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• W2056515822 abstract "Lovastatin promotes osteoblast differentiation by increasing bone morphogenetic protein-2 (BMP-2) expression. We demonstrate that lovastatin stimulates tyrosine phosphorylation of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), leading to an increase in its kinase activity in osteoblast cells. Inhibition of PI3K ameliorated expression of the osteogenic markers alkaline phosphatase, type I collagen, osteopontin, and BMP-2. Expression of dominant-negative PI3K and PTEN, an inhibitor of PI3K signaling, significantly attenuated lovastatin-induced transcription of BMP-2. Akt kinase was also activated in a PI3K-dependent manner. However, our data suggest involvement of an additional signaling pathway. Lovastatin-induced Erk1/2 activity contributed to BMP-2 transcription. Inhibition of PI3K abrogated Erk1/2 activity in response to lovastatin, indicating the presence of a signal relay between them. We provide, as a mechanism of this cross-talk, the first evidence that lovastatin stimulates rapid activation of Ras, which associates with and activates PI3K in the plasma membrane, which in turn regulates Akt and Erk1/2 to induce BMP-2 expression for osteoblast differentiation. Lovastatin promotes osteoblast differentiation by increasing bone morphogenetic protein-2 (BMP-2) expression. We demonstrate that lovastatin stimulates tyrosine phosphorylation of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), leading to an increase in its kinase activity in osteoblast cells. Inhibition of PI3K ameliorated expression of the osteogenic markers alkaline phosphatase, type I collagen, osteopontin, and BMP-2. Expression of dominant-negative PI3K and PTEN, an inhibitor of PI3K signaling, significantly attenuated lovastatin-induced transcription of BMP-2. Akt kinase was also activated in a PI3K-dependent manner. However, our data suggest involvement of an additional signaling pathway. Lovastatin-induced Erk1/2 activity contributed to BMP-2 transcription. Inhibition of PI3K abrogated Erk1/2 activity in response to lovastatin, indicating the presence of a signal relay between them. We provide, as a mechanism of this cross-talk, the first evidence that lovastatin stimulates rapid activation of Ras, which associates with and activates PI3K in the plasma membrane, which in turn regulates Akt and Erk1/2 to induce BMP-2 expression for osteoblast differentiation. Statins block cholesterol biosynthesis by competitively inhibiting the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase, which converts 3-hydroxy-3-methylglutaryl-CoA to mevalonate (1). Statins have recently been shown to reduce osteoclast activity and to stimulate osteoblast differentiation in vitro and bone formation in vivo (2.Maeda T. Matsunuma A. Kawane T. Horiuchi N. Biochem. Biophys. Res. Commun. 2001; 280: 874-877Crossref PubMed Scopus (357) Google Scholar, 3.Mundy G. Garrett R. Harris S. Chan J. Chen D. Rossini G. Boyce B. Zhao M. Gutierrez G. Science. 1999; 286: 1946-1949Crossref PubMed Scopus (1581) Google Scholar, 4.Oxlund H. Andreassen T.T. Bone. 2004; 34: 609-618Crossref PubMed Scopus (115) Google Scholar). The role of statins in increasing bone mineral density in experimental animals and their role in protecting against fractures in cross-sectional or retrospective case control studies have led to testing this group of drugs for osteoporosis management (3.Mundy G. Garrett R. Harris S. Chan J. Chen D. Rossini G. Boyce B. Zhao M. Gutierrez G. Science. 1999; 286: 1946-1949Crossref PubMed Scopus (1581) Google Scholar, 5.Chan K.A. Andrade S.E. Boles M. Buist D.S. Chase G.A. Donahue J.G. Goodman M.J. Gurwitz J.H. LaCroix A.Z. Platt R. Lancet. 2000; 355: 2185-2188Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 6.Chung Y.S. Lee M.D. Lee S.K. Kim H.M. Fitzpatrick L.A. J. Clin. Endocrinol. Metab. 2000; 85: 1137-1142Crossref PubMed Scopus (171) Google Scholar, 7.Edwards C.J. Hart D.J. Spector T.D. Lancet. 2000; 355: 2218-2219Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 8.Meier C.R. Schlienger R.G. Kraenzlin M.E. Schlegel B. Jick H. J. Am. Med. Assoc. 2000; 283: 3205-3210Crossref PubMed Scopus (464) Google Scholar, 9.Wang P.S. Solomon D.H. Mogun H. Avorn J. J. Am. Med. Assoc. 