Insulin-like Growth Factor-I Inhibits Transcriptional Responses of Transforming Growth Factor-β by Phosphatidylinositol 3-Kinase/Akt-dependent Suppression of the Activation of Smad3 but Not Smad2

Insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) have been shown to be oncogenic and tumor suppressive, respectively, on prostate epithelial cells. Here we show that IGF-I inhibits the ability of TGF-β to regulate expression of several genes in the non-tumorigenic rat prostatic epithelial line, NRP-152. In these cells, IGF-I also inhibits TGF-β-induced transcriptional responses, as shown by several promoter reporter constructs, suggesting that IGF-I intercepts an early step in TGF-β signaling. We show that IGF-I does not down-regulate TGF-β receptor levels, as determined by both receptor cross-linking and Western blot analyses. However, Western blot analysis reveals that IGF-I selectively inhibits the TGF-β-triggered activation Smad3 but not Smad2, while not altering expression of total Smads 2, 3, or 4. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY29004 reverses the ability of IGF-I to inhibit TGF-β-induced transcriptional responses and the activation of Smad3, suggesting that the suppression of TGF-β signaling by IGF-I is mediated through activation of PI3K. Moreover, we show that enforced expression of dominant-negative PI3K (DN-p85α) or phosphatidylinositol 3-phosphate-phosphatase, PTEN, also reverse the suppressive effect of IGF-I on TGF-β-induced 3TP-luciferase reporter activity, whereas constitutively active PI3K (p110αCAAX) completely blocks TGF-β-induced 3TP-luciferase reporter activity. Further transfection experiments including expression of constitutively active and dominant-negative Akt and rapamycin treatment suggest that suppression of TGF-β signaling/Smad3 activation by IGF-I occurs downstream of Akt and through mammalian target of rapamycin activation. In summary, our data suggest that IGF-I inhibits TGF-β transcriptional responses through selective suppression of Smad3 activation via a PI3K/Akt-dependent pathway. Insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) have been shown to be oncogenic and tumor suppressive, respectively, on prostate epithelial cells. Here we show that IGF-I inhibits the ability of TGF-β to regulate expression of several genes in the non-tumorigenic rat prostatic epithelial line, NRP-152. In these cells, IGF-I also inhibits TGF-β-induced transcriptional responses, as shown by several promoter reporter constructs, suggesting that IGF-I intercepts an early step in TGF-β signaling. We show that IGF-I does not down-regulate TGF-β receptor levels, as determined by both receptor cross-linking and Western blot analyses. However, Western blot analysis reveals that IGF-I selectively inhibits the TGF-β-triggered activation Smad3 but not Smad2, while not altering expression of total Smads 2, 3, or 4. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY29004 reverses the ability of IGF-I to inhibit TGF-β-induced transcriptional responses and the activation of Smad3, suggesting that the suppression of TGF-β signaling by IGF-I is mediated through activation of PI3K. Moreover, we show that enforced expression of dominant-negative PI3K (DN-p85α) or phosphatidylinositol 3-phosphate-phosphatase, PTEN, also reverse the suppressive effect of IGF-I on TGF-β-induced 3TP-luciferase reporter activity, whereas constitutively active PI3K (p110αCAAX) completely blocks TGF-β-induced 3TP-luciferase reporter activity. Further transfection experiments including expression of constitutively active and dominant-negative Akt and rapamycin treatment suggest that suppression of TGF-β signaling/Smad3 activation by IGF-I occurs downstream of Akt and through mammalian target of rapamycin activation. In summary, our data suggest that IGF-I