Wnt Signaling Protects 3T3-L1 Preadipocytes from Apoptosis through Induction of Insulin-like Growth Factors

Abstract

Ectopic expression of Wnt-1 in 3T3-L1 preadipocytes stabilizes β-catenin, activates TCF-dependent gene transcription, and blocks adipogenesis. Here we report that upon serum withdrawal, Wnt-1 causes 3T3-L1 cells to resist apoptosis through a mechanism that is partially dependent on phosphatidylinositol 3-kinase. Although activation of Wnt signaling by inhibition of GSK-3 activity or ectopic expression of dominant stable β-catenin blocks apoptosis, inhibition of Wnt signaling through expression of dominant negative TCF-4 increases apoptosis. Wnt-1 stimulates 3T3-L1 preadipocytes to secrete factors that increase PKB/Akt phosphorylation at levels comparable with treatment with 10% serum. With DNA microarrays, we identified several secreted antiapoptotic genes that are induced by Wnt-1, notably insulin-like growth factor I (IGF-I) and IGF-II. Consistent with IGFs mediating the antiapoptotic effects of Wnt-1 in preadipocytes, conditioned medium from Wnt-1 expressing 3T3-L1 cells was unable to promote protein kinase B phosphorylation after the addition of recombinant IGFBP-4. Thus, we demonstrated that Wnt-1 induces expression of antiapoptotic genes in 3T3-L1 preadipocytes such as IGF-I and IGF-II, which allows these cells to resist apoptosis in response to serum deprivation. Ectopic expression of Wnt-1 in 3T3-L1 preadipocytes stabilizes β-catenin, activates TCF-dependent gene transcription, and blocks adipogenesis. Here we report that upon serum withdrawal, Wnt-1 causes 3T3-L1 cells to resist apoptosis through a mechanism that is partially dependent on phosphatidylinositol 3-kinase. Although activation of Wnt signaling by inhibition of GSK-3 activity or ectopic expression of dominant stable β-catenin blocks apoptosis, inhibition of Wnt signaling through expression of dominant negative TCF-4 increases apoptosis. Wnt-1 stimulates 3T3-L1 preadipocytes to secrete factors that increase PKB/Akt phosphorylation at levels comparable with treatment with 10% serum. With DNA microarrays, we identified several secreted antiapoptotic genes that are induced by Wnt-1, notably insulin-like growth factor I (IGF-I) and IGF-II. Consistent with IGFs mediating the antiapoptotic effects of Wnt-1 in preadipocytes, conditioned medium from Wnt-1 expressing 3T3-L1 cells was unable to promote protein kinase B phosphorylation after the addition of recombinant IGFBP-4. Thus, we demonstrated that Wnt-1 induces expression of antiapoptotic genes in 3T3-L1 preadipocytes such as IGF-I and IGF-II, which allows these cells to resist apoptosis in response to serum deprivation. glycogen synthase kinase insulin-like growth factor insulin-like growth factor-binding protein lymphoid enhancer factor multidrug resistance protein-1 phosphatidylinositol 3-kinase protein kinase B T-cell factor TdT-mediated dUTP nick end labeling Wnt-induced secreted protein The Wnts are a family of proteins that affect cell fate and differentiation, including adipogenesis, myogenesis, neurogenesis, and mammary development (1Fukumoto S. Hsieh C.M. Maemura K. Layne M.D. Yet S.F. Lee K.H. Matsui T. Rosenzweig A. Taylor W.G. Rubin J.S. Perrella M.A. Lee M.E. J. Biol. Chem. 2001; 276: 17479-17483Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 2Lane T.F. Leder P. Oncogene. 1997; 15: 2133-2144Crossref PubMed Scopus (122) Google Scholar, 3Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Science. 2000; 289: 950-953Crossref PubMed Scopus (1539) Google Scholar, 4Buckingham M. Curr. Opin. Genet. Dev. 2001; 11: 440-448Crossref PubMed Scopus (353) Google Scholar). The Wnt-1 gene was first identified as an insertion site for mouse mammary tumor virus in mouse mammary carcinoma (5Nusse R. Varmus H.E. Cell. 1992; 69: 1073-1087Abstract Full Text PDF PubMed Scopus (833) Google Scholar). Wnts are secreted glycoproteins that interact with seven transmembrane frizzled receptors and low density lipoprotein receptor-related protein co-receptors (6Miller J.R. Hocking A.M. Brown J.D. Moon R.T. Oncogene. 