Akt Promotes BMP2-Mediated Osteoblast Differentiation and Bone Development

716 Research Article Akt promotes BMP2-mediated osteoblast differentiation and bone development

Aditi Mukherjee and Peter Rotwein* Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA *Author for correspondence (e-mail: [email protected])

Accepted 10 November 2008 Journal of Cell Science 122, 716-726 Published by The Company of Biologists 2009 doi:10.1242/jcs.042770

Summary Signaling through the IGF-I receptor by locally synthesized stages of osteoblast maturation, dominant-negative Akt IGF-I or IGF-II is crucial for normal skeletal development and prevented accumulation of bone-specific alkaline phosphatase for bone remodeling. Osteogenesis is primarily regulated by and reduced mineralization, and more significantly inhibited bone morphogenetic proteins (BMPs), which activate gene the longitudinal growth of metatarsal bones in primary culture expression programs driven by bone-specific transcription by interfering with both chondrocyte and osteoblast factors. In a mesenchymal stem cell model of osteoblast development and function. We conclude that an intact IGF- commitment and differentiation controlled by BMP2, we show induced PI3-kinase–Akt signaling cascade is essential for BMP2- that an inhibitor of PI3-kinase or a dominant-negative Akt were activated osteoblast differentiation and maturation, bone as potent in preventing osteoblast differentiation as the IGF development and growth, and suggest that manipulation of this binding protein IGFBP5, whereas a Mek inhibitor was pathway could facilitate bone remodeling and fracture repair. ineffective. Conversely, an adenovirus encoding an inducible- active Akt was able to overcome the blockade of differentiation caused by IGFBP5 or the PI3-kinase inhibitor, and could Supplementary material available online at restore normal osteogenesis. Inhibition of PI3-kinase or Akt did http://jcs.biologists.org/cgi/content/full/122/5/716/DC1 not block BMP2-mediated signaling, because the Smad- responsive genes Sox9 and JunB were induced normally under Key words: Bone development, Bone morphogenetic factors, Insulin- all experimental conditions. When activated during different like growth factors, PI3-kinase–Akt pathway, Akt, Osteoblast

Introduction also collaborate with Runx2 to induce other genes in differentiating

Journal of Cell Science Bone remodeling occurs throughout life to maintain bone mass and osteoblasts, including osterix (Osx/Sp7), another bone-specific integrity, and involves the dynamic interplay of two opposing transcription factor (Lian et al., 2006). processes: resorption by osteoclasts and deposition by osteoblasts The IGF family consists of two secreted growth factors, IGF-I (Hadjidakis and Androulakis, 2006; Khosla et al., 2008; Raisz, 2005; and IGF-II (official protein symbols IGF1 and IGF2), two receptors Zaidi, 2007). In the adult skeleton, both phases of remodeling are and six high-affinity binding proteins. Actions of both IGFs are coupled temporally and spatially, and take place within a specialized mediated by the IGF-I receptor, a ligand-activated tyrosine protein environment termed the bone multicellular unit (Khosla et al., 2008; kinase that uses a series of intracellular adaptor molecules, including Raisz, 2005; Zaidi, 2007). Bone remodeling also requires regulated the insulin receptor substrate proteins IRS1 and IRS2, to engage interactions between local and systemically derived signals mediated downstream signaling pathways (Nakae et al., 2001). IGF binding by hormones, growth factors and cytokines, and genetically-defined proteins function primarily to modulate the bioavailability of IGFs, hierarchical programs of bone-specific transcription factors (Raisz, but might have other IGF-independent effects (Bach et al., 2005; 2005; Zaidi, 2007). Among growth factors with positive actions on Duan and Xu, 2005). Studies in experimental animals have bone formation are the bone morphogenetic proteins (BMPs) (Li concluded that action of IGF is essential for normal bone formation, and Cao, 2006), and the insulin-like growth factors (IGFs) (Li and growth and maintenance. Mice globally lacking the IGF-I receptor Cao, 2006; Raisz, 2005; Zaidi, 2007). have retarded skeletal development accompanied by delayed BMPs are central regulators of osteoblast differentiation, and were ossification, as well as other severe systemic defects that contribute named originally for their ability to promote ectopic bone formation to their neonatal death (Liu et al., 1993). Targeted loss of the IGF- (Wozney, 1992). Like other members of the TGFβ superfamily, I receptor exclusively in osteoblasts also has a bone phenotype, in BMPs signal through heteromeric Type I and Type II serine- which total trabecular thickness and number were reduced because threonine kinase receptors, and activate the intracellular signaling of a decline in mineral apposition rate (Zhang et al., 2002). In molecules, Smad1, Smad5 and Smad8, through their serine agreement with these conclusions, individual knockouts of IRS1 phosphorylation (Herpin and Cunningham, 2007). Activated Smad and IRS2 also caused osteopenia, with defects seen in both cortical proteins form heterodimers with the co-Smad, Smad4, and and trabecular bone (Akune et al., 2002; Ogata et al., 2000). translocate to the nucleus, where they regulate target gene In contrast to the deficits secondary to loss of IGF signaling, transcription (Herpin and Cunningham, 2007). BMP2 stimulates increased expression of IGF-I appears to stimulate bone growth and transcription of Runx2, the master regulator of osteoblast mineralization. Targeting IGF-I to mature osteoblasts in transgenic commitment (Lian et al., 2006), and BMP2-activated Smad proteins mice caused enhanced bone formation and mineralization, and Akt in bone development 717