inhibits TGF-β transcriptional responses through selective suppression of Smad3 activation via a PI3K/Akt-dependent pathway. TGF-β, 1The abbreviations used are: TGF-β, transforming growth factor-β;TβR, TGF-β1 receptor; DMEM/F-12, Dulbecco's modified Eagle's medium/Ham's F-12; IGF-I, insulin-like growth hormone factor-I; LR3-IGF-I, long R3 IGF-I; IGF-IR, IGF-I receptor; DN, dominant negative; PI3K, phosphatidylinositol 3-kinase; CA, constitutively active; SARA, Smad anchor for receptor activation; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SRE, serum response element; SRF, serum response factor; NF-κB, nuclear factor κB; PH, pleckstrin homology; SH2, Src homology; SBE, Smad binding element; AP1, activator protein 1; CMV, cytomegalovirus; PI3P, phosphatidylinositol 3-phosphate; PI, phosphatidylinositol.1The abbreviations used are: TGF-β, transforming growth factor-β;TβR, TGF-β1 receptor; DMEM/F-12, Dulbecco's modified Eagle's medium/Ham's F-12; IGF-I, insulin-like growth hormone factor-I; LR3-IGF-I, long R3 IGF-I; IGF-IR, IGF-I receptor; DN, dominant negative; PI3K, phosphatidylinositol 3-kinase; CA, constitutively active; SARA, Smad anchor for receptor activation; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SRE, serum response element; SRF, serum response factor; NF-κB, nuclear factor κB; PH, pleckstrin homology; SH2, Src homology; SBE, Smad binding element; AP1, activator protein 1; CMV, cytomegalovirus; PI3P, phosphatidylinositol 3-phosphate; PI, phosphatidylinositol. for which there are three mammalian isoforms, is a multifunctional autocrine/paracrine growth regulator belonging to a large TGF-β superfamily (1Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. The Transforming Growth Factor Beta. Handbook of Experimental Pharmacology: Peptide Growth Factors and Their Receptors. Springer-Verlag New York Inc., New York1990: 419-472Google Scholar, 2Massague J. Cell. 1992; 69: 1067-1070Abstract Full Text PDF PubMed Scopus (639) Google Scholar). TGF-β functions as a tumor suppressor of the prostate (3Guo Y. Kyprianou N. Cancer Res. 1999; 59: 1366-1371PubMed Google Scholar, 4Tang B. de Castro K. Barnes H.E. Parks W.T. Stewart L. Bottinger E.P. Danielpour D. Wakefield L.M. Cancer Res. 1999; 59: 4834-4842PubMed Google Scholar) through a mechanism that is likely dependent on its ability to induce apoptosis and growth arrest (3Guo Y. Kyprianou N. Cancer Res. 1999; 59: 1366-1371PubMed Google Scholar, 5Hsing A.Y. Kadomatsu K. Bonham M.J. Danielpour D. Cancer Res. 1996; 56: 5146-5149PubMed Google Scholar, 6Chipuk J.E. Bhat M. Hsing A.Y. Ma J. Danielpour D. J. Biol. Chem. 2001; 276: 26614-26621Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). A number of studies support that TGF-β plays an important role in the mechanism and regulation of androgen action (7Kyprianou N. Isaacs J.T. Endocrinology. 1988; 123: 2124-2131Crossref PubMed Scopus (153) Google Scholar, 8Kim I.Y. Ahn H.J. Zelner D.J. Park L. Sensibar J.A. Lee C. Mol. Endocrinol. 1996; 10: 107-115Crossref PubMed Scopus (71) Google Scholar, 9Brodin G. ten Dijke P. Funa K. Heldin C.H. Landstrom M. Cancer Res. 1999; 59: 2731-2738PubMed Google Scholar, 10Lucia M.S. Sporn M.B. Roberts A.B. Stewart L.V. Danielpour D. J. Cell. Physiol. 1998; 175: 184-192Crossref PubMed Scopus (42) Google Scholar, 11Chipuk J.E. Cornelius S.C. Pultz N.J. Jorgensen J.S. Bonham M.J. Kim S.J. Danielpour D. J. Biol. Chem. 2002; 277: 1240-1248Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The pattern of cross-talk between androgen and TGF-β, which occurs through multiple steps (8Kim I.Y. Ahn H.J. Zelner D.J. Park L. Sensibar J.A. Lee C. Mol. Endocrinol. 