Statin-induced Ras Activation Integrates the Phosphatidylinositol 3-Kinase Signal to Akt and MAPK for Bone Morphogenetic Protein-2 Expression in 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)(3)(4). The role of statins in increasing bone mineral density in experimental animals and their role in protecting against fractures in cross-sec-tional or retrospective case control studies have led to testing this group of drugs for osteoporosis management (3,(5)(6)(7)(8)(9).
The lipophilic statins, viz. lovastatin, fluvastatin, simvastatin, and mevastatin, specifically activate the bone morphogenetic protein-2 (BMP-2) 3 gene promoter (3). The more water-soluble pravastatin does not, however, induce BMP-2 promoter activity or BMP-2 mRNA and protein levels (10). Pravastatin does not stimulate new bone formation in neonatal murine calvaria (3). 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). Although the expression of Cbfa-1/Runx2 was found to be unchanged by simvastatin treatment in the previous study (11), an earlier report demonstrated that lovastatin increases Cbfa-1/Runx2 expression while stimulating osteogenic differentiation of bone marrow mesenchymal cells (12). 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 PROCEDURES
Materials-We purchased statins and FTI-277 from Calbiochem. Phenylmethylsulfonyl fluoride, Na 3 VO 4 , 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 Ser 473 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 (pSR␣⌬p85), 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)(14)(15)(16). The DN RasN17 expression plasmid was a kind gift from Dr. Julian Downward (Imperial Cancer Research Foundation, London, UK). Adenoviral vectors expressing the ⌬p85 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 ␣-minimal essential medium with 10% fetal bovine serum as described (17). 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).
Preparation of Membranes-Solubilization buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1 mM Na 3 VO 4 , 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).
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 (17,19). 20 g 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). Northern analysis was repeated three times with different RNA isolations.
RNase Protection Assay-RNase protection assay was performed essentially as described (20). In brief, a 32 P-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 g 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-3indolyl phosphate and nitro blue tetrazolium essentially as described previously (17,21). 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).
Transfection and Luciferase Assay-The BMP-2-LUC reporter plasmid, in which the firefly luciferase gene is driven by 2.7 kb of 5Ј-flanking sequence of the BMP-2 gene, has been described previously (14,17,20). Cells were transfected with different expression plasmids using Lipofectamine Plus reagent as described (14,17,19,20). Luciferase activity was determined using a luciferase assay kit. The data were plotted as mean luciferase activity/g of protein as arbitrary units Ϯ S.E. as described (13,19).
Akt Kinase and MAPK Assay-Immune complex kinase assays for Akt kinase and MAPK were performed essentially as described (14,22).
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 Ͻ0.05 was considered significant.

Lovastatin-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). 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). We demonstrated recently the importance of PI3K signaling in BMP-2-induced osteoblast differentiation (14). 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 immuno-precipitates, 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). 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). 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 FIGURE 1. PI3K regulates lovastatin-induced BMP-2 expression in 2T3 osteoblasts. A, serum-deprived 2T3 cells were exposed to different concentrations of lovastatin for 5 min. Cleared cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, followed by PI3K assay as described under "Experimental Procedures." PI-3-P (arrow) was separated by TLC. B, 200 g of lysates of 2T3 cells incubated with lovastatin were immunoprecipitated (I.P.) with anti-p85 antibody, followed by anti-phosphotyrosine (anti-p-Tyr) immunoblotting (I.B.). C, 2T3 cells were incubated with 12.5 M LY294002 for 1 h, followed by treatment with lovastatin for 5 min. Equal amounts of cell lysates were immunoprecipitated with anti-phosphotyrosine antibody, followed by PI3K assay as described under "Experimental Procedures." The arrow indicates the position of PI-3-P. D, upper panel, total RNA was isolated from 2T3 cells treated with 12.5 M LY294002, followed by incubation with lovastatin. RNAs from untreated 2T3 cells were used as a control (lane 1). Total RNAs were analyzed by RNase protection assay using a BMP-2-specific probe as described under "Experimental Procedures." Lower panel, shown is the expression of 36B4, which was used as a loading control. E, serum-deprived 2T3 cells were incubated with 250 ng/ml noggin prior to incubation with lovastatin for 10 min. PI3K activity was measured as described for A.