resulted in increased trabecular bone volume (Zhao et al., 2000). C3H10T1/2 mesenchymal stem cells with recombinant BMP2 in Targeting IGF-I to osteoblast precursors also gave rise to a robust osteogenic medium. Under these conditions, BMP2 treatment was bone phenotype in mice, and led to increases in femur length, accompanied by the rapid and sustained stimulation of intracellular cortical width and cross-sectional area (Jiang et al., 2006). Thus, signaling via BMP receptors, as indicated by serine phosphorylation regardless of the timing of IGF-I overexpression in bone of of Smad1, Smad5 and Smad8 in protein extracts observed by day transgenic mice, net bone formation and mass were enhanced. 1 and maintained for up to 7 days (Fig. 1A), and by rapid and Therefore, based on several types of evidence, IGF action via the sustained upregulation of Dlx5 and Runx2 mRNA (Fig. 1B), two IGF-I receptor is crucial for normal bone development and osteoblast-specific transcription factors whose genes are well- mineralization. known targets of BMP2 (Lee et al., 2003; Phimphilai et al., 2006). IGF-mediated stimulation of the IGF-I receptor triggers receptor Subsequent events included accumulation of transcripts encoding autophosphorylation to create docking sites at phosphorylated the bone transcription factor osterix (Osx), and for the secreted tyrosine residues for adaptor molecules (Nakae et al., 2001). This osteoblast protein osteocalcin (Ocn) (Fig. 1B), followed by activity initiates a series of protein-protein interactions that lead to activation of bone-specific alkaline phosphatase, and mineralization of of intracellular signal transduction pathways (Nakae et al., 2001). extracellular matrix, the latter measured by Alizarin red staining Although several signaling pathways mediate IGF action in bone, (Fig. 1C,D). None of these biological effects were observed in cells as well as in other tissues (Giustina et al., 2008), a growing literature incubated in osteogenic medium without BMP2 (Fig. 1A-D). supports the idea that the PI3-kinase–Akt network is critical for To block potential IGF-regulated signaling cascades, we treated both osteoblast differentiation and bone growth (Fujita et al., 2004; confluent C3H10T1/2 cells with either the Mek inhibitor UO126 Ghosh-Choudhury et al., 2002; Liu et al., 2007; Osyczka and Leboy, or the PI3-kinase inhibitor LY294002, in the presence of BMP2 2005; Peng et al., 2003; Raucci et al., 2008), yet the biochemical and osteogenic medium, because both the Grb-Sos-Mek-Erk and or molecular mechanisms through which the IGF-stimulated PI3- PI3-kinase–Akt pathways have been shown to be activated by the kinase–Akt pathway increases osteoblast development and function IGF-I receptor via the adaptor molecules IRS1 and IRS2 in bone have not been elucidated. Fujita and colleagues have postulated an cells (Akune et al., 2002; Kadowaki et al., 1996; Ogata et al., 2000). interaction with Runx2, because the PI3-kinase inhibitor LY294002 Addition of UO126 had no effect on BMP2-mediated signaling, or reduced both its DNA-binding activity and its ability to stimulate on the rate or extent of osteoblast gene expression or differentiation target gene transcription (Fujita et al., 2004). Qiao and co-workers (Fig. 2A-D), although at the concentration used (10 μM), it have reached similar conclusions (Qiao et al., 2004), whereas others completely inhibited IGF-I-stimulated Erk phosphorylation in have suggested collaboration at the level of nuclear translocation C3H10T1/2 cells (supplementary material Fig. S1). By contrast, of BMP2-stimulated Smad proteins (Ghosh-Choudhury et al., LY294002 (20 μM), which blocked IGF-induced Akt 2002). phosphorylation (supplementary material Fig. S1), impaired Here, we define a key role for the IGF-activated PI3-kinase–Akt expression of osteoblast differentiation genes (Fig. 2B) and pathway in BMP-mediated osteoblast differentiation of completely prevented induction of alkaline phosphatase activity and uncommitted mesenchymal precursor cells and their subsequent mineralization (Fig. 2C,D), although like UO126, it also did not maturation. We also find that Akt-regulated signaling is crucial for inhibit BMP2-activated Smad phosphorylation (Fig. 2A).

