on a set of Smad3-activated genes was attenuated by hypoxia, and this was reversed by chemical PP2A inhibition. Our data demonstrate the existence of a Smad3-specific phosphatase and identify a novel role for PP2A. Moreover, our data implicate a novel mechanism by which hypoxia regulates growth factor responses. IntroductionTGF-β 2The abbreviations used are: TGF-βtransforming growth factor-βTGF-βRITGF type 1 receptorBMPbone morphogenetic proteinsiRNAsmall interfering RNAOAokadaic acidVEGFvascular endothelial growth factorHAhemagglutinint-Smadtotal Smadp-Smadphospho-specific SmadHIFhypoxia-inducible factorQ-RT-PCRquantitative reverse transcription-PCR. is a multifunctional growth factor that is required for normal embryonic development, is involved in immunological responses, and maintains epithelial homeostasis as well as restricts the growth of normal cells and limits tumor formation (1Siegel P.M. Massagué J. Nat. Rev. Cancer. 2003; 3: 807-821Crossref PubMed Scopus (1332) Google Scholar, 2Derynck R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Crossref PubMed Scopus (1939) Google Scholar). However, at later stages of cancer development, cells become resistant to the growth inhibitory properties of TGF-β and begin to exploit it as a growth-promoting cytokine (1Siegel P.M. Massagué J. Nat. Rev. Cancer. 2003; 3: 807-821Crossref PubMed Scopus (1332) Google Scholar, 3Cui W. Fowlis D.J. Bryson S. Duffie E. Ireland H. Balmain A. Akhurst R.J. Cell. 1996; 86: 531-542Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 4Bierie B. Moses H.L. Nat. Rev. Cancer. 2006; 6: 506-520Crossref PubMed Scopus (1087) Google Scholar). TGF-β operates by activating TGF-β receptor complexes on the cell surface. TGF-β brings type 1 receptor (TGF-βRI, i.e. ALK5) to close proximity of the type 2 receptor that activates the type 1 receptor, subsequently leading to the activation of R-Smads (Smad2 and -3) by phosphorylation of serine residues at the C-terminal ends (5Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar, 6Shi Y. Massagué J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4737) Google Scholar). R-Smads bind to a common Smad4 mediator, are accumulated in the nucleus, and activate transcriptional responses (5Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar, 7ten Dijke P. Hill C.S. Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar, 8Massagué J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1899) Google Scholar).The TGF-β signaling through Smad2/3 is antagonized by several mechanisms, such as the activation of inhibitory Smads (I-Smads, Smad6 and -7) and dephosphorylation of R-Smads and TGF-β receptor. I-Smads operate by preventing R-Smad binding to the activated receptor, by competing with R-Smad binding to Smad4, and by inducing internalization and proteosomal degradation of TGF-βRI resulting in the attenuation of TGF-β signaling (7ten Dijke P. Hill C.S. Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar, 8Massagué J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1899) Google Scholar, 9Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4199) Google Scholar). Smad7 has also been shown to mediate PP1C (protein phosphatase 1C) binding to the activated TGF-βRI and subsequent inactivation of the receptor by dephosphorylation (10Shi W. Sun C. He B. Xiong W. Shi X. Yao D. Cao X. J. Cell Biol. 2004; 164: 291-300Crossref PubMed Scopus (206) Google Scholar). Independently from I-Smads, PP2A can inhibit the TGF-β superfamily receptors and BMP R-Smad (11Batut J. Schmierer B. Cao J. Raftery L.A. Hill C.S. Howell M. Development. 