2000; 283: 3211-3216Crossref PubMed Scopus (420) Google Scholar).The lipophilic statins, viz. lovastatin, fluvastatin, simvastatin, and mevastatin, specifically activate the bone morphogenetic protein-2 (BMP-2) 3The abbreviations used are: BMP-2, bone morphogenetic protein-2; PI3K, phosphatidylinositol 3-kinase; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; siRNA, small interfering RNA; DN, dominant-negative; Ad, adenoviral; PI, phosphatidylinositol.3The abbreviations used are: BMP-2, bone morphogenetic protein-2; PI3K, phosphatidylinositol 3-kinase; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; siRNA, small interfering RNA; DN, dominant-negative; Ad, adenoviral; PI, phosphatidylinositol. gene promoter (3.Mundy G. Garrett R. Harris S. Chan J. Chen D. Rossini G. Boyce B. Zhao M. Gutierrez G. Science. 1999; 286: 1946-1949Crossref PubMed Scopus (1581) Google Scholar). The more water-soluble pravastatin does not, however, induce BMP-2 promoter activity or BMP-2 mRNA and protein levels (10.Sugiyama M. Kodama T. Konishi K. Abe K. Asami S. Oikawa S. Biochem. Biophys. Res. Commun. 2000; 271: 688-692Crossref PubMed Scopus (280) Google Scholar). Pravastatin does not stimulate new bone formation in neonatal murine calvaria (3.Mundy G. Garrett R. Harris S. Chan J. Chen D. Rossini G. Boyce B. Zhao M. Gutierrez G. Science. 1999; 286: 1946-1949Crossref PubMed Scopus (1581) Google Scholar). Transient exposure of bone cultures to lipophilic statins is sufficient to initiate the cascade resulting in bone formation, most probably because of the local production of BMP-2. Simvastatin-induced differentiation of MC3T3-E1 cells is accompanied by an increase in mRNA expression of BMP-2, vascular endothelial growth factor, alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (11.Maeda T. Matsunuma A. Kurahashi I. Yanagawa T. Yoshida H. Horiuchi N. J. Cell. Biochem. 2004; 92: 458-471Crossref PubMed Scopus (215) Google Scholar). Although the expression of Cbfa-1/Runx2 was found to be unchanged by simvastatin treatment in the previous study (11.Maeda T. Matsunuma A. Kurahashi I. Yanagawa T. Yoshida H. Horiuchi N. J. Cell. Biochem. 2004; 92: 458-471Crossref PubMed Scopus (215) Google Scholar), an earlier report demonstrated that lovastatin increases Cbfa-1/Runx2 expression while stimulating osteogenic differentiation of bone marrow mesenchymal cells (12.Li X. Cui Q. Kao C. Wang G.J. Balian G. Bone. 2003; 33: 652-659Crossref PubMed Scopus (147) Google Scholar). These studies clearly demonstrate a role for statins in osteoblast differentiation.Here we report that lovastatin stimulates osteoblast differentiation by activating phosphatidylinositol 3-kinase (PI3K) signaling. We further show a contribution of Erk1/2 MAPK to statin-induced signal trafficking. Interestingly, we identified a cross-talk between these two signaling pathways in osteoblasts. Finally, a central role of Ras activation by statins has been identified as a master regulator for the PI3K and Erk1/2 signaling pathways.EXPERIMENTAL PROCEDURESMaterials−We purchased statins and FTI-277 from Calbiochem. Phenylmethylsulfonyl fluoride, Na3VO4, and Nonidet P-40 were from Sigma. Aprotinin was obtained from Bayer. Antibodies against the p85 and p110 subunits of PI3K, Erk1/2 MAPK, Akt, hemagglutinin, and tubulin were from Santa Cruz Biotechnology, Inc., and anti-phosphotyrosine antibody and a Ras binding assay kit with anti-Ras antibody were from Upstate. Anti-phospho-Akt Ser473 and anti-phospho-Erk1/2 antibodies were obtained from Cell Signaling Technology. Scrambled and Ras-targeted small interfering (siRNA) oligonucleotides were obtained from Dharmacon. Tissue culture reagents and Lipofectamine were obtained from Invitrogen. A Dual-Luciferase assay kit was purchased from Promega. The plasmids expressing the dominant-negative (DN) p85 subunit of PI3K (pSRαΔp85), DN Akt (Akt(K179M)), DN Erk2, Gal4-Elk-1, and Gal4-luciferase and adenoviral (Ad) vectors expressing PTEN (Ad PTEN) and hemagglutinin-tagged DN Akt were described previously (13.Mahimainathan L. Ghosh-Choudhury N. Venkatesan B.A. Danda R.S. Choudhury G.G. Am. J. Physiol. 2005; 289: F72-F82Crossref PubMed Scopus (31) Google Scholar, 14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 15.Choudhury G.G. J. Biol. Chem. 2004; 279: 27399-27409Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 16.Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2004; 279: 15258-15268Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The DN RasN17 expression plasmid was a kind gift from Dr. Julian Downward (Imperial Cancer Research Foundation, London, UK). Adenoviral vectors expressing the Δp85 subunit of PI3K and DN RasN17 were kindly provided by Dr. Harold Franch (Emory University) and Dr. Yasushi Oshima (University of Tokyo School of Medicine), respectively. Recombinant noggin was a kind gift from Dr. Richard Harland (University of California, Berkeley, CA).Cell Culture−2T3 cells and 2T3 cells stably transfected with the 2.7-kb BMP-2 promoter-driven firefly luciferase expression plasmid (2T3-Luc cells) were grown in α-minimal essential medium with 10% fetal bovine serum as described (17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar). The cells were serum-deprived for 24 h, followed by treatment with lovastatin, simvastatin, or pravastatin. The cells were infected with the adenoviral vectors as described (14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar).Preparation of Membranes−Solubilization buffer (20 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, and 0.1% aprotinin) was added to the cell monolayer after treatment with lovastatin. The cells were collected by scrapping and lysed by 20 brisk strokes in a Dounce homogenizer. The nuclear pellet was removed by low speed centrifugation. The membrane fraction was purified from the supernatant essentially as we described previously (18.Choudhury G.G. Karamitsos C. Hernandez J. Gentilini A. Bardgette J. Abboud H.E. Am. J. Physiol. 1997; 273: F931-F938PubMed Google Scholar). RNA Preparation and Northern Analysis−RNA was isolated from 2T3 cells treated with lovastatin for 24 h in the presence or absence of LY294002. RNA was isolated using 5 ml of RNAzol B, followed by chloroform extraction and precipitation of RNA with isopropyl alcohol (17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar, 19.Ghosh-Choudhury N. Singha P.K. Woodruff K. St Clair P. Bsoul S. Werner S.L. Choudhury G.G. J. Biol. Chem. 2006; 281: 20160-20170Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). 20 μg of RNA were separated by electrophoresis on denaturing agarose gels and transferred to nylon filters. The filters were hybridized with type I collagen, osteopontin, and 36B4 cDNA probes (17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar). Northern analysis was repeated three times with different RNA isolations.RNase Protection Assay−RNase protection assay was performed essentially as described (20.Ghosh-Choudhury N. Choudhury G.G. Harris M.A. Wozney J. Mundy G.R. Abboud S.L. Harris S.E. Biochem. Biophys. Res. Commun. 2001; 286: 101-108Crossref PubMed Scopus (59) Google Scholar). In brief, a 32P-labeled cRNA probe for BMP-2 was synthesized using T7 polymerase and a template plasmid containing a BMP-2 genomic DNA fragment. This probe was hybridized to 5 μg of total RNA isolated from 2T3 cells treated with lovastatin in the presence or absence of LY294002, followed by RNase A and RNase T1 digestion and treatment with proteinase K. The reaction mixture was extracted with phenol/chloroform and ethanol-precipitated. The products were analyzed on 6% polyacrylamide gel containing 7 m urea.Alkaline Phosphatase Assay−After treatment, 2T3 cells were fixed in 10% formalin and stained using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium essentially as described previously (17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar, 21.Ghosh-Choudhury N. Harris M.A. Wozney J. Mundy G.R. Harris S.E. Biochem. Biophys. Res. Commun. 1997; 231: 196-202Crossref PubMed Scopus (19) Google Scholar). Stained structures were photographed with a Nikon digital camera attached to a microscope. Lysates from 2T3 cells were assayed for alkaline phosphatase activity using p-nitrophenyl phosphate as substrate essentially as described (21.Ghosh-Choudhury N. Harris M.A. Wozney J. Mundy G.R. Harris S.E. Biochem. Biophys. Res. Commun. 1997; 231: 196-202Crossref PubMed Scopus (19) Google Scholar).Transfection and Luciferase Assay−The BMP-2-LUC reporter plasmid, in which the firefly luciferase gene is driven by 2.7 kb of 5′-flanking sequence of the BMP-2 gene, has been described previously (14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar, 20.Ghosh-Choudhury N. Choudhury G.G. Harris M.A. Wozney J. Mundy G.R. Abboud S.L. Harris S.E. Biochem. Biophys. Res. Commun. 