Journal of Cell Science longitudinal bone growth and that it exerts positive actions on both chondrocyte and osteoblast differentiation and function in developing bone. Based on these observations, we conclude that the IGF-stimulated PI3-kinase–Akt pathway is a central component in an interactive osteogenic signaling network that is necessary for both bone development and remodeling.

Results Inhibition of the PI3-kinase–Akt pathway blocks BMP2- mediated osteoblast differentiation We previously demonstrated that both BMP2-stimulated osteoblast differentiation of mouse mesenchymal stem cells and growth and mineralization of mouse metatarsal bones, could be blocked by IGFBP5 (Mukherjee and Rotwein, 2008). In these studies, the inhibitory effects of IGFBP5 on osteogenesis appeared to depend on its ability to bind IGF-I with high affinity, thereby sequestering IGF-I from its cell-surface receptor, and leading to impaired IGF- Fig. 1. BMP2 promotes osteoblast differentiation. Results are shown of I receptor activity (Mukherjee and Rotwein, 2008). Since an IGF- experiments in which C3H10T1/2 cells were incubated in osteogenic media I analog with diminished affinity for IGFBPs but normal affinity (OM) without or with BMP2 (200 ng/ml) for up to 7 days. (A) Immunoblots for the IGF-I receptor could restore BMP2-mediated osteogenesis of whole-cell protein lysates for serine-phosphorylated Smad1, Smad5 and Smad8 (pSmad1,5,8), total Smads, Akt phosphorylated at Ser473 (pAktS473) in the presence of otherwise inhibitory concentrations of IGFBP5 and total Akt. (B) Results of RT-PCR assays showing expression of (Mukherjee and Rotwein, 2008), our results indicated that sustained osteoblast-specific genes Dlx5, Runx2, osterix (Osx) and osteocalcin (Ocn), IGF action is required for osteoblast differentiation and bone growth. and control gene S17 after incubation for up to 7 days in osteogenic medium The focus of current experiments was to identify and characterize with or without BMP2. (C) Representative images of qualitative alkaline phosphatase (AP) staining in cells after incubation in osteogenic medium the pertinent IGF-activated signaling pathways. with or without BMP2 for 7 days. (D) Measurement of mineralization To define the IGF-mediated mechanisms involved in regulation assessed by Alizarin red staining 7 days after incubation in osteogenic of osteoblast development and function, we first incubated confluent medium with or without BMP2. 718 Journal of Cell Science 122 (5)