2008; 135: 2927-2937Crossref PubMed Scopus (64) Google Scholar, 12Bengtsson L. Schwappacher R. Roth M. Boergermann J.H. Hassel S. Knaus P. J. Cell Sci. 2009; 122: 1248-1257Crossref PubMed Scopus (39) Google Scholar). Moreover, Smad2 and -3 activity may be attenuated by a negative feedback mechanism independently from the TGF-βRI activity through dephosphorylation of the C-terminal serines by the PPM1A (PP2C) phosphatase (13Lin X. Duan X. Liang Y.Y. Su Y. Wrighton K.H. Long J. Hu M. Davis C.M. Wang J. Brunicardi F.C. Shi Y. Chen Y.G. Meng A. Feng X.H. Cell. 2006; 125: 915-928Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). The PPM1A phosphatase is a general inhibitor of TGF-β and BMP signaling. It may inhibit the activity of several Smad proteins either by dephosphorylation (Smad1–3) (13Lin X. Duan X. Liang Y.Y. Su Y. Wrighton K.H. Long J. Hu M. Davis C.M. Wang J. Brunicardi F.C. Shi Y. Chen Y.G. Meng A. Feng X.H. Cell. 2006; 125: 915-928Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 14Duan X. Liang Y.Y. Feng X.H. Lin X. J. Biol. Chem. 2006; 281: 36526-36532Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) or by regulating their degradation (Smad1, -5, and -8) (15Kokabu S. Nojima J. Kanomata K. Ohte S. Yoda T. Fukuda T. Katagiri T. J. Bone Miner. Res. 2010; (in press)PubMed Google Scholar). Noticeably, in each case the attenuation of TGF-βRI signaling results in the attenuation of both Smad2 and -3 phosphorylation. PPM1A belongs to a different class of phosphatases than PP1C and PP2A. PPM1A is a monomer, and PP1C and PP2A multimers. PP2A (protein phosphatase 2A) belongs to the same conserved gene family as PP1C, but it has distinct substrate specificity and expression pattern and is differentially inhibited by phosphatase inhibitors such as okadaic acid (16Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar, 17Cohen P.T. Trends Biochem. Sci. 1997; 22: 245-251Abstract Full Text PDF PubMed Scopus (453) Google Scholar). Their requirement on divalent cations differs (17Cohen P.T. Trends Biochem. Sci. 1997; 22: 245-251Abstract Full Text PDF PubMed Scopus (453) Google Scholar). The substrate specificity of PP2A multimer complex depends on regulatory β-subunits. PP2A may form ∼70 different forms and thereby may differentially target a number of substrates. Accordingly, PP2A has been reported to exert different activity toward the TGF-β superfamily receptors (11Batut J. Schmierer B. Cao J. Raftery L.A. Hill C.S. Howell M. Development. 2008; 135: 2927-2937Crossref PubMed Scopus (64) Google Scholar).The lack of sufficient tissue oxygenation, i.e. hypoxia, occurs during development and is a common feature of ischemic diseases and tumors. The best characterized mechanisms by which hypoxia signals are mediated are through the hypoxia-inducible factor (HIF). HIF is stabilized upon hypoxia and activates the transcription of a wide range of genes required to counteract the reduced oxygen availability (18Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3828) Google Scholar, 19Masson N. Willam C. Maxwell P.H. Pugh C.W. Ratcliffe P.J. EMBO J. 2001; 20: 5197-5206Crossref PubMed Scopus (846) Google Scholar, 20Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim Av Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4367) Google Scholar). Solid tumors contain hypoxic regions because of aberrant or limited amounts of vasculature. Hypoxia in tumors occurs at later stages of tumor growth, at the time when the tumors reach the size of ∼1 mm. Similarly to TGF-β, hypoxia acts as a progression factor in carcinomas, and the occurrence of hypoxia coincides with the conversion of the TGF-β response (1Siegel P.M. Massagué J. Nat. Rev. Cancer. 2003; 3: 807-821Crossref PubMed Scopus (1332) Google Scholar). Cooperation between hypoxia and TGF-β signaling has been reported to occur at least by two mechanisms. Hypoxia activates the expression of TGF-β1 (21Li J.J. Lu J. Kaur C. Sivakumar V. Wu C.Y. Ling E.A. Neuroscience. 