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• W1997806989 abstract "This study explores the relationship between anti-proliferative signaling by transforming growth factor-β (TGF-β) and insulin-like growth factor-binding protein-3 (IGFBP-3) in human breast cancer cells. In MCF-7 cells, the expression of recombinant IGFBP-3 inhibited proliferation and sensitized the cells to further inhibition by TGF-β1. To investigate the mechanism, we used T47D cells that lack type II TGF-β receptor (TGF-βRII) and are insensitive to TGF-β1. After introducing the TGF-βRII by transfection, the basal proliferation rate was significantly decreased. Exogenous TGF-β1 caused no further growth inhibition, but immunoneutralization of endogenous TGF-β1 restored the proliferation rate almost to the control level. The addition of IGFBP-3 did not inhibit the proliferation of control cells but caused dose-dependent inhibition in TGF-βRII-expressing cells when exogenous TGF-β1 was also present. Similarly, receptor-expressing cells showed dose-dependent sensitivity to exogenous TGF-β1 only in the presence of exogenous IGFBP-3. This indicates that in these cells, anti-proliferative signaling by exogenous IGFBP-3 requires both the TGF-βRII and exogenous TGF-β1. To investigate this synergism, the phosphorylation of TGF-β signaling intermediates, Smad2 and Smad3, was measured. Phosphorylation of each Smad was stimulated by TGF-β1 and, independently, by IGFBP-3 with the two agents together showing a cumulative effect. These data suggest that IGFBP-3 inhibitory signaling requires an active TGF-β signaling pathway and implicate Smad2 and Smad3 in IGFBP-3 signal transduction. This study explores the relationship between anti-proliferative signaling by transforming growth factor-β (TGF-β) and insulin-like growth factor-binding protein-3 (IGFBP-3) in human breast cancer cells. In MCF-7 cells, the expression of recombinant IGFBP-3 inhibited proliferation and sensitized the cells to further inhibition by TGF-β1. To investigate the mechanism, we used T47D cells that lack type II TGF-β receptor (TGF-βRII) and are insensitive to TGF-β1. After introducing the TGF-βRII by transfection, the basal proliferation rate was significantly decreased. Exogenous TGF-β1 caused no further growth inhibition, but immunoneutralization of endogenous TGF-β1 restored the proliferation rate almost to the control level. The addition of IGFBP-3 did not inhibit the proliferation of control cells but caused dose-dependent inhibition in TGF-βRII-expressing cells when exogenous TGF-β1 was also present. Similarly, receptor-expressing cells showed dose-dependent sensitivity to exogenous TGF-β1 only in the presence of exogenous IGFBP-3. This indicates that in these cells, anti-proliferative signaling by exogenous IGFBP-3 requires both the TGF-βRII and exogenous TGF-β1. To investigate this synergism, the phosphorylation of TGF-β signaling intermediates, Smad2 and Smad3, was measured. Phosphorylation of each Smad was stimulated by TGF-β1 and, independently, by IGFBP-3 with the two agents together showing a cumulative effect. These data suggest that IGFBP-3 inhibitory signaling requires an active TGF-β signaling pathway and implicate Smad2 and Smad3 in IGFBP-3 signal transduction. transforming growth factor-β type I TGF-β receptor type II TGF-β receptor type V TGF-β receptor insulin-like growth factor insulin-like growth factor-binding protein human IGFBP Tris-buffered saline Transforming growth factor-β (TGF-β)1 is a member of a family of structurally homologous dimeric proteins, which are multifunctional growth factors (1Wakefield L.M. Colletta A.A. McCune B.K. Sporn M.B. Cancer Treat. Res. 1992; 61: 97-136Crossref PubMed Google Scholar). TGF-β has been shown to display a variety of biological activities including the negative and positive regulations of cell growth, stimulation of extracellular matrix formation, stimulation of angiogenesis, and