Fig. 2. Inhibition of PI3-kinase activity blocks BMP2- induced osteoblast differentiation. Results are shown of experiments in which C3H10T1/2 cells were incubated in osteogenic media (OM) containing BMP2 for up to 10 days without (Con, control) or with the MEK inhibitor UO126 (UO) (10 μM) or the PI3-kinase inhibitor, LY294002 (LY) (20 μM), as described in the Materials and Methods. (A) Immunoblots of whole-cell protein lysates for Akt phosphorylated at Ser473 (pAktS473), total Akt, tyrosine and serine phosphorylated Erk1 and Erk2 (pErk1/2), total Erks, serine phosphorylated Smad1, Smad5 and Smad8 (pSmad1,5,8), total Smads and α-tubulin. (B) Results of RT-PCR assays showing expression of osteoblast-specific genes encoding Dlx-5, Runx2, osterix (Osx) and osteocalcin (Ocn), and the control gene S17 after incubation for up to 7 days in osteogenic medium with or without UO126 or LY294002. (C) Representative images of qualitative alkaline phosphatase (AP) staining in cells after incubation in osteogenic medium with or without UO126 or LY294002 for 5, 7 or 10 days. The graph depicts measurement of alkaline phosphatase activity in lysates of cells incubated for 5 or 10 days in osteogenic medium with or without UO126 or LY294002 (mean ± s.d., n=3; *P<0.01, **P<0.001 vs cells incubated without LY294002). (D) Measurement of osteoblast-mediated mineralization assessed by Alizarin red staining at days 5, 7 and 10 after incubation in osteogenic medium with or without UO126 or LY294002. The graph shows calculation of mineralized area at day 10 (mean ± s.d., n=5; **P<0.01 vs cells incubated without LY294002).

We next considered whether BMP2 could activate the PI3- prevented (Fig. 4C-E), even though BMP2-activated Smad kinase–Akt pathway, and whether LY294002 could impair BMP2- phosphorylation and Sox9 and JunB gene expression appeared to be mediated signaling. To address the first question, we incubated normal (Fig. 4B,C). In other mesenchymal cell derivatives, including C3H10T1/2 cells with BMP2 in serum-free medium for up to 60 skeletal muscle, inhibition of signaling through Akt can block minutes, and measured Akt phosphorylation on Ser473 as an differentiation by promoting cell death (Fujio et al., 2001; Lawlor indicator of PI3-kinase activation. Addition of BMP2 stimulated

Journal of Cell Science serine phosphorylation of Smad1, Smad5 and Smad8 within 15 minutes, but had no effect on Akt. Conversely, IGF-I induced phosphorylation of Akt but not that of Smad proteins (Fig. 3A). To examine effects of PI3-kinase inhibition on the acute actions of BMP2, we measured expression of Smad target genes Sox9 and JunB (Chalaux et al., 1998; Zehentner et al., 1999) after addition of BMP2 with or without LY294002 to confluent C3H10T1/2 cells in osteogenic medium. Under these conditions, BMP2 stimulated the progressive accumulation of both mRNAs starting at 12 hours, as well as inducing transcripts for Dlx5 and Runx2 (Fig. 3B). Addition of LY294002 had no effect on the kinetics of Sox9 or JunB gene expression, or mRNA abundance, but completely prevented accumulation of Dlx5 or Runx2 mRNA. Thus, LY294002 interferes selectively with BMP2-induced Smad-regulated gene activation. To expand these observations, we next used adenovirus-mediated gene transfer to deliver a regulated dominant-negative version of the serine-threonine protein kinase Akt (AktDN) to C3H10T1/2 cells, to assess whether inhibition of Akt activity also could block BMP2- mediated osteoblast differentiation (see Fig. 4A). Synthesis of AktDN in our adenoviral delivery system was prevented by the antibiotic DN Fig. 3. Acute effects of BMP2 on signaling and gene expression. (A) BMP2 doxycycline (Dox). In the presence of Dox, no Akt was produced activates Smads but not Akt. Immunoblots of whole-cell protein lysates for (Fig. 4B) and osteoblast-specific genes were induced, alkaline serine phosphorylated Smad1, Smad5 and Smad8 (pSmad1,5,8), total Smads, phosphatase activity accumulated and bone matrix mineralization Akt phosphorylated at Ser473 (pAktS473), and total Akt after incubation of proceeded normally (Fig. 4C-E). However, when Dox was omitted C3H10T1/2 cells in serum-free medium with BMP2 (200 ng/ml), 10 nM IGF-I DN or both growth factors for 0, 15, 30 or 60 minutes. (B) Results of RT-PCR from the culture medium, Akt accumulated in the cells (Fig. 4B), experiments for mRNA encoding Sox9, JunB, Dlx-5, Runx2 and S17 after and as a result, the expression of osteoblast genes was impaired, incubation for 0, 6, 12, or 24 hours in osteogenic medium without BMP2, with alkaline phosphatase activity was eliminated and mineralization BMP2 or with BMP2 plus 20 μM LY294002. Akt in bone development 719