BMP-2 expression. To evaluate the role of PI3K in lovastatininduced 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 statininduced expression of the genes necessary for osteoblast differentiation.
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) (3,17). 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 simvastatininduced 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 (⌬p85) (14) 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 ⌬p85 due to inefficient transient transfection.
Lovastatin Induces Akt Kinase Activity in Osteoblasts-To elucidate the signaling pathway downstream of PI3K in lovastatin-treated 2T3 cells, we tested Akt kinase activation using immune complex kinase assay (14). Lovastatin rapidly stimulated Akt kinase activity (Fig. 4A). To rule out the possible involvement of lovastatin-induced expression of BMP-2 in the activation of Akt, the 2T3 cells were treated with noggin prior to lovastatin treatment, and the phosphorylation of Akt was determined as a measure of its activation. Noggin treatment failed to inhibit lovastatin-induced Akt phosphorylation (Fig.  4B), indicating that BMP-2 is not indirectly involved in stimulation of the PI3K/Akt signaling in response to lovastatin. To test whether statin-induced Akt activation requires PI3K signaling, we treated 2T3 cells with the PI3K inhibitor LY294002

. Inhibition 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 (17,21). B, lysates from 2T3 cells incubated with noggin and lovastatin were assayed for alkaline phosphatase (ALP) activity using p-nitrophenyl phosphate as described previously (17,32). The means Ϯ S.E. of triplicate measurements are shown. *, p Ͻ 0.005 versus the control; **, p Ͻ 0.005 versus lovastatin alone. C, 2T3 cells were incubated with 12.5 M LY294002 prior to lovastatin treatment. Alkaline phosphatase activity was measured in the cell lysates as described for B. The means Ϯ S.E. of triplicate measurements are shown. *, p Ͻ 0.005 versus lovastatin-stimulated samples; **, p Ͻ 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.
prior to incubation with lovastatin. LY294002 abolished lovastatin-induced Akt kinase activity (Fig. 4C, compare lanes 2 and  4), indicating that Akt activation is dependent on PI3K. Next, we tested the involvement of Akt in lovastatin-induced BMP-2 promoter activity. 2T3-Luc cells were transiently transfected with a plasmid expressing a kinase-dead mutant of Akt kinase (Akt(K179M)) and were subsequently treated with lovastatin. The data show that DN Akt kinase modestly inhibited lovastatin-induced BMP-2 promoter activity (Fig. 4D). To critically examine the involvement of Akt, we used an adenoviral vector encoding DN Akt. This vector infects Ͼ90% of 2T3 cells (14). The expression of DN Akt from the viral vector partially inhibited lovastatin-induced activation of the BMP-2 promoter (Fig.  4E). These data suggest that Akt kinase partially contributes to transcription of the BMP-2 gene in response to lovastatin.
Lovastatin Induces MAPK Activation in Osteoblasts-Our results showing inhibition of BMP-2 expression in response to lovastatin by blocking PI3K signaling (Figs. 1D and 3, D-H) demonstrate a significant role of the lipid kinase in this process. In contrast, partial inhibition of BMP-2 transcription was obtained by blocking Akt kinase signaling (Fig. 4E), indicating the involvement of yet another signaling pathway in statin-induced BMP-2 gene expression. A role of Erk1/2 MAPK has been implicated in BMP-2 signal transduction in osteoblasts (28). We examined the effect of statin on MAPK activity in 2T3 cells. Immune complex kinase assay showed increased MAPK activity in response to both lovastatin and simvastatin (Fig. 5A, left and right panels). This observation was confirmed using anti-phospho-Erk1/2 antibody, which recognizes the activated form of the kinase. Both lovastatin and simvastatin increased the phosphorylation of MAPK (Fig. 5B). To investigate the role of MAPK in BMP-2 transcription, we expressed DN Erk2 in 2T3-Luc cells. The expression of DN Erk2 inhibited lovastatininduced transcription of BMP-2 (Fig. 5C). Together, these data indicate that, along with Akt, MAPK also regulates BMP-2 expression in response to lovastatin.