2001; 286: 101-108Crossref PubMed Scopus (59) Google Scholar). Cells were transfected with different expression plasmids using Lipofectamine Plus reagent as described (14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar, 19.Ghosh-Choudhury N. Singha P.K. Woodruff K. St Clair P. Bsoul S. Werner S.L. Choudhury G.G. J. Biol. Chem. 2006; 281: 20160-20170Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 20.Ghosh-Choudhury N. Choudhury G.G. Harris M.A. Wozney J. Mundy G.R. Abboud S.L. Harris S.E. Biochem. Biophys. Res. Commun. 2001; 286: 101-108Crossref PubMed Scopus (59) Google Scholar). Luciferase activity was determined using a luciferase assay kit. The data were plotted as mean luciferase activity/μg of protein as arbitrary units ± S.E. as described (13.Mahimainathan L. Ghosh-Choudhury N. Venkatesan B.A. Danda R.S. Choudhury G.G. Am. J. Physiol. 2005; 289: F72-F82Crossref PubMed Scopus (31) Google Scholar, 19.Ghosh-Choudhury N. Singha P.K. Woodruff K. St Clair P. Bsoul S. Werner S.L. Choudhury G.G. J. Biol. Chem. 2006; 281: 20160-20170Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).Immunoprecipitation and Immunoblotting−Cells were lysed in radioimmune precipitation assay buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 0.05% aprotinin, and 1% Nonidet P-40). Protein concentration was determined in the cleared cell lysate, and an equal amount of protein was immunoprecipitated with the respective antibodies (14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 19.Ghosh-Choudhury N. Singha P.K. Woodruff K. St Clair P. Bsoul S. Werner S.L. Choudhury G.G. J. Biol. Chem. 2006; 281: 20160-20170Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 20.Ghosh-Choudhury N. Choudhury G.G. Harris M.A. Wozney J. Mundy G.R. Abboud S.L. Harris S.E. Biochem. Biophys. Res. Commun. 2001; 286: 101-108Crossref PubMed Scopus (59) Google Scholar, 22.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). For immunoblotting, equal amounts of cleared cell lysates were separated by SDS-PAGE, followed by transfer of proteins to polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibody. The washed blot was developed using enhanced chemiluminescence reagent (14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 22.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 23.Ghosh-Choudhury N. Choudhury G.G. Celeste A. Ghosh P.M. Moyer M. Abboud S.L. Kreisberg J. Biochim. Biophys. Acta. 2000; 1497: 186-196Crossref PubMed Scopus (76) Google Scholar, 24.Ghosh-Choudhury N. Woodruff K. Qi W. Celeste A. Abboud S.L. Choudhury G.G. Biochem. Biophys. Res. Commun. 2000; 272: 705-711Crossref PubMed Scopus (78) Google Scholar).PI3K Assay−PI3K assay was performed using the anti-phosphotyrosine or anti-Ras immunoprecipitates using phosphatidylinositol (PI) as substrate in the presence of [γ-32P]ATP as described (14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 22.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar).Akt Kinase and MAPK Assay−Immune complex kinase assays for Akt kinase and MAPK were performed essentially as described (14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 22.Ghosh-Choudhury N. Abboud S.L. Mahimainathan L. Chandrasekar B. Choudhury G.G. J. Biol. Chem. 2003; 278: 21998-22005Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar).Ras Activity Assay−Ras activation assay was performed according to the procedures recommended by Upstate. Lysates from cells stimulated with statins were precipitated with the Raf-1 Ras-binding domain bound to agarose beads, and the bead-associated proteins were analyzed by SDS gel electrophoresis, followed by immunoblotting using anti-Ras antibody.Data Analysis−The significance of the data was determined by analysis of variance, followed by Student-Newman-Keuls analysis. A p value <0.05 was considered significant.RESULTSLovastatin-stimulated PI3K Is Necessary for BMP-2 Expression−The murine osteoblast cell line 2T3 undergoes differentiation into mature osteoblasts upon treatment with BMP-2 (17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar). The screening of a library of small molecular compounds using this cell line resulted in the discovery of statin as an inducer of BMP-2 and new bone formation in vivo (3.Mundy G. Garrett R. Harris S. Chan J. Chen D. Rossini G. Boyce B. Zhao M. Gutierrez G. Science. 