induction of differentiation of several cell lineages (2Roberts A.B. Flanders K.C. Kondaiah P. Thompson N.L. van Obberghen-Schilling E.V. Wakefield L. Rossi P. de Crombrugghe B. Heine V. Sporn M.B. Recent Prog. Horm. Res. 1988; 44: 157-197PubMed Google Scholar, 3Moses H.L. Yang E.Y. Pieterol J.A. Cell. 1990; 63: 245-247Abstract Full Text PDF PubMed Scopus (876) Google Scholar). All human breast tumor cell lines secrete all three isoforms of TGF-β, namely TGF-β1, TGF-β2, and TGF-β3 (4Valverius E.M. Walker-Jones D. Bates S.E. Stampfer M.R. Clark R. McCormick F. Dickson R.B. Lippman M.E. Cancer Res. 1989; 49: 6269-6274PubMed Google Scholar, 5Fynan T.M. Reiss M. Crit. Rev. Oncog. 1993; 4: 493-540PubMed Google Scholar), and levels are elevated with increased malignancy (6Daly R.J. King R.J. Darbre P.D. J. Cell. Biochem. 1990; 43: 199-211Crossref PubMed Scopus (46) Google Scholar).TGF-β is synthesized and secreted as a high molecular weight latent complex that restricts its in vivo availability (7Pircher R. Lawrence D.A. Jullien P. Cancer Res. 1984; 44: 5538-5543PubMed Google Scholar, 8Pircher R. Jullien P. Lawrence D.A. Biochem. Biophys. Res. Commun. 1986; 136: 30-37Crossref PubMed Scopus (185) Google Scholar). TGF-β must be released from this complex before it can exert its actions, which is an important regulatory step in the action of this growth factor. Biological activities of TGF-β are believed to be mediated through specific cell surface receptors (9Tucker R.F. Branum E.L. Shipley G.D. Ryan R.J. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6757-6761Crossref PubMed Scopus (171) Google Scholar). A number of different size receptors have been identified in cultured cells and tissues, which include types I, II, III, IV, V, and VI receptors (10Cheifetz S. Ling N. Guillemin R. Massague J. J. Biol. Chem. 1988; 263: 17225-17228Abstract Full Text PDF PubMed Google Scholar, 11Cheifetz S. Andres J.L. Massague J. J. Biol. Chem. 1988; 263: 16984-16991Abstract Full Text PDF PubMed Google Scholar). Of them, only type I receptor (TGF-βRI) and type II receptor (TGF-βRII) have been shown to be directly involved in signal transduction (12Attisano L. Wrana J.L. Lopez-Caillas F. Massague J. Annu. Rev. Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (2995) Google Scholar). This is supported by the observation that the lack of response to TGF-β in some types of cancer cell lines correlates with the loss or low expression levels of TGF-βRI and/or TGF-βRII (13Sun L. Wu G. Willson J.K.V. Zborowska E. Yang J. Rajkarunanayake Wang J. Gentry L.E. Wang X.-F. Brattain M.G. J. Biol. Chem. 1994; 269: 26449-26455Abstract Full Text PDF PubMed Google Scholar, 14Wang J. Han W. Zborowska E. Liang J. Wang X.-F. Willson J.K.V. Sun L. Brattain M.G. J. Biol. Chem. 1996; 271: 17366-17371Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).Smads are molecules of relative molecular mass 42–60 kDa with two regions of homology at the NH2- and COOH-terminals termed Mad homology domains (MH1 and MH2, respectively), connected with a proline-rich linker sequence (15Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar). They fall into three classes based on sequence similarity and function. Class I Smads or pathway-restricted Smads couple to different receptors. Of these, Smad2 and Smad3 are phosphorylated after stimulation by TGF-β (16Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (901) Google Scholar) or activin (17Chen Y. Lebrun J.J. Vale W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12992-12997Crossref PubMed Scopus (143) Google Scholar), whereas Smad1 and Smad5 are involved in bone morphogenetic protein signaling (18Kretzschmar M. Liu F. Hata A. Doody J. Massague J. Genes Dev. 1997; 11: 984-995Crossref PubMed Scopus (476) Google Scholar). In the COOH-terminal region, pathway-restricted Smads have a characteristic Ser-Ser-X-Ser motif, the two most COOH-terminal serine residues of which are phosphorylated by activated TGF-βRI (18Kretzschmar M. Liu F. Hata A. Doody J. Massague J. Genes Dev. 1997; 11: 984-995Crossref PubMed Scopus (476) Google Scholar, 19Souchelnytskyi S. Tamaki K. Engstrom U. Wernstedt C. ten Dijke P. Heldin C.