2008; 156: 662-672Crossref PubMed Scopus (16) Google Scholar). Moreover, TGF-β has been reported to cooperate with hypoxia, and hypoxic signaling is affected by Smad3, which binds the α-subunit of the HIF complex and thereby enhances the hypoxic gene expression (22Sánchez-Elsner T. Botella L.M. Velasco B. Langa C. Bernabéu C. J. Biol. Chem. 2002; 277: 43799-43808Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 23Sánchez-Elsner T. Botella L.M. Velasco B. Corbí A. Attisano L. Bernabéu C. J. Biol. Chem. 2001; 276: 38527-38535Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 24Zhang H. Akman H.O. Smith E.L. Zhao J. Murphy-Ullrich J.E. Batuman O.A. Blood. 2003; 101: 2253-2260Crossref PubMed Scopus Google Scholar). However, hypoxia may TGF-β signaling under we show that hypoxia to in TGF-β response as hypoxia the TGF-β-induced phosphorylation of Smad3 Smad2 phosphorylation. is due to dephosphorylation of Smad3 by phosphatase under we have shown that hypoxia the TGF-β-induced Smad3 phosphorylation but the Smad2 phosphorylation this is due to dephosphorylation of Smad3 by PP2A. our this is the of a Smad phosphatase that is to leading to a of only of the TGF-β TGF-β activated R-Smad may be inhibited by mechanisms, the inhibition of R-Smad receptor as well as receptor degradation by this type of inhibition and subsequent attenuation in R-Smad phosphorylation is well Moreover, PP1C can TGF-βRI (10Shi W. Sun C. He B. Xiong W. Shi X. Yao D. Cao X. J. Cell Biol. 2004; 164: 291-300Crossref PubMed Scopus (206) Google Scholar). have been shown to occur on several in the TGF-β superfamily signaling BMP and TGF-β signaling are by which target the C-terminal phosphorylation of and the phosphorylation of Smad2 and -3 M. C. Massagué J. Sci. 2006; PubMed Scopus Google Scholar, K.H. D. Long J. Lin X. Feng X.H. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). BMP receptor with form of PP2A leading to dephosphorylation of BMP in and different PP2A forms can different receptors of the TGF-β superfamily and (11Batut J. Schmierer B. Cao J. Raftery L.A. Hill C.S. Howell M. Development. 2008; 135: 2927-2937Crossref PubMed Scopus (64) Google Scholar, 12Bengtsson L. Schwappacher R. Roth M. Boergermann J.H. Hassel S. Knaus P. J. Cell Sci. 2009; 122: 1248-1257Crossref PubMed Scopus (39) Google Scholar). L. Schwappacher R. Roth M. Boergermann J.H. Hassel S. Knaus P. J. Cell Sci. 2009; 122: 1248-1257Crossref PubMed Scopus (39) Google Scholar) show that PP2A and only the in also reported that the dephosphorylation to nuclear of in Noticeably, in the by (11Batut J. Schmierer B. Cao J. Raftery L.A. Hill C.S. Howell M. Development. 2008; 135: 2927-2937Crossref PubMed Scopus (64) Google the TGF-β signaling is to be attenuated as by Smad2 C-terminal phosphorylation. that PP2A regulates TGF-β and BMP signaling both at the receptor and R-Smad under conditions. However, our results show that the in Smad3 C-terminal phosphorylation under hypoxia is independently from the receptor activity and that it is for The data demonstrate that TGF-βRI is not affected by hypoxia and that receptor expression not the hypoxic attenuation in Smad3 phosphorylation. Smad3 phosphorylation to be inhibited by hypoxia in the of a with Smad3 phosphorylation was by a chemical TGF-βRI hypoxia the dephosphorylation the in essential receptor or the of receptor Smad2 and -3 may also be by However, of the mechanism of both Smad2 and -3 are affected (13Lin X. Duan X. Liang Y.Y. Su Y. Wrighton K.H. Long J. Hu M. Davis C.M. Wang J. Brunicardi F.C. Shi Y. Chen Y.G. Meng A. Feng X.H. Cell. 2006; 125: 915-928Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). Smad2 and -3 reported to be dephosphorylated under TGF-β at time by PPM1A (13Lin X. Duan X. Liang Y.Y. Su Y. Wrighton K.H. Long J. Hu M. Davis C.M. Wang J. Brunicardi F.C. Shi Y. Chen Y.G. Meng A. Feng X.H. Cell. 2006; 125: 915-928Abstract Full Text Full Text PDF PubMed Scopus (377) Google with our data that the phosphatases a role in regulating TGF-β responses independently of receptor we not to dephosphorylation of Smad3 by PPM1A or it is that not have a on the hypoxic Smad3 dephosphorylation TGF-β at time PPM1A is to okadaic