Lovastatin Stimulates Cross-talk between the PI3K and MAPK Signaling Pathways-To investigate any role of PI3K in regulating MAPK activity, we used the PI3K inhibitor LY294002. 2T3 cells were treated with LY294002, followed by incubation with lovastatin for different periods of time. LY294002 inhibited lovastatin-stimulated activating phosphorylation of MAPK at all time points (Fig. 6A). To confirm this observation, we used Ad PTEN to inhibit PI3K signaling. The expression of PTEN inhibited lovastatin-induced activation of MAPK (Fig. 6B). These data indicate that PI3K contributes to MAPK activity in response to lovastatin in osteoblasts.
Increased MAPK activity results in transcriptional activation of the ETS family transcription factor Elk-1 (29,30). Direct phosphorylation of the C-terminal domain of Elk-1 by MAPK increases its transcriptional activity. Thus, the functional consequences of MAPK activation can be measured by determining the transcriptional activation of Elk-1. To test the role of PI3K in the functional consequences of MAPK activation, 2T3 cells were cotransfected with an expression vector encoding the Elk-1 C-terminal transactivation domain fused to the Gal4 DNA-binding domain and with a firefly luciferase reporter plasmid under the control of the Gal4 DNA element (31,32). Lovastatin increased the Elk-1-dependent expression of the reporter gene (Fig. 6C). Incubation of transfected cells with LY294002 significantly inhibited lovastatin-induced reporter gene transcription (Fig. 6C), indicating activation of MAPK. To confirm this observation, we used a DN p85 subunit of PI3K in the cotransfection assay. The expression of DN PI3K blocked lovastatin-induced reporter gene transcription (Fig. 6D). These data demonstrate that PI3K regulates MAPK-dependent transcriptional activation in response to lovastatin.
Lovastatin Activates Ras-mediated Transcription of BMP-2-Canonical MAPK signaling is initiated by membrane activation of the small G-protein Ras. We therefore tested the effect of lovastatin on the activation of this upstream signaling molecule in 2T3 cells. Because Ras is known to be in the membrane upon activation, we first tested the membrane localization of Ras in lovastatin-stimulated 2T3 cells. Lovastatin stimulated membrane association of Ras in a time-dependent manner (Fig. 7A). Only GTP-bound Ras interacts with Raf-1 kinase to activate the  latter to initiate stimulation of the MAPK cascade. Therefore, the level of GTP-bound Ras also represents activated Ras. Using a pulldown affinity binding assay with the Ras-binding domain from Raf-1, we examined the GTP loading of Ras in 2T3 cells. Lovastatin increased the binding of Ras to the Raf-1 Ras-binding domain in a time-dependent manner (Fig. 7B). These data indicate that lovastatin stimulates Ras activation.
To test the role of Ras in lovastatin-induced MAPK activation, we used the farnesyltransferase inhibitor FTI-277, which has been used to inhibit Ras activity (33). Treatment of 2T3 cells with FTI-277 blocked lovastatin-stimulated MAPK activity (Fig. 7C), suggesting a role for Ras in this process. To confirm this observation, we used the DN RasN17 mutant. The expression of DN RasN17 in 2T3 cells inhibited lovastatin-induced activating phosphorylation of MAPK (Fig. 7D). We showed above that DN Erk2 inhibited BMP-2 transcription (Fig. 5C). Because Ras regulates lovastatin-induced MAPK activity, the effect of DN RasN17 on BMP-2 transcription was tested. 2T3-Luc cells were infected with an adenoviral vector expressing RasN17 prior to incubation with lovastatin. DN Ras completely inhibited lovastatin-induced transcription of BMP-2 (Fig. 7E). These data demonstrate that the activation of Ras/MAPK signaling plays a part in BMP-2 expression in response to lovastatin. To confirm the involvement of Ras in lovastatin-induced BMP-2 transcription, we used an siRNA targeting Ras. 2T3 cells were transfected with Ras siRNA. Immunoblotting of the cell lysates with anti-Ras antibody showed effective inhibition of Ras protein expression (Fig. 7F, compare lanes 1 and 2 with lane  3). Next, we examined the role of Ras in lovastatin-induced BMP-2 transcription. 2T3-Luc cells were transfected with Ras siRNA, followed by incubation with lovastatin. The results show that the Ras siRNA, but not the scrambled siRNA, significantly inhibited lovastatin-induced BMP-2 transcription (Fig.  7G). Together, these results confirm that Ras plays an important role in transcription of the BMP-2 gene in response to lovastatin.