-H. J. Biol. Chem. 1997; 272: 28107-28115Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Class II Smads that are represented by Smad4 (20Hahn S.A. Schutte M. Shamsul Hoque A.T.M. Moskaluk C.A. da Costa L.T. Rozenblum E. Weinstein C.L. Fischer A. Yeo C.J. Hruban R.H. Kern S.E. Science. 1996; 271: 350-353Crossref PubMed Scopus (2157) Google Scholar) appear to be a general partner for the pathway-restricted Smads by bringing the cytoplasmic Smads into the nucleus where they can activate transcriptional responses (16Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (901) Google Scholar, 21Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (806) Google Scholar). Class III Smads, which include Smad6 and Smad7, are known as the inhibitory Smads because they bind to TGF-βRI and interfere with the phosphorylation of the pathway-restricted Smads (22Imamura T. Takase M. Nishihara A. Oeda E. Hanai J.I. Kawabata M. Miyazono K. Nature. 1997; 389: 622-626Crossref PubMed Scopus (865) Google Scholar, 23Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.-H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar).After the binding of TGF-β to TGF-βRII, a constitutively active serine-threonine kinase, TGF-βRI is recruited into the complex where it is phosphorylated by the type II receptor. The activated TGF-βRI then interacts with and phosphorylates Smad2 and Smad3, thus inducing their association with Smad4 followed by the translocation of the heteromeric complex to the nucleus where they can potentiate the transcription of target genes (21Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (806) Google Scholar, 24Wu R.Y. Zhang Y. Feng X.H. Derynck R. Mol. Cell. Biol. 1997; 17: 2521-2528Crossref PubMed Scopus (186) Google Scholar).Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is a member of a family of six well characterized IGF-binding proteins, IGFBP-1 to IGFBP-6 (25Baxter R.C. Horm. Res. 1994; 42: 140-144Crossref PubMed Scopus (299) Google Scholar). IGFBP-3 is the most abundant IGFBP in the circulation, serves as a storage depot for IGFs (26Baxter R.C. Martin J.L. Prog. Growth Factor Res. 1989; 1: 49-68Abstract Full Text PDF PubMed Scopus (557) Google Scholar), and has been shown to have both IGF-dependent and IGF-independent effects on cell proliferation. In its IGF-dependent actions, IGFBP-3 modulates the interaction between IGFs and their cell surface receptors, resulting in either inhibition or stimulation of cellular growth (27DeMellow J.S.M. Baxter R.C. Biochem. Biophys. Res. Commun. 1988; 156: 199-204Crossref PubMed Scopus (488) Google Scholar, 28Cohen P. Lamson G. Okajima T. Rosenfeld R.G. Mol. Endocrinol. 1993; 7: 380-386Crossref PubMed Scopus (128) Google Scholar). IGFBP-3 has also been shown to act as a growth inhibitor in the absence of IGFs (29Oh Y. Muller H.L. Lamson G. Rosenfeld R.G. J. Biol. Chem. 1993; 268: 14964-14971Abstract Full Text PDF PubMed Google Scholar), an effect that may be mediated by putative IGFBP-3 receptor(s) on the cell surface. No signaling receptor for IGFBP-3 has yet been unequivocally identified.MCF-7 human breast cancer cells express intact TGF-β signaling machinery and have been shown to be responsive to TGF-β growth inhibition. On the other hand, T47D human breast cancer cells lack TGF-βRII and are unresponsive to TGF-β treatment (30Kalkhoven E. Roelen B.A. de Winter J.P. Mummery C.L. van den Eijnden-van Raaij A.J.M. van der Saag P.T. van der Burg B. Cell Growth Differ. 1995; 6: 1151-1161PubMed Google Scholar). We have found that T47D cells also fail to respond to exogenous IGFBP-3, although we previously showed that transfection with IGFBP-3 is growth inhibitory to these cells (31Firth S.M. Fanayan S. Baxter R.C. Biochem. Biophys. Res. Commun. 