1999; 18: 7860-7872Crossref PubMed Scopus (607) Google Scholar, 7Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1103) Google Scholar). In the canonical Wnt signaling pathway, inhibition of GSK-31 prevents phosphorylation and targeted degradation of β-catenin. In the absence of Wnt signaling, hypophosphorylated β-catenin accumulates in the cytoplasm, enters the nucleus, and activates TCF/LEF-dependent gene transcription (8Kuhl M. Sheldahl L.C. Park M. Miller J.R. Moon R.T. Trends Genet. 2000; 16: 279-283Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar). We previously reported that both Wnt-1 and Wnt-10b block adipogenic conversion of 3T3-L1 preadipocytes through stabilization of β-catenin and inhibition of C/EBPα and peroxisome proliferator-activated receptor γ expression (3Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Science. 2000; 289: 950-953Crossref PubMed Scopus (1539) Google Scholar). Inhibition of Wnt signaling with dominant negative TCF-4 or with soluble frizzled-related proteins (sFRP) causes spontaneous differentiation (3Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Science. 2000; 289: 950-953Crossref PubMed Scopus (1539) Google Scholar, 9Bennett C.N. Ross S.E. Longo K.A. Bajnok L. Hemati N. Johnson K.W. Harrison S.D. MacDougald O.A. J. Biol. Chem. 2002; 277: 30998-31004Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar), indicating that an endogenous Wnt feeds back to repress adipogenesis. Wnt-10b is the best candidate for the endogenous inhibitor because Wnt-10b stabilizes free cytosolic β-catenin, inhibits adipogenesis, and is expressed in preadipocytes and stromovascular cells but not in adipocytes (3Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Science. 2000; 289: 950-953Crossref PubMed Scopus (1539) Google Scholar, 9Bennett C.N. Ross S.E. Longo K.A. Bajnok L. Hemati N. Johnson K.W. Harrison S.D. MacDougald O.A. J. Biol. Chem. 2002; 277: 30998-31004Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar). Suppression of Wnt-10b in response to elevated cAMP promotes expression of adipogenic transcription factors and proteins involved in carbohydrate and lipid metabolism (3Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Science. 2000; 289: 950-953Crossref PubMed Scopus (1539) Google Scholar, 9Bennett C.N. Ross S.E. Longo K.A. Bajnok L. Hemati N. Johnson K.W. Harrison S.D. MacDougald O.A. J. Biol. Chem. 2002; 277: 30998-31004Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 10MacDougald O.A. Mandrup S. Trends Endocrinol. Metab. 2002; 13: 5-11Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 11Ross S.E. Erickson R.L. Gerin I. DeRose P.M. Bajnok L. Longo K.A. Misek D.E. Kuick R. Hanash S.M. Atkins K.B. Mahle S. Nebb H.I. Madsen L. Kristiansen K. MacDougald O.A. Mol. Cell. Biol. 2002; 22: 5989-5999Crossref PubMed Scopus (216) Google Scholar). In addition to playing a key role in adipogenesis, Wnt signaling protects against apoptosis in cells exposed to cellular or chemical stress (12Bournat J.C. Brown A.M. Soler A.P. J. Neurosci. Res. 2000; 61: 21-32Crossref PubMed Scopus (95) Google Scholar, 13Chen S. Guttridge D.C. You Z. Zhang Z. Fribley A. Mayo M.W. Kitajewski J. Wang C.Y. J. Cell Biol. 2001; 152: 87-96Crossref PubMed Scopus (363) Google Scholar, 14You Z. Saims D. Chen S. Zhang Z. Guttridge D.C. Guan K.L. MacDougald O.A. Brown A.M. Evan G. Kitajewski J. Wang C.Y. J. Cell Biol. 2002; 157: 429-440Crossref PubMed Scopus (189) Google Scholar). For example, ectopic expression of Wnt-1 in Rat-1 cells inhibits apoptosis in response to vincristine or vinblastine through a PKB/Akt-independent mechanism (13Chen S. Guttridge D.C. You Z. Zhang Z. Fribley A. Mayo M.W. Kitajewski J. Wang C.Y. J. Cell Biol. 2001; 152: 87-96Crossref PubMed Scopus (363) Google Scholar). Furthermore, low serum conditions fail to induce apoptosis in PC-12 cells that express Wnt-1 (12Bournat J.C. Brown A.M. Soler A.P. J. Neurosci. Res. 2000; 61: 21-32Crossref PubMed Scopus (95) Google Scholar). Inhibitors of GSK-3 and PI3K each partially reversed this effect, suggesting that the cytoprotective effects of Wnt-1 are mediated through direct Wnt signaling and Wnt-induced gene expression (12Bournat J.C. Brown A.M. Soler A.P. J. Neurosci. Res. 2000; 61: 21-32Crossref PubMed Scopus (95) Google Scholar). The response of 3T3-L1 cells to cellular stress is differentiation-dependent. Serum-starved 3T3-L1 preadipocytes undergo apoptosis, but fully differentiated adipocytes are resistant, perhaps because of increased expression of Bcl-2 and neuronal apoptosis inhibitory protein (15Magun R. Boone D.L. Tsang B.K. Sorisky A. Int. J. Obes. Relat. Metab. Disord. 