1996; 10: 107-115Crossref PubMed Scopus (71) Google Scholar, 9Brodin G. ten Dijke P. Funa K. Heldin C.H. Landstrom M. Cancer Res. 1999; 59: 2731-2738PubMed Google Scholar, 11Chipuk J.E. Cornelius S.C. Pultz N.J. Jorgensen J.S. Bonham M.J. Kim S.J. Danielpour D. J. Biol. Chem. 2002; 277: 1240-1248Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 12Kyprianou N. Isaacs J.T. Mol. Endocrinol. 1989; 3: 1515-1522Crossref PubMed Scopus (393) Google Scholar), is likely controlled by androgen receptor co-regulators and other growth modulators such as IGF-I (5Hsing A.Y. Kadomatsu K. Bonham M.J. Danielpour D. Cancer Res. 1996; 56: 5146-5149PubMed Google Scholar, 13Stewart L.V. Song K. Hsing A.Y. Danielpour D. Exp. Cell Res. 2003; 284: 301-313Crossref Scopus (10) Google Scholar). Our laboratory has reported previously that IGF-I is a potent inhibitor of the apoptosis of the NRP-152 rat prostate epithelial cell line induced by TGF-β but not by a variety of other apoptosis inducers (5Hsing A.Y. Kadomatsu K. Bonham M.J. Danielpour D. Cancer Res. 1996; 56: 5146-5149PubMed Google Scholar), suggesting that IGF-I blocks early TGF-β signals that lead to apoptosis. TGF-β1 signals mainly through a cooperative interaction with cell surface signaling receptors, TβRI, and TβRII (2Massague J. Cell. 1992; 69: 1067-1070Abstract Full Text PDF PubMed Scopus (639) Google Scholar, 14ten Dijke P. Miyazono K. Heldin C.H. Curr. Opin. Cell Biol. 1996; 8: 139-145Crossref PubMed Scopus (234) Google Scholar). TGF-β1 first associates with TβRII, which recruits TβRI to form a ligand/receptor heteromeric complex. The constitutively active kinase domain of TβRII then activates TβRI by transphosphorylation of the GS box in the cytoplasmic domain (15Wieser R. Wrana J.L. Massague J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (592) Google Scholar). The activated TβRI then transmits TGF-β signals by binding and activating Smads 2 and 3 through transphosphorylation of their C-terminal SSXS serines (16Abdollah S. Macias-Silva M. Tsukazaki T. Hayashi H. Attisano L. Wrana J.L. J. Biol. Chem. 1997; 272: 27678-27685Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar), a process that requires accessory proteins such as SARA (17Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar), Hrs (18Miura S. Takeshita T. Asao H. Kimura Y. Murata K. Sasaki Y. Hanai J.I. Beppu H. Tsukazaki T. Wrana J.L. Miyazono K. Sugamura K. Mol. Cell. Biol. PubMed Scopus Google Scholar), EMBO J. 2001; PubMed Scopus Google Scholar), and receptor H. N. M. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). Smads and Y. R. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar) and then to the nuclear with or J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), or through with Smad response S. Heldin C.H. ten Dijke P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J.L. 1998; PubMed Scopus Google Scholar). TGF-β also of the protein kinase through which with activated Smads are for the of growth arrest and apoptosis induced by TGF-β Y. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). IGF-I, which is a potent inhibitor of to and activates the receptor kinase IGF-IR, a receptor shown to be for by a number of R. Exp. Cell Res. 1999; PubMed Scopus Google Scholar, R. J. Cell. 1999; PubMed Google Scholar, R. Cancer Res. 1995; Google Scholar, R. M. M. B. 1997; PubMed Scopus Google Scholar). serum IGF-I is to be a of prostate and be in the of Res. 1999; PubMed Scopus Google Scholar, P. S. C. H. L. G. R. J. Cancer PubMed Scopus Google Scholar, J. K. L. S. J. C. S. PubMed Scopus Google Scholar, S. P. Cancer Res. 1999; 59: Google Scholar). studies show that the IGF-I is and be the to T. M. H. Endocrinology. 