1998; 246: 325-329Crossref PubMed Scopus (46) Google Scholar). In this study, we demonstrate that restoration of active TGF-β signaling is required for T47D cells to respond to exogenous IGFBP-3 and that IGFBP-3 stimulates the phosphorylation of the signaling intermediates Smad2 and Smad3, suggesting a previously unrecognized pathway of IGFBP-3 signaling.DISCUSSIONIntracellular signals from TGF-β are transduced by a mechanism that involves the transmembrane serine-threonine kinase receptors, TGF-βRI and TGF-βRII (38Massague J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). TGF-β binds primarily to the TGF-βRII followed by the recruitment of TGF-βRI to form a ternary complex that allows TGF-βRII to phosphorylate TGF-βRI. This activation of the type I receptor kinase is the first necessary step in transducing the TGF-β signal downstream.There is now considerable evidence that the loss of TGF-βRII expression occurs in a variety of human neoplasms, resulting in a lack of response to TGF-β growth inhibition in these cells. Loss of TGF-βRII expression is caused by various mechanisms including homozygous gene losses, gross gene rearrangements, and truncated transcripts of this receptor (13Sun L. Wu G. Willson J.K.V. Zborowska E. Yang J. Rajkarunanayake Wang J. Gentry L.E. Wang X.-F. Brattain M.G. J. Biol. Chem. 1994; 269: 26449-26455Abstract Full Text PDF PubMed Google Scholar, 30Kalkhoven E. Roelen B.A. de Winter J.P. Mummery C.L. van den Eijnden-van Raaij A.J.M. van der Saag P.T. van der Burg B. Cell Growth Differ. 1995; 6: 1151-1161PubMed Google Scholar, 39Park K. Kim S.-J. Bang Y.-J. Park J.-G. Kim N.K. Roberts A.B. Sporn M.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8772-8776Crossref PubMed Scopus (426) Google Scholar, 40De Jonge R.R. Garrigue-Antar L. Vellucci V.F. Reiss M. Oncol. Res. 1997; 9: 89-98PubMed Google Scholar). Inactivation of the TGF-β pathway has also been shown to be caused by the mutation or loss of TGF-βRI (41Kim I.Y. Ahn H.J. Zelner D.J. Shaw J.W. Sensibar J.A. Kim J.H. Kato M. Lee C. Cancer Res. 1996; 56: 44-48PubMed Google Scholar, 42Baldwin R.L. Friess H. Yokoyama M. Lopez M.E. Kobrin M.S. Buchler M.W. Korc M. Int. J. Cancer. 1996; 67: 283-288Crossref PubMed Scopus (116) Google Scholar). Furthermore, the deletion or mutation of Smad2, Smad3, or Smad4 has also been demonstrated in several cancer cell lines (43Pouliot F. Labrie C. Int. J. Cancer. 1999; 81: 98-103Crossref PubMed Scopus (40) Google Scholar). Any of these mechanisms provides a selective advantage by allowing the cells to escape from TGF-β-mediated growth control.In this study, we have investigated possible interactions between TGF-β and IGFBP-3 signaling pathways. We first showed that the overexpression of IGFBP-3 in MCF-7 human breast cancer cells, which normally express low levels of the protein (44Martin J.L. Coverley J.A. Pattison S.T. Baxter R.C. Endocrinology. 1995; 136: 1219-1226Crossref PubMed Google Scholar), led to a decreased proliferation rate. Exogenous IGFBP-3 is known to inhibit MCF-7 cell proliferation (45Pratt S.E. Pollak M.N. Biochem. Biophys. Res. Commun. 1994; 198: 292-297Crossref PubMed Scopus (73) Google Scholar), and the growth inhibitory effect of the anti-estrogen ICI 182780 (46Huynh H. Yang X.F. Pollak M. J. Biol. Chem. 1996; 271: 1016-1021Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) and vitamin D (47Colston K.W. Perks C.M. Xie S.P. Holly J.M.P. J. Mol. Endocrinol. 1998; 20: 157-162Crossref PubMed Scopus (131) Google Scholar) on these cells is believed to be mediated in part by the induction of IGFBP-3 gene expression. In contrast, transfection of MCF-7 cells with IGFBP-3 cDNA was reported by Chen et al. (48Chen J.-C. Shao Z.-M. Sheikh M.S. Hussain A. LeRoith D. Roberts C.T. Fontana J.A. J. Cell. Physiol. 