1999; 286: 1946-1949Crossref PubMed Scopus (1581) Google Scholar). We demonstrated recently the importance of PI3K signaling in BMP-2-induced osteoblast differentiation (14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). To elucidate the underlying mechanism of lovastatin-induced bone formation, we investigated the role of PI3K in response to lovastatin in 2T3 cells. PI3K activity was determined in the antiphosphotyrosine immunoprecipitates. Lovastatin significantly increased PI3K activity in the anti-phosphotyrosine immunoprecipitates in a dose-dependent manner (Fig. 1A). Because PI3K activity was detected in anti-phosphotyrosine immunoprecipitates, we tested the tyrosine phosphorylation of the p85 regulatory subunit of PI3K in response to lovastatin. Anti-p85 immunoprecipitates were immunoblotted with anti-phosphotyrosine antibody. Lovastatin increased the tyrosine phosphorylation of the p85 subunit of PI3K (Fig. 1B). These data indicate that, in osteoblasts, lovastatin stimulates the tyrosine phosphorylation of the p85 regulatory subunit of PI3K, resulting in its activation.Lovastatin has been shown to increase BMP-2 mRNA (3.Mundy G. Garrett R. Harris S. Chan J. Chen D. Rossini G. Boyce B. Zhao M. Gutierrez G. Science. 1999; 286: 1946-1949Crossref PubMed Scopus (1581) Google Scholar). To investigate the role of PI3K in BMP-2 expression, we used the PI3K inhibitor LY294002, which blocked lovastatin-induced PI3K activity (Fig. 1C). RNase protection assay was used to examine BMP-2 expression. Lovastatin increased the expression of BMP-2 mRNA (Fig. 1D). Inhibition of the lipid kinase by LY294002 abrogated the lovastatin-induced expression of BMP-2 (Fig. 1D, compare lanes 2 and 3). These data indicate for the first time that PI3K regulates the expression of BMP-2 in response to lovastatin. To examine the possibility that the lovastatin-induced expression of BMP-2 in turn activates the PI3K signaling pathway, PI3K activity was measured in 2T3 cells treated with lovastatin in the presence of a BMP-2 antagonist, noggin. Noggin treatment had no effect on lovastatin-induced PI3K activity in 2T3 osteoblast (Fig. 1E), indicating direct activation of PI3K activity by lovastatin in 2T3 osteoblasts.Lovastatin-induced Osteoblast Differentiation Is Mediated via the PI3K Pathway−It was reported previously that lovastatin-induced osteoblast differentiation is associated with the expression of osteoblast-specific genes (3.Mundy G. Garrett R. Harris S. Chan J. Chen D. Rossini G. Boyce B. Zhao M. Gutierrez G. Science. 1999; 286: 1946-1949Crossref PubMed Scopus (1581) Google Scholar). To examine whether lovastatin induces osteoblast differentiation through induction of BMP-2, we examined the expression of the osteoblast marker protein alkaline phosphatase in the presence of the BMP-2 antagonist noggin. 2T3 cells were treated with lovastatin in the presence of noggin, and the activity of alkaline phosphatase was determined by a colorimetric staining method as well as by in vitro enzyme assay. Noggin treatment effectively inhibited lovastatin-induced expression and activity of alkaline phosphatase (Fig. 2, A and B), indicating that the late action of lovastatin on osteoblast differentiation is mediated through BMP-2 expression. To evaluate the role of PI3K in lovastatin-induced osteoblast differentiation, we tested the effect of the PI3K inhibitor LY294002 on the lovastatin-induced expression of osteoblast differentiation markers in 2T3 cells. Pretreatment of 2T3 cells with LY294002 blocked lovastatin-induced alkaline phosphatase activity (Fig. 2C). We further tested the expression of two other markers of osteoblast differentiation, type I collagen and osteopontin. Northern analysis showed induction of both these mRNAs by lovastatin (Fig. 2D). Preincubation of 2T3 cells with LY294002 inhibited the lovastatin-induced expression of type I collagen and osteopontin (Fig. 2D, compare lanes 2 and 3). These data indicate that PI3K regulates the statin-induced expression of the genes necessary for osteoblast differentiation.FIGURE 2Inhibition of PI3K blocks lovastatin-stimulated expression of osteoblast-specific markers. A, 2T3 cells were exposed to 250 ng/ml noggin prior to incubation with lovastatin. After fixation with formalin, the cells were stained with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium for alkaline phosphatase as described (17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar, 21.Ghosh-Choudhury N. Harris M.A. Wozney J. Mundy G.R. Harris S.E. Biochem. Biophys. Res. Commun. 