1998; PubMed Scopus Google Scholar, C. J. G. H. Cancer Res. Google Scholar, Y. K. B. G. Cancer Res. Google Scholar, S. C. S. 2001; PubMed Scopus Google Scholar, L. S. C. EMBO J. 2002; PubMed Scopus Google Scholar). kinase which of a of three mammalian isoforms, and plays a role in not of IGF-I on tumor growth and of apoptosis Cell. 2002; 14: PubMed Scopus Google Scholar). three have an a kinase domain with an activation a and a the Cell. 2002; 14: PubMed Scopus Google Scholar). activated by receptor or via PI3K a the of phosphatidylinositol to which is for the anchor of Akt through the also a domain protein that requires for Akt whereas a kinase Akt of phosphatidylinositol the via PI3K, an domain protein that associates to activated receptor Akt growth and inhibit apoptosis has been by its in cell growth and including protein kinase protein kinase in in mTOR, and the androgen receptor S. C. S. 2001; PubMed Scopus Google Scholar, P. T. 2001; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar). PTEN, a tumor is a phosphatase in including prostate J. 1999; PubMed Scopus Google Scholar, H. Cancer Res. 1997; Google Scholar). of of to of with cell and apoptosis. In we the first that IGF-I multiple TGF-β the NRP-152 epithelial cell We show that IGF-I receptor signaling selectively the ability of TGF-β to Smad3 but not Our data suggest a role for PI3K and Akt in the mechanism by which IGF-I blocks TGF-β-induced PI3K inhibitor dominant-negative PI3K and the PTEN, inhibit transcriptional activation of the activator promoter also reverses the of Smad3 activation by Moreover, transfection of or but not IGF-I in TGF-β-induced transcriptional or rapamycin inhibit the suppression of TGF-β-induced suggesting that suppression by IGF-I is of Akt and and their as TGF-β1 Inc., C. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google number and and Inc., IGF-I and and serum and growth factor and and rapamycin number and J. Curr. Opin. Cell Biol. 1998; 10: PubMed Scopus Google Scholar), and Inc., serum Inc., Cell and prostatic epithelial cell in with growth and in The in a and a of experiments serum in with and NRP-152 by the total through a and and by of the and in and then by action to for with and and the and D. J. Cell. Physiol. 1996; PubMed Scopus Google Scholar). The of with with of a and Western a of of in to or the of and in the or of TGF-β1 for to with and with S. Danielpour D. R. Hsing A.Y. H. T. Roberts A.B. Cell Growth Google Scholar), and for and by the protein TGF-β receptor Western by with to the of or of for in through and to for in and and and with the and the used and by C. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The used to protein the NRP-152 a of in with and for then with or for to TGF-β1 by cell in of and as by the The of of for of and of of the through a in for 2 The and to and and a of or 2 in or constructs, with of reporter a 3 and in for to in and then with for by TGF-β1 for experiments to for 2 transfection and with or for 2 by or for and then with TGF-β1 for and a in with 2 or for and with binding with serum and with of the binding or with a of TGF-β1 D. Mol. Biol. Google Scholar). A of but used to cell number a The then on a for 2 with 2 of serum and with of in on a for with 2 and with a cell in the of of 2 and to a for 2 in of for on a by for and by through a We have shown previously (5Hsing A.Y. Kadomatsu K. Bonham M.J. Danielpour D. Cancer Res. 1996; 56: 5146-5149PubMed Google Scholar) that TGF-β1 apoptosis of non-tumorigenic NRP-152 in of these is dependent on and IGF-I whereas the ability of TGF-β1 to induce their apoptosis (5Hsing A.Y. Kadomatsu K. Bonham M.J. Danielpour D. Cancer Res. 1996; 56: 5146-5149PubMed Google Scholar). the of IGF-I that are of which are by these cells, Danielpour and M. M. we used an of IGF-I that to the IGF-I receptor with as IGF-I but to is active IGF-I in TGF-β-induced apoptosis and apoptosis by of TGF-β used In to in NRP-152 is to apoptosis by inducers of apoptosis on these (5Hsing A.Y. Kadomatsu K. Bonham M.J. Danielpour D. Cancer Res. 1996; 56: 5146-5149PubMed Google