1994; 158: 69-78Crossref PubMed Scopus (153) Google Scholar) to enhance IGF-I-stimulated proliferation. Although the different responses to IGFBP-3 in different studies are not easily explained, they may be related to the presence of IGFBP-3 proteolytic activity secreted by MCF-7 cells (49Grimes R.W. Manni A. Hammond J.M. Breast Cancer Res. Treat. 1996; 39: 187-196Crossref PubMed Scopus (4) Google Scholar), which has recently been shown to be stimulated by estrogen and inhibited by TGF-β (50Salahifar H. Baxter R.C. Martin J.L. Endocrinology. 2000; 141: 3104-3110Crossref PubMed Scopus (15) Google Scholar).We found that in IGFBP-3-transfected MCF-7 cells, sensitivity to growth inhibition by TGF-β was unexpectedly increased, raising the possibility of an interaction between TGF-β and IGFBP-3 inhibitory signaling. Late passage MCF-7 cells are reported to be less sensitive to TGF-β than early passage MCF-7 cells, a difference associated with a 3-fold reduction in TGF-βRII expression (51Ko Y. Banerji S.S. Liu Y. Li W. Liang J. Soule H.D. Pauley R.J. Willson J.K. Zborowska E. Brattain M.G. J. Cell. Physiol. 1998; 176: 424-434Crossref PubMed Scopus (30) Google Scholar). To investigate further how TGF-β signaling might be influenced by IGFBP-3, we turned to the T47D breast cancer cell line, which is reported in some studies to totally lack the TGF-βRII expression. In fact, there are conflicting reports regarding the level of TGF-βRII in T47D cells and the sensitivity of these cells to TGF-β treatment. TGF-β inhibition of T47D cell proliferation has been demonstrated by several groups (52Roberts A.B. Anzano M.A. Wakefield L.M. Roche N.S. Stern D.F. Sporn M.B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 119-123Crossref PubMed Scopus (967) Google Scholar, 53Knabbe C. Lippman M.E. Wakefield L.M. Flanders K.C. Kasid A. Derynck R. Dickson R.B. Cell. 1987; 48: 417-428Abstract Full Text PDF PubMed Scopus (842) Google Scholar, 54Jakowlew S.B. Moody T.W. Mariano J.M. Anticancer Res. 1997; 17: 1849-1860PubMed Google Scholar), whereas others (55Arteaga C.L. Tandon A.K. von Hoff D.D. Osborne C.K. Cancer Res. 1988; 48: 3898-3904PubMed Google Scholar, 56Murphy C.L. Dotzlaw H. Mol. Endocrinol. 1989; 3: 611-617Crossref PubMed Scopus (78) Google Scholar) have demonstrated a resistance to TGF-β effects in these cells. Kalkhoven et al. (30Kalkhoven E. Roelen B.A. de Winter J.P. Mummery C.L. van den Eijnden-van Raaij A.J.M. van der Saag P.T. van der Burg B. Cell Growth Differ. 1995; 6: 1151-1161PubMed Google Scholar) have shown that resistance to TGF-β growth inhibition was due to the lack of sufficient TGF-βRII expression. Pouliot and Labrie (43Pouliot F. Labrie C. Int. J. Cancer. 1999; 81: 98-103Crossref PubMed Scopus (40) Google Scholar) have also shown that T47D cells lack TGF-βRII but express mRNAs for Smad2, Smad3, and Smad4. The differences in the TGF-β responsiveness of T47D cell lines reported by various groups may reflect the variation between clonal lines used in different studies.In the present study, we used a T47D cell line with demonstrable resistance to TGF-β1 due to lack of TGF-βRII. Transfection of these cells with a TGF-βRII expression plasmid resulted in a significantly lower basal proliferation rate compared with control vector-transfected cells. This observation suggested that other components in the TGF-β signal transduction pathway are intact and that the loss of TGF-βRII expression is the mechanism by which these T47D cells escape TGF-β growth inhibition. Treatment with exogenous TGF-β1 alone did not have any further growth inhibitory effect on the T47D/TGF-βRII cells over 4 days, although it increased the basal phosphorylation levels of both Smad2 and Smad3 rapidly (within 10–15 min) and transiently. The lack of effect of exogenous TGF-β1 on 4-day growth inhibition may be due to the presence of a sustained level of endogenous TGF-β1, neutralization of which was able to stimulate cell growth almost to the level of TGF-βRII-negative control cells.IGFBP-3 has been