acid but has requirement for or PP2A is not on divalent but is inhibited by okadaic the of our data show that the hypoxic Smad3 dephosphorylation is okadaic at Moreover, in hypoxia Smad3 phosphatase activity in the of a of PPM1A by not the hypoxic Smad3 was in to the of PP2A by which did inhibit the Smad3 The inhibition occurred in hypoxia but did not have a on Smad3 phosphorylation in for the of PP2A in Smad3 Moreover, the data a of PP2A and Smad3 and that the occurs only under hypoxic but not in the data that the PP2A did not with the Smad2 with PP2A activity with Smad3 but not with and PP2A dephosphorylated Smad3 in specifically under hypoxic of demonstrate that Smad2 and -3 have show that Smad2 and -3 are not Yang X. M. M. T. C. Nat. Cell Biol. PubMed Scopus Google Scholar, J. D. M. E. W. R. Sci. PubMed Scopus Google Scholar). Moreover, Smad2 and -3 have been reported to distinct in transcriptional responses E. J. D. M. C. R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) and T. A. M. C. Heldin C.H. Souchelnytskyi S. Biol. Cell. 2005; PubMed Scopus Google Scholar). Our data on the hypoxic regulation of R-Smad activity a mechanism as to differential regulation of Smad2 and -3 may the data may to such as the of Smad3 with of Yang X. M. M. T. C. Nat. Cell Biol. PubMed Scopus Google Scholar) as well as the of tumor progression in Smad3 Y. J.M. Cell. Full Text Full Text PDF PubMed Scopus Google our PP2A is the phosphatase specificity between Smad2 and PP2A has been in a number of homeostasis and in the of it is as a tumor (16Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar, M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). least the different regulatory and two scaffold for PP2A may different the PP2A and phosphatase with in the PP2A regulatory have been reported to affect the of PP2A to cell W. R. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google and different forms can inhibit different TGF-β superfamily receptors (11Batut J. Schmierer B. Cao J. Raftery L.A. Hill C.S. Howell M. Development. 2008; 135: 2927-2937Crossref PubMed Scopus (64) Google Scholar). It is that hypoxia to the PP2A by regulatory and thereby PP2A target because hypoxia is known to the expression of a different proteins and also to activate it is that the proteins may be involved in the of PP2A to hypoxia has been reported to the expression of TGF-β (21Li J.J. Lu J. Kaur C. Sivakumar V. Wu C.Y. Ling E.A. Neuroscience. 2008; 156: 662-672Crossref PubMed Scopus (16) Google Scholar, A. L. A. H. C. M. M. J. 2002; PubMed Scopus Google and has been shown to cooperate with Smad3 and to the transcriptional responses H. Akman H.O. Smith E.L. Zhao J. Murphy-Ullrich J.E. Batuman O.A. Blood. 2003; 101: 2253-2260Crossref PubMed Scopus Google Scholar). The of the PP2A complex as well as HIF may also bind to the complex to be in of hypoxic Smad3 dephosphorylation by PP2A on our we that hypoxia has a on the epithelial through hypoxia to expression of nuclear that was by PP2A although the nuclear accumulation of was not affected by hypoxia. at least a of genes that epithelial and such as and by hypoxia that in was reversed by PP2A inhibition. under hypoxia the Smad2 may and normal responses in hypoxia M. Akhurst R.J. Balmain A. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar). in hypoxia PP2A the C-terminal serines of Smad3 and the phosphorylation at the may and of Smad3 IntroductionTGF-β 2The abbreviations used are: TGF-βtransforming growth factor-βTGF-βRITGF type 1 receptorBMPbone morphogenetic proteinsiRNAsmall interfering RNAOAokadaic acidVEGFvascular endothelial growth factorHAhemagglutinint-Smadtotal Smadp-Smadphospho-specific SmadHIFhypoxia-inducible factorQ-RT-PCRquantitative reverse transcription-PCR. is a multifunctional growth factor that is required for normal embryonic development, is involved in immunological responses, and maintains epithelial homeostasis as well as restricts the growth of normal cells and limits tumor formation (1Siegel P.M. Massagué J. Nat. Rev. Cancer. 