Scholar). data suggest that IGF-I functions by early signals important to the of apoptosis by our that IGF-I is to inhibit TGF-β we determined IGF-I also the ability of TGF-β to regulate the expression of several genes in NRP-152 cells. We that the of the 13Stewart L.V. Song K. Hsing A.Y. Danielpour D. Exp. Cell Res. 2003; 284: 301-313Crossref Scopus (10) Google and and the of and not expression by TGF-β1 IGF-I intercepts TGF-β signaling in NRP-152 cells, we determined the of TGF-β1 on NRP-152 with the activator promoter reporter and a promoter TGF-β1 treatment with LR3-IGF-I, and and TGF-β1 to to in transfection and cell by these our that inhibits activator promoter induced by TGF-β1 in NRP-152 We in and rat prostatic cell line, The rat prostatic cell line that apoptosis by but that is not by IGF-I (5Hsing A.Y. Kadomatsu K. Bonham M.J. Danielpour D. Cancer Res. 1996; 56: 5146-5149PubMed Google Scholar) of 3TP-luciferase by TGF-β1 in not by We the effect of on the activation by TGF-β1 of several promoter response AP1, SRE, SRF, reporter to 3TP-luciferase activity, the activation by TGF-β of the Smad binding and these suggest that IGF-I early TGF-β signaling steps to the activation of SBE, AP1, SRF, SRE, and promoter response suggesting that TGF-β or Smads are likely of suppression by IGF-I TGF-β receptor we first for TGF-β receptor expression on NRP-152 with or 2 or for with for and the to total cell to and by TGF-β TβRI, TβRII, and not but by treatment The effect of on expression of TβRI and TβRII in NRP-152 also determined by Western blot analysis that of with 2 not inhibit expression of these signaling but expression of TβRI Smads are the signaling of TGF-β receptors, we the expression of of these proteins or their activation by TGF-β by NRP-152 with or 2 for and then with TGF-β1 for 2, and and for expression of both total and Smads 2 and 3 by Western blot analysis In these both Smads 2 and 3 activated by TGF-β1 with of expression these the of Smad3 but not by TGF-β with 2 LR3-IGF-I, whereas the of total Smads 3 and 2 not In we the suppression of Smad3 by of TGF-β whereas of by TGF-β1 by such treatment not the expression of total Smads 2, 3, or 4. expression of total Smad3 by of treatment with and of total Smad3 not by In of on the completely by that IGF-I blocks the ability of TGF-β1 to selectively Smad3 but not have been reported to responses of IGF-I Cell. 2002; 14: PubMed Scopus Google Scholar). We IGF-I inhibits TGF-β responses through a a inhibitor of PI3K, L. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). We first by Western blot analysis of that NRP-152 with activation of Akt by 2 LR3-IGF-I, whereas of activation. We determined also reverse suppression of TGF-β 3TP-luciferase and blot expression of and as The of of NRP-152 with to in these experiments of cell with of treatment with in these experiments NRP-152 with or 2 to TGF-β1 and responses of TGF-β1 the suppression of expression by that the ability of to reverse the IGF-I suppression of TGF-β signaling through PI3K, we these with or with the and with LR3-IGF-I, with and for expression of TGF-β-induced 3TP-luciferase reporter activity, whereas TGF-β-induced reporter activity. of are the regulation of not PI3K but also PTEN, a phosphatase that the de J.L. C. Sasaki T. J. Cell. 1998; 95: Full Text Full Text PDF PubMed Scopus Google Scholar). expression of is to to in of TGF-β-induced our we the ability of to inhibit TGF-β-induced 3TP-luciferase by NRP-152 with or to of or expression of completely the suppressive effect of on such TGF-β-induced We the role of Akt in the suppressive effect of in TGF-β signaling. to the with PTEN, with or expression Our show that constitutively active Akt inhibits TGF-β-induced 3TP-luciferase to the as treatment with and the active Akt is to inhibit 3TP-luciferase