Lovastatin-induced PI3K/Akt Signaling Is Ras-dependent-We showed above that both PI3K and Ras regulated MAPK (Figs. 6, A and B; and 7, C and D). A role of Ras in PI3K activation was reported previously (34 -36). We tested the involvement of Ras in PI3K activation in response to lovastatin. Treatment of 2T3 cells with FTI-277 blocked lovastatin-stimulated PI3K activity (Fig. 8A), indicating that Ras may regulate PI3K activation by lovastatin. Using a co-immunoprecipitation assay, we examined the binding of Ras to PI3K in 2T3 cells in response to lovastatin. Lysates of 2T3 cells incubated with lovastatin were immunoprecipitated with antibody against the p110 catalytic subunit of PI3K, followed by immunoblotting with anti-Ras antibody. Lovastatin increased the association of Ras with p110 (Fig. 8B). To confirm this association, we assayed PI3K activity in anti-Ras immunoprecipitates. Lovastatin significantly increased PI3K activity in anti-Ras immunoprecipitates  ; lanes 1-4), followed by lovastatin treatment for the indicated periods of time. The cell lysates were analyzed by immunoblotting using anti-phospho-Erk1/2 antibody (upper panel), anti-PTEN antibody (middle panel), or anti-Erk1/2 antibody (lower panel). C, 2T3 cells were transfected with Gal4-Elk-1 fusion protein and Gal4-luciferase reporter expression plasmids. The cells were incubated for 1 h with LY294002, followed by lovastatin treatment. Luciferase activity was measured in the cell lysates as described in the legend to Fig. 3. D, 2T3 cells were transiently transfected with plasmids expressing the Gal4-Elk-1 fusion protein, Gal4-luciferase, and the deletion mutant of the p85 subunit (Del p85) of PI3K, followed by lovastatin treatment. Luciferase activity was measured in the cell lysates as described in the legend to Fig. 3. In C and D, the means Ϯ S.E. of triplicate measurements are shown. *, p Ͻ 0.005 versus the control; **, p Ͻ 0.005 versus lovastatin only. FEBRUARY 16, 2007 • VOLUME 282 • NUMBER 7 (Fig. 8C). These results conclusively demonstrate that lovastatin induces cross-talk between Ras and PI3K and that Ras activation is necessary for PI3K activity. Because Akt is downstream of PI3K, we examined the role of Ras in its activation in response to lovastatin. 2T3 cells treated with FTI-277 were incubated with lovastatin. Kinase activity was measured in the anti-Akt immunoprecipitates. FTI-277 inhibited lovastatin-induced Akt kinase activity (Fig. 8D). To confirm this observation, we used DN RasN17. The expression of DN Ras attenuated lovastatin-stimulated activating phosphorylation of Akt. These data demonstrate that Ras contributes to PI3K-dependent Akt signal transduction along with MAPK in response to lovastatin in osteoblast cells.

DISCUSSION
In this study, we sought to unravel the intracellular signaling mechanism by which statins induce BMP-2 expression and osteoblast differentiation. We used BMP-2 transcription as an indicator in delineating the signaling mechanism triggered by lovastatin. We have show that lovastatin increased PI3K activity in the anti-phosphotyrosine immunoprecipitates (Fig. 1A). In COS cells, overexpression of insulin and platelet-derived growth factor receptors demonstrates the direct tyrosine phosphorylation of the p85 subunit by insulin and platelet-derived growth factor, respectively (37,38). Our results using co-immunoprecipitation revealed the tyrosine phosphorylation of the p85 regulatory subunit of PI3K in response to lovastatin (Fig.  1B). These data suggest that the activation of PI3K by lovastatin may require tyrosine phosphorylation. Furthermore, we have shown that the BMP-2 antagonist noggin did not inhibit lovastatin-induced PI3K activation. These data indicate that BMP-2 is not required for PI3K activation in response to lovastatin.