Fig. 4. Dominant-negative Akt (AktDN) blocks BMP2-stimulated osteoblast differentiation. C3H10T1/2 cells were infected with Ad-AktDN and Ad-tTA and incubated in osteogenic medium with BMP2 with or without doxycycline (Dox) for up to 7 days. (A) Experimental scheme. (B) Immunoblots of whole-cell protein lysates for Akt, AktDN, pSmad1,5,8, total Smads and α-tubulin. (C) Results of RT-PCR experiments for mRNA encoding Sox9, JunB, Dlx-5, Runx2, Osx, Ocn and S17. (D) Results of alkaline phosphatase staining on day 7. (E) Measurement of mineralization by Alizarin red staining on day 7. (F) Measurement of cell numbers after 1, 3, or 5 days in osteogenic medium.

and Rotwein, 2000), yet under the conditions of these experiments, inhibition of differentiation seen with either IGFBP5 or LY294002 cell numbers remained constant whether or not Akt signaling was (see Fig. 5A and Fig. 6A). Synthesis of iAkt by the adenoviral gene impaired by expression of AktDN (Fig. 4F). Thus, taken together, the delivery vehicle was prevented by Dox, and stimulated in its absence results in Figs 2-4 show that inhibition of either PI3-kinase or Akt (Fig. 5B and Fig. 6B), and full Akt enzymatic activity of the fusion

Journal of Cell Science activity blocked all aspects of BMP2-mediated osteogenic protein was induced by the selective estrogen receptor modulator 4- differentiation of cultured mesenchymal stem cells, apparently hydoxytamoxifen (4-HT), thus bypassing normal regulatory without interfering with Smad function or impairing cell viability. mechanisms (Tureckova et al., 2001). As shown in Fig. 5C, BMP2- mediated osteoblast-specific gene expression was blocked by IGFBP5 An inducible Akt promotes osteoblast differentiation in the but was restored by iAkt, as was accumulation of alkaline phosphatase presence of IGFBP5 or the PI3-kinase inhibitor LY294002 and mineralization (Fig. 5C-E, lanes ‘+IGFBP5, –Dox’). Similar To test the hypothesis that the PI3-kinase–Akt pathway has an results were observed in cells incubated with LY294002 (Fig. 6); once essential role in IGF-regulated osteogenic differentiation, we next iAkt was produced and activated, osteoblast differentiation proceeded asked whether an inducible-activated Akt (iAkt) could reverse the normally, even in the presence of the PI3-kinase inhibitor (Fig. 6C-

Fig. 5. Active Akt reverses the inhibitory effects of IGFBP5 on BMP2-mediated osteoblast differentiation. C3H10T1/2 cells were infected with Ad-iAkt and Ad-tTA, and incubated in osteogenic medium with BMP2, 4-hydroxytamoxifen (4-HT), purified mouse IGFBP-5 and with or without Dox for up to 7 days. (A) Experimental scheme. (B) Immunoblots of whole-cell protein lysates for Akt, iAkt, pSmad1,5,8 and total Smads, and immunoblot of conditioned medium for IGFBP5. (C) RT-PCR experiments for mRNA encoding Dlx-5, Runx2, Osx, Ocn and S17. (D) Alkaline phosphatase activity on day 7. (E) Assessment of mineralization by Alizarin red staining on day 7. 720 Journal of Cell Science 122 (5)