1998; 22: 567-571Crossref PubMed Scopus (45) Google Scholar). Supplementation of serum-free medium with IGF-I protects preadipocytes against apoptosis, indicating that this growth factor impacts this process (16Gagnon A. Dods P. Roustan-Delatour N. Chen C.S. Sorisky A. Endocrinology. 2001; 142: 205-212Crossref PubMed Scopus (37) Google Scholar). Wnt signaling has pleiotropic effects during development, including the regulation of cell fate and mitosis. Recently, it has been shown that this signaling pathway has an important antiapoptotic function. However, the molecular mechanism for the protective effects of Wnt have only been partially characterized. Herein, we demonstrate that Wnt-1 induces expression of antiapoptotic genes in 3T3-L1 preadipocytes such as IGF-I and IGF-II, which allows these cells to resist apoptosis in response to serum deprivation. The 3T3-L1 preadipocyte line was cultured in Dulbecco's modified Eagle's medium supplemented with 1 mm sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% calf serum (Sigma) (17Green H. Kehinde O. J. Cell. Physiol. 1979; 101: 169-171Crossref PubMed Scopus (234) Google Scholar). Cells were maintained in a subconfluent state unless otherwise indicated. Human hemagglutinin-tagged Wnt-1 in the retroviral vector pLNCX, compliments of Jan Kitajewski (Columbia University), was used to generate retroviral stocks in 293T cells for subsequent infection of 3T3-L1 cells. Pooled clones were selected with 400 μg/ml Geneticin (Invitrogen Life Technologies). Dominant negative TCF-4 (ΔN31 TCF-4) and dominant stable β-catenin (S33Y β-cat), each in the neomycin-resistant vector, pPGS, were provided by Eric Fearon (University of Michigan). Mouse recombinant IGF-I and IGF-II and human recombinant IGFBP-4 were purchased from R&D Systems. Wortmannin (Calbiochem) and LY294002 (New England Biolabs) were used to inhibit PI3K. Cells analyzed for apoptosis by TUNEL were grown on 18-m-square glass coverslips. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized in 0.1% sodium citrate, 1% Triton X-100, and nicked DNA was end-labeled with fluorescein-conjugated dNTPs (Roche Molecular Biochemicals for 1 h at 37 °C, washed with 1× phosphate-buffered saline, and then counter-stained with the nuclear dye Hoechst 33342 (Sigma). Coverslips were mounted on slides, and six random fields for each treatment were analyzed using a Nikon TE200 fluorescent microscope. The total cell number (Hoechst 33342-labeled nuclei) and apoptotic cell number (fluorescein-labeled nuclei) from each field were used to calculate the apoptotic index for each treatment, which is defined as: (no. of fluorescein-labeled nuclei)/(no. of Hoechst-labeled nuclei) × 100. Following TUNEL, 6–12 random microscopic fields/treatment were photographed digitally, and image analysis was performed on a Macintosh computer using the public domain NIH Image program (rsb.info.nih.gov/nih-image/). All experiments were performed in triplicate. Differences between treatment were with and were at The of DNA to gene expression in 3T3-L1 cells has been S.E. Erickson R.L. Gerin I. DeRose P.M. Bajnok L. Longo K.A. Misek D.E. Kuick R. Hanash S.M. Atkins K.B. Mahle S. Nebb H.I. Madsen L. Kristiansen K. MacDougald O.A. Mol. Cell. Biol. 2002; 22: 5989-5999Crossref PubMed Scopus (216) Google Scholar). was with from 3T3-L1 cells with or was with of and and of the mouse were performed to the were at mm using the levels for genes were as and the of Wnt-1 