Differentiation of osteoblasts to mature bone is associated with de novo expression of genes such as alkaline phosphatase, type I collagen, and osteopontin. Our results demonstrate that PI3K is necessary for the expression of all these genes in response to lovastatin (Fig. 2, C and D). These results represent FIGURE 7. Activation of Ras is required for lovastatin-induced BMP-2 expression. A, 2T3 cells were incubated with lovastatin for the indicated periods of time. Membranes were isolated from these cells essentially as described (18). Equal amounts of membrane proteins were immunoblotted with anti-Ras and anti-Na,K-ATPase antibodies. B, upper panel, Ras activation was measured with a pulldown assay using the Ras-binding domain of Raf-1 in cell lysates of serum-deprived 2T3 cells exposed to lovastatin for the indicated time periods as described under "Experimental Procedures." Lower panel, the results from immunoblot analysis of the same samples with anti-actin antibody are shown. C, serum-deprived 2T3 cells were treated with FTI-277 for 1 h, followed by lovastatin treatment. MAPK assay was performed in the cell lysates using myelin basic protein (MBP) as substrate as described in the legend to Fig. 5A. D, 2T3 cells were transfected with a plasmid expressing DN Ras (lanes 3 and 4) or vector only (lanes 1 and 2), followed by lovastatin treatment (lanes 2 and 4). the first demonstration of the requirement of the lipid kinase in lovastatin-induced osteoblast-specific gene expression. The osteogenic factor BMP-2 contributes significantly in lovastatinmediated new bone formation (2,3,11,39,40). Our data showing that noggin inhibited lovastatin-induced alkaline phosphatase expression (Fig. 2, A and B) support this observation. Moreover, lovastatin was shown to increase BMP-2 transcription in the 2T3 cell model (3). Our data show that lovastatin-stimulated transcription of BMP-2 utilizes PI3K signaling (Fig. 3).
The activation of PI3K produces the biologically active lipid PI 3,4,5-trisphosphate. PI 3,4,5-trisphosphate binds to the PH (pleckstrin homology) domain of Akt kinase, resulting in its translocation to the plasma membrane, where it undergoes phosphorylation by PDK1 at threonine 308 (41). Also Akt is phosphorylated at serine 473 by the mTOR-rictor complex, resulting in its full activation (42,43). In endothelial cells, Akt has been reported to be stimulated by simvastatin, which increases the translocation of Akt to the plasma membrane (44). Furthermore, simvastatin promotes Akt-dependent endothelial cell survival and angiogenesis in ischemic limbs of normocholesterolemic rabbits, similar to administration of vascular endothelial growth factor (45). We have shown that lovastatin stimulated the activation of Akt in 2T3 osteoblasts in a PI3K-dependent manner (Fig. 4, A and C). In addition, our results obtained by adenoviral expression of DN Akt demonstrate partial inhibition of BMP-2 transcription (Fig. 4E). The mechanism by which Akt kinase regulates BMP-2 transcription is not clear. One possibility may include the transcription factor NF-B, which has been shown to regulate BMP-2 gene expression in chondrocytes (46). Akt phosphorylates and activates IB kinase, resulting in the phosphorylation of IB, leading to its degradation and NF-B translocation to the nucleus (47,48). The involvement of Akt-regulated NF-B in BMP-2 transcription awaits further investigation.