in the of suggest that the effect of IGF-I on TGF-β signaling is mediated by the activation of Akt In to activated the suppressive effect of on transcriptional by TGF-β1 suggesting that suppressive effect of IGF-I is of activated these of not we to the role of signals in response by of Akt also reverse Our suggest that mTOR, which is activated by suppression of TGF-β which is a inhibitor of and inhibits by the of to G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), used to of NRP-152 with rapamycin completely the suppressive effect of on TGF-β-induced 3TP-luciferase suggesting that the IGF-I suppression is of Akt and through the activation of Moreover, rapamycin does not with TGF-β to 3TP-luciferase activity. We our that IGF-I blocks the activation of Smad3 through a we first a to the for 2 IGF-I to Smad3 activation of treatment with of with the activation of Smad3 by TGF-β1 The of 2 to the of to completely reverse effect on the activation of Smad3 and to inhibit activation of Akt by we which the IGF-I suppression of TGF-β-induced also reverse the suppression of Smad3 activation. rapamycin the effect of on suppression of TGF-β-induced Smad3 activation suggesting that selective suppression of Smad3 activation. Here we the first that IGF-I inhibits early signals that TGF-β transcriptional responses, as in NRP-152 and rat prostatic epithelial cell shown with the of the IGF-I LR3-IGF-I, which has for proteins J. Endocrinol. PubMed Scopus Google Scholar). our support that suppression is through signaling and not through of the of a is IGF-I also by the growth and of through an mechanism R. B. P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, our data show that of by the PI3K inhibitor, or by transfection of or expression reverses the suppression by on TGF-β whereas the (p110αCAAX) completely TGF-β Further transfection experiments with and or suppression of the of an Akt mTOR, by rapamycin treatment support that the suppression of TGF-β signaling by IGF-I is mediated through activated Akt A and our rapamycin data suggest is in the mechanism of suppression by to be by that is for the suppression of is that which suppression of by a with also TGF-β responses through the of to the GS of TβRI Massague J. EMBO J. 1997; PubMed Scopus Google Scholar, M. L. J. Massague J. Mol. Cell. 2001; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). However, activation of TβRI by rapamycin has been shown to activation of TβRI by enforced expression of Massague J. EMBO J. 1997; PubMed Scopus Google Scholar, M. L. J. Massague J. Mol. Cell. 2001; 8: Full Text Full Text PDF PubMed Scopus Google Scholar) and does not of rapamycin on TGF-β signals in a number of cell N. C. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). rapamycin reverses IGF-I suppression of TGF-β responses through the interaction of with TβRI in NRP-152 cells, then is likely that the expression of or its to is in our of PI3K including Akt and in activation of a regulator of cell Cell Biol. PubMed Scopus Google Scholar, G. Cell Res. 1995; PubMed Scopus Google Scholar, N. G. 1997; PubMed Scopus Google Scholar, L. J. Cancer 1996; Google Scholar, T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). kinase is reported to be by TGF-β through the activation of protein phosphatase of protein phosphatase to TβRI C. H. M. 14: PubMed Scopus Google Scholar, C. R. Mol. Cell. Biol. 1998; PubMed Google Scholar). the that activated by IGF-I, the activation of Smad3 by TGF-β suggest that the of protein phosphatase to the activated TβRI to Smad3 activation by suppression of is both our laboratory on NRP-152 (5Hsing A.Y. Kadomatsu K. Bonham M.J. Danielpour D. Cancer Res. 1996; 56: 5146-5149PubMed Google Scholar) and by other on 1998; PubMed Scopus Google Scholar) and prostate R. B. P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) that IGF-I blocks the of apoptosis by through In cells, IGF-I shown to not early TGF-β1 signals but to the activation of by TGF-β1 through a PI3K/Akt-dependent the other IGF-I reported to apoptosis in through the ability of induced by to apoptosis R. B. P. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In LR3-IGF-I, which is to to does not apoptosis induced by TGF-β1 in cells, that effect of through an The mechanism by which the of apoptosis is by between the by which IGF-I blocks TGF-β signals are likely to in cell between and NRP-152 cells, other is that the is whereas the is non-tumorigenic in Moreover, NRP-152 have a epithelial cell D. Kadomatsu K. Sporn M.B. Cancer Res. Google Scholar) and cell as by their ability to a D. J. Cell 1999; Google Scholar), and form prostatic in in the of Danielpour D. 1999; PubMed Scopus Google Scholar). TGF-β functions as a tumor suppressor in these cells, TGF-β signaling by expression of their B. de Castro K. Barnes H.E. Parks W.T. Stewart L. Bottinger E.P. Danielpour D. Wakefield L.M. Cancer Res. 1999; 59: 4834-4842PubMed Google Scholar, T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar). We show that treatment of NRP-152 with for does not expression of TGF-β or total Smads 2, 3, or 4. these selectively inhibit the activation of Smad3 but not the activation of or of Smad3 expression TGF-β1 Moreover, blocks suppression of Smad3 activated by Our that the intercepts activation of Smad3 by TGF-β through a target that to be The by which IGF-I inhibits Smad3 but not activation likely in in the by which these proteins are activated by SARA, a has been shown to to TβRI for activation (17Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar), that SARA is not for the activation of Smad3 D. H. Y. K. N. Res. 2001; PubMed Scopus Google Scholar) and also not be for TGF-β-induced activation of in S. S. Asao H. M. K. J. 2003; PubMed Scopus Google Scholar). domain with SARA to to TβRI (18Miura S. Takeshita T. Asao H. Kimura Y. Murata K. Sasaki Y. Hanai J.I. Beppu H. Tsukazaki T. Wrana J.L. Miyazono K. Sugamura K. Mol. Cell. Biol. PubMed Scopus Google Scholar). SARA and Hrs be by which Smads 2 and 3 are activated SARA and Hrs to the through the domain in the of (18Miura S. Takeshita T. Asao H. Kimura Y. Murata K. Sasaki Y. Hanai J.I. Beppu H. Tsukazaki T. Wrana J.L. Miyazono K. Sugamura K. Mol. Cell. Biol. PubMed Scopus Google Scholar). treatment of with of or of or is likely to of and SARA and Hrs activating and However, we show that or not inhibit TGF-β signaling or Smad3 activation and not that SARA Hrs is for the activation of Smad3 in NRP-152 cells. TGF-β signaling by these and suggesting that SARA and Hrs inhibit Smad3 activation in these cells. in with that in J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar), Akt activated by TGF-β through a mechanism that by or Moreover, also the activation of Smad2, and epithelial J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), through a pathway. Further by that has that Akt the ability of TGF-β to support of these through of an Akt in Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar). our data show that signaling not TGF-β-induced apoptosis but also TGF-β signaling early steps that the activation of Smad3 activation is a by which signaling through Akt is to of Moreover, our data support that Smads 2 and 3 are activated or in prostatic epithelial cells, with expression of 2 and 3 shown in C. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). support our that signaling is oncogenic for in suppression of Further in our laboratory in the mechanism by which IGF-I blocks Smad3 signaling is likely to on the mechanism by which IGF-I of prostatic We and for the and PI3K and constructs, respectively, Hsing for and and Sporn for support and