Fig. 6. Active Akt promotes osteoblast differentiation in the presence of a PI3-kinase inhibitor. C3H10T1/2 cells were infected with Ad-iAkt and Ad-tTA, and incubated in osteogenic medium with BMP2, LY294002, 4-hydroxytamoxifen (4-HT) with or without Dox for up to 7 days. (A) Experimental scheme. (B) Immunoblots of whole-cell protein lysates for Akt, iAkt, pSmad1,5,8 and total Smads. (C) Results of RT-PCR experiments for mRNA encoding Dlx-5, Runx2, Osx, Ocn and S17. (D) Alkaline phosphatase staining on day 7. (E) Measurement of mineralization by Alizarin red staining on day 7.

E, lanes ‘+LY294002, –Dox’). These latter data additionally show 2006), and proceeds with production of bone-specific proteins, that the dose of LY294002 used was not toxic, because its effects deposition of extracellular matrix and its subsequent mineralization could be reversed. Based on the results depicted in Figs 2, 4, 5 and (Balcerzak et al., 2003; Hoshi et al., 2000; Lian et al., 2006). As 6, we conclude that the IGF-stimulated PI3-kinase–Akt pathway is depicted in Figs 2, 4, 5 and 6, IGF-mediated Akt activity appears required for BMP2-mediated osteogenic differentiation of cultured to be a necessary collaborator with BMP2-stimulated signaling mesenchymal stem cells. Since both Smad phosphorylation and pathways for initiating osteoblast differentiation. To begin to activation of Smad-dependent genes appeared to be normal when address whether Akt actions also are needed for later events in bone IGF-stimulated PI3-kinase or Akt were blocked, it seems likely that cell maturation and function, we devised a way to activate AktDN the point of interaction between the two growth-factor-initiated by removal of Dox from the medium at different times during the signaling cascades is downstream of Smad action and upstream of course of BMP2-mediated osteogenesis of mesenchymal stem cells induction of osteoblast-specific gene expression. (see Fig. 7A). As shown in Fig. 7B, in the presence of Dox, no AktDN was synthesized, whereas its sustained production was seen Akt activity is required during all phases of osteoblast beginning 1 day after Dox removal from culture medium. Analysis differentiation and function of osteoblast gene expression revealed complete inhibition when Osteogenic differentiation is a multi-step process, which begins with AktDN was present at the onset of differentiation (Fig. 7C, Dox expression of osteoblast-specific transcription factors (Lian et al., removal on day 0; also see Fig. 4C). By contrast, Dlx5 and Runx2 Journal of Cell Science

Fig. 7. Continual Akt activity is necessary for osteoblast differentiation, maturation and function. C3H10T1/2 cells were infected with Ad-AktDN and Ad-tTA and incubated in osteogenic medium with BMP2 and Dox. Dox was removed sequentially at days 0, 2, 4 or 6 to induce expression of AktDN. (A) Experimental scheme. (B) Immunoblots of whole- cell protein lysates for Akt and AktDN. (C) Results of RT-PCR assays for mRNA encoding Dlx-5, Runx2, Osx, Ocn and S17 at day 3, 5, 7 and 10. (D) Results of alkaline phosphatase activity measured at day 3, 5, 7 and 10 by staining and by in vitro enzymatic assay (graph) after removal of Dox on different days (mean ± s.d., n=3 experiments; *P<0.001, **P<0.01 vs +Dox). (E) Mineralized area assessed by Alizarin red staining at day 10 after removal of Dox on different days (mean ± s.d., n=5 experiments; *P<0.001, **P<0.05 vs +Dox at day 10). Representative images are depicted above the graph. Akt in bone development 721

Fig. 8. A PI3-kinase inhibitor prevents growth of cultured neonatal mouse metatarsal bones. Neonatal mouse metatarsals were incubated in DMEM containing 0.5% BSA with or without 20 μM LY294002 for up to 10 days. (A) Representative images of metatarsal bones after incubation with or without LY294002 for 10 days. (B) Relative change in metatarsal bone length after incubation with or without LY294002 for 4, 7 or 10 days compared with day 0 (mean ± s.d., n=3 experiments; *P<0.001 vs +LY294002).