In contrast to the observation of partial regulation of BMP-2 transcription by Akt (Fig. 4E), the expression of PTEN (which dephosphorylates PI 3,4,5-trisphosphate, resulting in inhibition of PI3K-dependent biological activity) significantly inhibited lovastatin-induced transcription of BMP-2 (Fig. 3H). Furthermore, the expression of DN PI3K by an adenoviral vector also significantly blocked BMP-2 transcription in response to lovastatin (Fig. 3G). These results indicate the existence of a PI3K-regulated alternative signaling pathway, which may regulate BMP-2 transcription. To this end, we have demonstrated that statins increased Erk1/2 MAPK activity, which partially but significantly regulated lovastatin-induced transcription of BMP-2 (Fig. 5), similar to Akt kinase. We (13,18) and others (49,50) have shown previously that PI3K inhibition blocks MAPK activity by stimuli such as epidermal growth factor, platelet-derived growth factor, insulin-like growth factor-1, and lysophosphatidic acid. Furthermore, the downstream target of PI3K, PDK1 (which phosphorylates Akt at threonine 308), has also been shown to phosphorylate MEK (MAPK/Erk kinase), thus regulating the activation of MAPK (51). Our results showing inhibition of MAPK phosphorylation by LY294002 and PTEN conclusively demonstrate that lovastatinstimulated MAPK is PI3K-dependent (Fig. 6, A and B). This positive regulatory effect of PI3K on MAPK activity resulted in transactivation of the MAPK target Elk-1 transcription factor (Fig. 6, C and D). These data demonstrate for the first time the presence of a direct relation between PI3K and MAPK in lovastatin-mediated signal transduction in osteoblasts.
In addressing the mechanism of cross-talk between PI3K and MAPK, we considered Ras, which plays an important role in the activation of MAPK (52). The involvement of Ras in the activation of PI3K has been reported (34). GTP-bound Ras physically interacts with the p110 catalytic subunit of PI3K, resulting in an increase in its activity (35). Insulin was shown to increase PI3K activity in a Ras-dependent manner (36). In the present study, we have shown for the first time the membrane localization and GTP loading of Ras in  lanes 2 and 3). Akt kinase activity was measured in the cell lysates using histone H2B as substrate as described in the legend to Fig. 4. The arrow indicates the position of phosphorylated histone H2B. Lower panel, the immunoblot of the same samples using anti-Akt antibody is shown. E, 2T3 cells were transiently transfected with a plasmid expressing DN Ras (lanes 3 and 4) or vector only (lanes 1 and 2), followed by lovastatin treatment (lanes 2 and 4). Cleared cell lysates were immunoblotted with anti-phospho-Akt antibody (pAkt; upper panel), anti-Ras antibody (middle panel), or anti-Akt antibody (lower panel). response to lovastatin (Fig. 7, A and B), indicating activation of this small GTPase. We showed that MAPK regulated BMP-2 transcription (Fig. 5C) and that Ras regulated MAPK (Fig. 7, B and C). Therefore, one possibility is that Ras may regulate BMP-2 transcription. We showed inhibition of lovastatin-induced transcription of BMP-2 by DN Ras (Fig.  7E). These data are confirmed by our observation that the down-regulation of Ras using siRNA inhibited BMP-2 transcription by lovastatin (Fig. 7G).
Our data demonstrating inhibition of PI3K activity by FTI-277 places Ras upstream of the lipid kinase in lovastatin-stimulated osteoblasts (Fig. 8A). Furthermore, we have shown that lovastatin stimulated interaction of Ras with PI3K, resulting in its increased activity (Fig. 8, B and C). As Akt kinase is PI3K-dependent, Ras also regulated this kinase activity (Fig. 8, D and E). These data indicate a direct role of Ras in PI3K/Akt signaling in response to lovastatin. However, these results are in contrast to the observation that growth factors, including epidermal and nerve growth factors, do not stimulate PI3K activity in a Rasdependent manner (34).
In summary, we have demonstrated for the first time that PI3K regulates lovastatin-stimulated expression of osteoblastspecific genes, including BMP-2. We have shown that PI3K contributes to lovastatin-induced activation of MAPK, which, together with Akt kinase, regulates BMP-2 expression. Also, our data provide the first evidence of the direct activation of Ras by lovastatin, which results in interaction between Ras and PI3K, leading to its activation. Furthermore, we have demonstrated that the binding of Ras to PI3K increases PI3K/Akt signaling. A schema summarizing the results is presented in Fig. 9.