to was as the for each was from 3T3-L1 cells with or Wnt-1 expressing cells with and with was performed for IGF-I IGF-II and using for an °C, by °C, 1 °C, 1 °C, and a °C, were to the of these were by and with and Cells were washed with 1× phosphate-buffered and in the mm 0.1% mm and a of and of total protein were by and were performed with for and to and using cell that undergo apoptosis in response to cellular stress are by ectopic expression of Wnt-1 (12Bournat J.C. Brown A.M. Soler A.P. J. Neurosci. Res. 2000; 61: 21-32Crossref PubMed Scopus (95) Google Scholar, 13Chen S. Guttridge D.C. You Z. Zhang Z. Fribley A. Mayo M.W. Kitajewski J. Wang C.Y. J. Cell Biol. 2001; 152: 87-96Crossref PubMed Scopus (363) Google Scholar, 14You Z. Saims D. Chen S. Zhang Z. 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A. 2001; PubMed Scopus Google for genes by Wnt-1 and involved in apoptosis. gene is by by Wnt-1, and in a for genes by Wnt-1 and involved in apoptosis. gene is by by Wnt-1, and In addition to the several of apoptosis were induced including the multidrug resistance protein Wnt-induced secreted protein-1 and growth factor and and The of Wnt-1 to against apoptosis through of or genes involved in cellular differentiation, and IGF-I protects 3T3-L1 cells against apoptosis during serum (16Gagnon A. Dods P. Roustan-Delatour N. Chen C.S. Sorisky A. Endocrinology. 2001; 142: 205-212Crossref PubMed Scopus (37) Google Scholar, K. J. Endocrinol. 2000; PubMed Scopus Google Scholar), and Wnt-1 induces expression of IGF-I and IGF-II Thus, we that Wnt-1 inhibits apoptosis by expression and of which through an mechanism to the antiapoptotic PKB/Akt from Wnt-1 cells phosphorylation of PKB/Akt and of inhibits the activity of this transcription factor A. A. J. M.E. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of conditioned from Wnt-1 cells to phosphorylation of and was after of conditioned with indicating that IGFs the effects of Wnt-1 on PKB/Akt Consistent with this the of phosphorylation by Wnt-1 conditioned was to that induced by IGF-I Furthermore, IGF-II stimulates PKB/Akt phosphorylation in 3T3-L1 cells of PKB/Akt in and Wnt-1 cells that phosphorylation of PKB/Akt in serum-free medium is in preadipocytes, with phosphorylation as as h of IGF-I or IGF-II to the IGF-I receptor a signaling that activation of protein and PKB/Akt Trends 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). PKB/Akt several proteins activation or of cell promotes cell Trends 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the activity in conditioned of 3T3-L1 cells that express Wnt-1, we that the of these cells during serum Consistent with this 3T3-L1 cells that express Wnt-1 have elevated levels of apoptosis in the of the PI3K or LY294002 However, the levels of apoptosis in the PI3K cells that perhaps direct activation of antiapoptotic signaling by Wnt-1, We demonstrate that ectopic expression of Wnt-1 and activation of the canonical Wnt signaling pathway promotes the of 3T3-L1 cells during serum deprivation. to the mechanism of this antiapoptotic response was to express of the Wnt-1 signaling pathway in to or block the effects of Wnt-1 signaling on apoptosis, DNA to the expression of antiapoptotic genes is increased by Wnt-1 signaling, and the of genes to antiapoptotic in 3T3-L1 cells. We that activation of Wnt signaling by inhibition of GSK-3 or expression of a of β-catenin apoptosis. inhibition of Wnt signaling with a dominant negative TCF-4 apoptosis. the of the canonical Wnt signaling pathway in the regulation of preadipocyte apoptosis. analysis identified several candidate genes induced by Wnt-1 and that have effects on of these IGF-I were The of IGFs by 3T3-L1 cells that express Wnt-1, and the of IGFs to the PKB/Akt pathway was using the IGFBP-4. a role for the IGF-I signaling pathway in the of Wnt-1 to inhibit apoptosis was using PI3K The of Wnt-1 to inhibit apoptosis has been in several cell (12Bournat J.C. Brown A.M. Soler A.P. J. Neurosci. Res. 2000; 61: 21-32Crossref PubMed Scopus (95) Google Scholar, 13Chen