2003; 3: 807-821Crossref PubMed Scopus (1332) Google Scholar, 2Derynck R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Crossref PubMed Scopus (1939) Google Scholar). However, at later stages of cancer development, cells become resistant to the growth inhibitory properties of TGF-β and begin to exploit it as a growth-promoting cytokine (1Siegel P.M. Massagué J. Nat. Rev. Cancer. 2003; 3: 807-821Crossref PubMed Scopus (1332) Google Scholar, 3Cui W. Fowlis D.J. Bryson S. Duffie E. Ireland H. Balmain A. Akhurst R.J. Cell. 1996; 86: 531-542Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 4Bierie B. Moses H.L. Nat. Rev. Cancer. 2006; 6: 506-520Crossref PubMed Scopus (1087) Google Scholar). TGF-β operates by activating TGF-β receptor complexes on the cell surface. TGF-β brings type 1 receptor (TGF-βRI, i.e. ALK5) to close proximity of the type 2 receptor that activates the type 1 receptor, subsequently leading to the activation of R-Smads (Smad2 and -3) by phosphorylation of serine residues at the C-terminal ends (5Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar, 6Shi Y. Massagué J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4737) Google Scholar). R-Smads bind to a common Smad4 mediator, are accumulated in the nucleus, and activate transcriptional responses (5Moustakas A. Souchelnytskyi S. Heldin C.H. J. Cell Sci. 2001; 114: 4359-4369Crossref PubMed Google Scholar, 7ten Dijke P. Hill C.S. Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar, 8Massagué J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1899) Google Scholar).The TGF-β signaling through Smad2/3 is antagonized by several mechanisms, such as the activation of inhibitory Smads (I-Smads, Smad6 and -7) and dephosphorylation of R-Smads and TGF-β receptor. I-Smads operate by preventing R-Smad binding to the activated receptor, by competing with R-Smad binding to Smad4, and by inducing internalization and proteosomal degradation of TGF-βRI resulting in the attenuation of TGF-β signaling (7ten Dijke P. Hill C.S. Trends Biochem. Sci. 2004; 29: 265-273Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar, 8Massagué J. Seoane J. Wotton D. Genes Dev. 2005; 19: 2783-2810Crossref PubMed Scopus (1899) Google Scholar, 9Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4199) Google Scholar). Smad7 has also been shown to mediate PP1C (protein phosphatase 1C) binding to the activated TGF-βRI and subsequent inactivation of the receptor by dephosphorylation (10Shi W. Sun C. He B. Xiong W. Shi X. Yao D. Cao X. J. Cell Biol. 2004; 164: 291-300Crossref PubMed Scopus (206) Google Scholar). Independently from I-Smads, PP2A can inhibit the TGF-β superfamily receptors and BMP R-Smad (11Batut J. Schmierer B. Cao J. Raftery L.A. Hill C.S. Howell M. Development. 2008; 135: 2927-2937Crossref PubMed Scopus (64) Google Scholar, 12Bengtsson L. Schwappacher R. Roth M. Boergermann J.H. Hassel S. Knaus P. J. Cell Sci. 2009; 122: 1248-1257Crossref PubMed Scopus (39) Google Scholar). Moreover, Smad2 and -3 activity may be attenuated by a negative feedback mechanism independently from the TGF-βRI activity through dephosphorylation of the C-terminal serines by the PPM1A (PP2C) phosphatase (13Lin X. Duan X. Liang Y.Y. Su Y. Wrighton K.H. Long J. Hu M. Davis C.M. Wang J. Brunicardi F.C. Shi Y. Chen Y.G. Meng A. Feng X.H. Cell. 2006; 125: 915-928Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). The PPM1A phosphatase is a general inhibitor of TGF-β and BMP signaling. It may inhibit the activity of several Smad proteins either by dephosphorylation (Smad1–3) (13Lin X. Duan X. Liang Y.Y. Su Y. Wrighton K.H. Long J. Hu M. Davis C.M. Wang J. Brunicardi F.C. Shi Y. Chen Y.G. Meng A. Feng X.H. Cell. 2006; 125: 915-928Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 14Duan X. Liang