shown to mediate the growth inhibitory effects of a number of anti-proliferative agents on breast cancer cells. Growth inhibition by TGF-β (57Oh Y. Muller H.L. Ng L. Rosenfeld R.G. J. Biol. Chem. 1995; 270: 13589-13592Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 58Rajah R. Valentinis B. Cohen P. J. Biol. Chem. 1997; 272: 12181-12188Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar), retinoic acid (59Gucev Z.S. Oh Y. Kelley K.M. Rosenfeld R.G. Cancer Res. 1996; 56: 1545-1550PubMed Google Scholar), anti-estrogens (46Huynh H. Yang X.F. Pollak M. J. Biol. Chem. 1996; 271: 1016-1021Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), and the tumor suppressor p53 (60Buckbinder L. Talbott R. Velasco-Miguel S. Takenaka I. Faha B. Seizinger B.R. Kley N. Nature. 1995; 377: 646-649Crossref PubMed Scopus (804) Google Scholar) has been shown to correlate with the induction of IGFBP-3 at both the transcriptional and translational levels. However, the mechanism by which IGFBP-3 can mediate these growth inhibitory effects is not well understood. In a previous study, we showed that T47D cells transfected with an IGFBP-3 expression plasmid were growth-inhibited at low passage numbers post-transfection but became refractory to the IGFBP-3 growth inhibitory effect at higher passage numbers (31Firth S.M. Fanayan S. Baxter R.C. Biochem. Biophys. Res. Commun. 1998; 246: 325-329Crossref PubMed Scopus (46) Google Scholar). The mechanism by which the early passage cells producing endogenous IGFBP-3 were sensitive to the IGFBP-3 growth inhibitory effect is not clear. In the light of the present study, this requires further investigation because we have shown here that exogenous IGFBP-3 is not inhibitory to either T47D/vector or T47D/TGF-βRII cells in the absence of exogenous TGF-β. This finding suggests that IGFBP-3 expressed within the cell may be able to bypass the step that requires TGF-β interaction with TGF-βRII at the cell surface to sensitize the cells to exogenous IGFBP-3.In the presence of an active TGF-β signaling pathway, T47D/TGF-βRII cells are responsive to exogenous IGFBP-3 and TGF-β1, whereas the same combined treatment is inactive in T47D/vector cells. This finding suggests that both active TGF-β signaling pathway and exogenous IGFBP-3 are required to cause growth inhibition in T47D/TGF-βRII cells and that there is synergism between IGFBP-3 and TGF-β1 in their growth inhibitory actions.In investigating the mechanism of this synergism, we have shown the ability of exogenous IGFBP-3 as well as TGF-β1 to potentiate Smad2 and Smad3 phosphorylation. This is the first report of phosphorylation of intracellular signaling intermediates known to be involved in growth inhibitory signals in response to IGFBP-3. The synergistic effect of IGFBP-3 and TGF-β1 was also evident in their potentiation of Smad2 and Smad3 phosphorylation, which was greater in the presence of both effectors than either agent alone. This provides evidence for some commonality in the inhibitory signaling pathways used by TGF-β1 and IGFBP-3. We recently reported that the sensitivity to IGFBP-3 in MCF-10A mammary epithelial cells was abrogated by the expression of oncogenic ras and restored when mitogen-activated protein kinase phosphorylation was blocked by the inhibitor PD98059 (61Martin J.L. Baxter R.C. J. Biol. Chem. 1999; 274: 16407-16411Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Whether the mitogen-activated protein kinase pathway provides a link between the inhibitory effects of TGF-β1 and IGFBP-3 in T47D cells remains to be determined.The identity of the proteins on the T47D cell surface or elsewhere that interact with IGFBP-3 to facilitate the intracellular TGF-β signaling cascade remains elusive. Several cell-associated proteins that bind IGFBP-3 have been described (58Rajah R. Valentinis B. Cohen P. J. Biol. Chem. 1997; 272: 12181-12188Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 62Oh Y. Muller H.L. Pham H.M. Rosenfeld R.G. J. Biol. Chem. 1993; 268: 26045-26048Abstract Full Text PDF PubMed Google Scholar, 63Hodgkinson S. Fowke P. Al Somai N. McQuoid M. J. Endocrinol. 