1997; 231: 196-202Crossref PubMed Scopus (19) Google Scholar). B, lysates from 2T3 cells incubated with noggin and lovastatin were assayed for alkaline phosphatase (ALP) activity using p-nitrophenyl phosphate as described previously (17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar, 32.Choudhury G.G. Kim Y.S. Simon M. Wozney J. Harris S. Ghosh-Choudhury N. Abboud H.E. J. Biol. Chem. 1999; 274: 10897-10902Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The means ± S.E. of triplicate measurements are shown. *, p < 0.005 versus the control; **, p < 0.005 versus lovastatin alone. C, 2T3 cells were incubated with 12.5 μm LY294002 prior to lovastatin treatment. Alkaline phosphatase activity was measured in the cell lysates as described for B. The means ± S.E. of triplicate measurements are shown. *, p < 0.005 versus lovastatin-stimulated samples; **, p < 0.005 versus lovastatin alone. D, total RNAs isolated from 2T3 cells treated with lovastatin with or without prior exposure to LY294002 were analyzed for the expression of type I collagen and osteopontin transcripts by Northern blotting using specific cDNA probes. 36B4 transcripts were analyzed in the same RNA samples to demonstrate loading.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Lovastatin and Simvastatin Induce BMP-2 Promoter Activity in a PI3K-dependent Mechanism−To test the role of PI3K in statin-induced BMP-2 transcription, we used 2T3 cells stably expressing the firefly luciferase reporter plasmid under the control of the 2.7-kb BMP-2 promoter (2T3-Luc cells) (3.Mundy G. Garrett R. Harris S. Chan J. Chen D. Rossini G. Boyce B. Zhao M. Gutierrez G. Science. 1999; 286: 1946-1949Crossref PubMed Scopus (1581) Google Scholar, 17.Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar). These cells were treated with lovastatin or simvastatin. Luciferase activity was assayed in the cleared cell lysates. Both lovastatin and simvastatin dose-dependently increased BMP-2 promoter activity in 2T3 cells, whereas pravastatin did not have any effect (Fig. 3, A-C). To test the involvement of PI3K in the activity of the BMP-2 promoter induced by statins, 2T3-Luc cells were treated with increasing doses of the PI3K inhibitor LY294002, followed by incubation with lovastatin or simvastatin. The results show that both lovastatin- and simvastatin-induced BMP-2 promoter activities were dose-dependently blocked by LY294002 (Fig. 3, D and E). To confirm this observation, the 2T3-Luc cells were transiently transfected with a deletion mutant of the p85 regulatory subunit of PI3K that acts as a DN kinase (Δp85) (14.Ghosh-Choudhury N. Abboud S.L. Nishimura R. Celeste A. Mahimainathan L. Choudhury G.G. J. Biol. Chem. 2002; 277: 33361-33368Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) prior to lovastatin stimulation. The expression of DN PI3K in 2T3 osteoblasts partly inhibited lovastatin-induced activation of BMP-2 transcription (Fig. 3F), possibly because of insufficient expression of Δp85 due to inefficient transient transfection. To overcome this problem, 2T3-Luc cells were infected with an adenovirus expressing the Δp85 subunit of PI3K. The expression of DN PI3K from the adenoviral vector completely inhibited lovastatin-induced BMP-2 transcription (Fig. 3G). PI3K signaling is inhibited by the tumor suppressor protein PTEN, which dephosphorylates PI 3,4,5-trisphosphate (25.Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2571) Google Scholar, 26.Myers M.P. Pass I. Batty I.H. Van der Kaay J. Stolarov J.P. Hemmings B.A. Wigler M.H. Downes C.P. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13513-13518Crossref PubMed Scopus (995) Google Scholar, 27.Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar). Therefore, to confirm the involvement of PI3K, we used the adenoviral vector encoding PTEN (Ad PTEN). Infection of 2T3-Luc cells with Ad PTEN significantly inhibited lovastatin-induced BMP-2 promoter activity (Fig. 3H). These results demonstrate that the PI3K signaling pathway mediates statin-induced BMP-2 promoter activity in 2T3 osteoblasts.FIGURE 3Statin-induced BMP-2 transcription is regulated by PI3K signaling. A and B, 2T3-Luc" @default.
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• W2056515822 title "Statin-induced Ras Activation Integrates the Phosphatidylinositol 3-Kinase Signal to Akt and MAPK for Bone Morphogenetic Protein-2 Expression in Osteoblast Differentiation" @default.
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