Y.Y. Feng X.H. Lin X. J. Biol. Chem. 2006; 281: 36526-36532Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) or by regulating their degradation (Smad1, -5, and -8) (15Kokabu S. Nojima J. Kanomata K. Ohte S. Yoda T. Fukuda T. Katagiri T. J. Bone Miner. Res. 2010; (in press)PubMed Google Scholar). Noticeably, in each case the attenuation of TGF-βRI signaling results in the attenuation of both Smad2 and -3 phosphorylation. PPM1A belongs to a different class of phosphatases than PP1C and PP2A. PPM1A is a monomer, and PP1C and PP2A multimers. PP2A (protein phosphatase 2A) belongs to the same conserved gene family as PP1C, but it has distinct substrate specificity and expression pattern and is differentially inhibited by phosphatase inhibitors such as okadaic acid (16Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1511) Google Scholar, 17Cohen P.T. Trends Biochem. Sci. 1997; 22: 245-251Abstract Full Text PDF PubMed Scopus (453) Google Scholar). Their requirement on divalent cations differs (17Cohen P.T. Trends Biochem. Sci. 1997; 22: 245-251Abstract Full Text PDF PubMed Scopus (453) Google Scholar). The substrate specificity of PP2A multimer complex depends on regulatory β-subunits. PP2A may form ∼70 different forms and thereby may differentially target a number of substrates. Accordingly, PP2A has been reported to exert different activity toward the TGF-β superfamily receptors (11Batut J. Schmierer B. Cao J. Raftery L.A. Hill C.S. Howell M. Development. 2008; 135: 2927-2937Crossref PubMed Scopus (64) Google Scholar).The lack of sufficient tissue oxygenation, i.e. hypoxia, occurs during development and is a common feature of ischemic diseases and tumors. The best characterized mechanisms by which hypoxia signals are mediated are through the hypoxia-inducible factor (HIF). HIF is stabilized upon hypoxia and activates the transcription of a wide range of genes required to counteract the reduced oxygen availability (18Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3828) Google Scholar, 19Masson N. Willam C. Maxwell P.H. Pugh C.W. Ratcliffe P.J. EMBO J. 2001; 20: 5197-5206Crossref PubMed Scopus (846) Google Scholar, 20Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim Av Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4367) Google Scholar). Solid tumors contain hypoxic regions because of aberrant or limited amounts of vasculature. Hypoxia in tumors occurs at later stages of tumor growth, at the time when the tumors reach the size of ∼1 mm. Similarly to TGF-β, hypoxia acts as a progression factor in carcinomas, and the occurrence of hypoxia coincides with the conversion of the TGF-β response (1Siegel P.M. Massagué J. Nat. Rev. Cancer. 2003; 3: 807-821Crossref PubMed Scopus (1332) Google Scholar). Cooperation between hypoxia and TGF-β signaling has been reported to occur at least by two mechanisms. Hypoxia activates the expression of TGF-β1 (21Li J.J. Lu J. Kaur C. Sivakumar V. Wu C.Y. Ling E.A. Neuroscience. 2008; 156: 662-672Crossref PubMed Scopus (16) Google Scholar). Moreover, TGF-β has been reported to cooperate with hypoxia, and hypoxic signaling is affected by Smad3, which binds the α-subunit of the HIF complex and thereby enhances the hypoxic gene expression (22Sánchez-Elsner T. Botella L.M. Velasco B. Langa C. Bernabéu C. J. Biol. Chem. 2002; 277: 43799-43808Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 23Sánchez-Elsner T. Botella L.M. Velasco B. Corbí A. Attisano L. Bernabéu C. J. Biol. Chem. 2001; 276: 38527-38535Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 24Zhang H. Akman H.O. Smith E.L. Zhao J. Murphy-Ullrich J.E. Batuman O.A. Blood. 2003; 101: 2253-2260Crossref PubMed Scopus Google Scholar). However, hypoxia may TGF-β signaling under we show that hypoxia to in TGF-β response as hypoxia the TGF-β-induced phosphorylation of Smad3 Smad2 phosphorylation. is due to dephosphorylation of Smad3 by phosphatase under hypoxia.