1995; 145: R1-R6Crossref PubMed Scopus (19) Google Scholar), although the specificity of the binding and whether these IGFBP-3-binding proteins play a functional role in the growth inhibition by IGFBP-3 have yet to be confirmed. The type V TGF-β receptor (TGF-βRV) has recently been described as having a role in mediating the IGF-independent growth inhibitory effect of IGFBP-3 in mink lung cells (64Leal S.M. Liu Q.L. Huang S.S. Huang J.S. J. Biol. Chem. 1997; 272: 20572-20576Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Interestingly, phosphorylation of Smad2 and Smad3 was not stimulated by IGFBP-3 in this cell line, despite an effect of TGF-β on Smad phosphorylation (65Leal S.M. Huang S.S. Huang J.S. J. Biol. Chem. 1999; 274: 6711-6717Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Thus, the functional link of the reported signaling of IGFBP-3 through TGF-βRV and the synergy between IGFBP-3 and TGF-β, which is dependent on the presence of TGF-βRII that we have described in T47D cells, is not clear.In conclusion, we have demonstrated that the well recognized TGF-β signaling pathway requiring the presence of TGF-βRII and involving the phosphorylation of receptor-associated Smads can be activated by IGFBP-3 in a way that apparently increases the sensitivity toward TGF-β, thus leading to an enhancement of growth inhibition in the presence of both agents. The level at which IGFBP-3 interacts and the mechanism by which T47D cells transfected with IGFBP-3 cDNA may bypass the early steps of the TGF-β signaling cascade remain important areas for investigation. Transforming growth factor-β (TGF-β)1 is a member of a family of structurally homologous dimeric proteins, which are multifunctional growth factors (1Wakefield L.M. Colletta A.A. McCune B.K. Sporn M.B. Cancer Treat. Res. 1992; 61: 97-136Crossref PubMed Google Scholar). TGF-β has been shown to display a variety of biological activities including the negative and positive regulations of cell growth, stimulation of extracellular matrix formation, stimulation of angiogenesis, and induction of differentiation of several cell lineages (2Roberts A.B. Flanders K.C. Kondaiah P. Thompson N.L. van Obberghen-Schilling E.V. Wakefield L. Rossi P. de Crombrugghe B. Heine V. Sporn M.B. Recent Prog. Horm. Res. 1988; 44: 157-197PubMed Google Scholar, 3Moses H.L. Yang E.Y. Pieterol J.A. Cell. 1990; 63: 245-247Abstract Full Text PDF PubMed Scopus (876) Google Scholar). All human breast tumor cell lines secrete all three isoforms of TGF-β, namely TGF-β1, TGF-β2, and TGF-β3 (4Valverius E.M. Walker-Jones D. Bates S.E. Stampfer M.R. Clark R. McCormick F. Dickson R.B. Lippman M.E. Cancer Res. 1989; 49: 6269-6274PubMed Google Scholar, 5Fynan T.M. Reiss M. Crit. Rev. Oncog. 1993; 4: 493-540PubMed Google Scholar), and levels are elevated with increased malignancy (6Daly R.J. King R.J. Darbre P.D. J. Cell. Biochem. 1990; 43: 199-211Crossref PubMed Scopus (46) Google Scholar). TGF-β is synthesized and secreted as a high molecular weight latent complex that restricts its in vivo availability (7Pircher R. Lawrence D.A. Jullien P. Cancer Res. 1984; 44: 5538-5543PubMed Google Scholar, 8Pircher R. Jullien P. Lawrence D.A. Biochem. Biophys. Res. Commun. 1986; 136: 30-37Crossref PubMed Scopus (185) Google Scholar). TGF-β must be released from this complex before it can exert its actions, which is an important regulatory step in the action of this growth factor. Biological activities of TGF-β are believed to be mediated through specific cell surface receptors (9Tucker R.F. Branum E.L. Shipley G.D. Ryan R.J. Moses H.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6757-6761Crossref PubMed Scopus (171) Google Scholar). 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