S. Guttridge D.C. You Z. Zhang Z. Fribley A. Mayo M.W. Kitajewski J. Wang C.Y. J. Cell Biol. 2001; 152: 87-96Crossref PubMed Scopus (363) Google Scholar). Ectopic expression of dominant negative or a of β-catenin, has effects on the of 3T3-L1 cells and Rat-1 cells (13Chen S. Guttridge D.C. You Z. Zhang Z. Fribley A. Mayo M.W. Kitajewski J. Wang C.Y. J. Cell Biol. 2001; 152: 87-96Crossref PubMed Scopus (363) Google with as of of the family of transcription is involved in and in In have increased apoptosis, perhaps as a of increased and expression T. M. R. K. R. R. 2000; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). with increased of was unable to induce cell in from T. M. R. K. R. R. 2000; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). these a mechanism by which gene expression both and in a of cell DNA we have identified several candidate genes that are induced by Wnt-1 and are antiapoptotic We on of these IGF-I In IGF-I through a mechanism to 3T3-L1 cells from apoptosis during serum (16Gagnon A. Dods P. Roustan-Delatour N. Chen C.S. Sorisky A. Endocrinology. 2001; 142: 205-212Crossref PubMed Scopus (37) Google Scholar). IGFs antiapoptotic in a number of cell and The antiapoptotic kinase PKB/Akt is and in response to IGF-I in a suggesting that PKB/Akt to in 3T3-L1 cells (16Gagnon A. Dods P. Roustan-Delatour N. Chen C.S. Sorisky A. Endocrinology. 2001; 142: 205-212Crossref PubMed Scopus (37) Google Scholar). We have demonstrated that IGF-II stimulates PKB/Akt phosphorylation at to of both IGF-I and IGF-II have been shown to β-catenin through inhibition of phosphorylation and gene expression R. C. J.P. L. Oncogene. 2001; PubMed Scopus Google Scholar, D. S. A. 2000; PubMed Scopus Google Scholar), suggesting that Wnt-1 have both effects and effects on gene is an of synthase that the conversion of to PubMed Scopus Google Scholar). expression of is with tumor through several including inhibition of apoptosis C. A. J. Res. 2001; PubMed Scopus Google Scholar). The regulation of by Wnt signaling was first in mouse mammary cells K. 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Cell Biol. 2002; 157: 429-440Crossref PubMed Scopus (189) Google Scholar), the that Wnt-1 induce the expression of several proteins that promote cell through the of apoptotic signaling induced by Wnt-1, is a that was first identified in cells. expression is with the of these cells to treatment with R.L. PubMed Scopus Google Scholar). that increased has been as a of resistance in cells A. 1999; PubMed Scopus Google Scholar). the regulation expression has in the field of The that Wnt-1 cellular resistance and in response to the vincristine and vinblastine has been reported (13Chen S. Guttridge D.C. You Z. Zhang Z. Fribley A. Mayo M.W. Kitajewski J. Wang C.Y. J. Cell Biol. 2001; 152: 87-96Crossref PubMed Scopus (363) Google Scholar). The mechanism of this resistance is suggesting that it is to gene expression the direct activation of antiapoptotic by Wnt-1 signaling (13Chen S. Guttridge D.C. You Z. Zhang Z. Fribley A. Mayo M.W. Kitajewski J. Wang C.Y. J. 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Guan K.L. MacDougald O.A. Brown A.M. Evan G. Kitajewski J. Wang C.Y. J. Cell Biol. 2002; 157: 429-440Crossref PubMed Scopus (189) Google Scholar, D. M.A. Lee J. J. M. C. R.L. P. D. S. A. 1998; PubMed Scopus Google Scholar). factor have effects on the of cells in otherwise Nature. 1992; PubMed Scopus Google Scholar, Trends Endocrinol. Metab. 2002; 13: Full Text Full Text PDF PubMed Google Scholar, J.C. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). Furthermore, the between cell growth and apoptosis is for the of Z. Saims D. Chen S. Zhang Z. Guttridge D.C. Guan K.L. MacDougald O.A. Brown A.M. Evan G. Kitajewski J. Wang C.Y. J. Cell Biol. 2002; 157: 429-440Crossref PubMed Scopus (189) Google Scholar). of this to or a of cell or a of the is a key to of for the several has been the of genes that development and apoptosis. We have and previously reported genes in which expression is increased by Wnt-1 and which in both and