mRNA was fully induced even when AktDN was present from day 3 onwards (Fig. 7C, Dox removal on day 2), although expression of genes encoding Osx and Ocn was diminished by ~50-60%, and only reached maximal values when normal differentiation conditions were sustained for a longer interval (Fig. 7C, Dox removal on day 4). Similarly, alkaline phosphatase enzymatic activity, a measure of differentiated osteoblast function, was inhibited by >85% if AktDN was present by day 3, but was fully induced in cells if the inhibitor Fig. 9. AktDN inhibits growth of cultured neonatal mouse metatarsal bones. did not appear until day 5 (Fig. 7D, compare Dox removal on day Neonatal mouse metatarsals were infected with Ad-EGFP and Ad-tTa, or Ad- 2 and day 4). Taken together, the results in Fig. 7C,D indicate a AktDN and Ad-tTa, and were incubated in DMEM containing 0.5% BSA with requirement for continuous IGF-stimulated Akt activity for at least or without Dox for up to 10 days. (A) Representative image of metatarsal bone the first few days of BMP2-directed osteoblast differentiation in for EGFP expression at 10 days after infection with Ad-EGFP and Ad-tTa order for sufficient bone-specific mRNAs and proteins to be without addition of Dox. In the presence of Dox no EGFP was detected. (B) Immunoblot showing induction of AktDN in the absence of Dox, and produced to sustain differentiated functions. However, matrix expression of endogenous Akt (lower band) and α-tubulin in tissue lysates mineralization, a later event in the process of osteoblast maturation, from metatarsals after 10 days of culture. (C) Representative images of appears to have a qualitatively different set of requirements, because metatarsal bones after incubation with Ad-EGFP or Ad-AktDN with or without DN Dox, or no adenovirus (Con) for 10 days. (D) Relative change in metatarsal Journal of Cell Science the presence of Akt from day 5 onward prevented 50% of full DN DN bone length after infection with Ad-Akt for 4, 7, or 10 days with or without mineralization by day 10, and expression of Akt from day 7 Dox treatment compared with day 0 (mean ± s.d., n=3 experiments; *P<0.001, onwards still blocked ~25% of the normal accumulation of **P<0.01 vs +Dox). mineralized matrix at day 10 (Fig. 7E, compare Dox removal on day 4 and day 6 with +Dox), even though at these time points AktDN had no inhibitory effects on expression of bone genes or on alkaline Inhibition of Akt activity impairs cartilage growth and osteoblast phosphatase activity. Our provisional interpretation of the results development and function in isolated mouse metatarsal bones in Fig. 7 is that there are several temporally distinct Akt targets in We analyzed histological sections of mouse metatarsal bones differentiating osteoblasts that govern different aspects of bone cell incubated ex vivo for different intervals to address potential development and function. mechanisms by which Akt signaling was required for bone growth. At the start of the 10-day culture period, proliferating cartilage made Signaling through the PI3-kinase–Akt pathway is required for up nearly half of the bone length and, together with hypertrophic growth of isolated mouse metatarsal bones cartilage, comprised 85% of the total, with the mid-diaphyseal Short-term culture of neonatal mouse metatarsal bones has been mineralizing zone comprising the remaining 15% (Fig. 10, top used previously to study bone growth and endochondral ossification panel). By day 10, this central mineralizing zone had increased to (Krishnan et al., 2003; Mukherjee et al., 2005). As we have shown almost 30% of the now longer bone, with proliferating cartilage recently, these bones increased in length by >35% in serum-free remaining proportionately constant (54%), and the hypertrophic medium over a 10-day culture period, but growth was blocked by zone decreasing from 39% to 18% (Fig. 10, second panel). We IGFBP5 (Mukherjee and Rotwein, 2008). As also depicted in Fig. observed nearly identical results on day 10 of culture with bones 8, a single addition of 20 μM LY294002 at 1 day after plating infected with Ad-AktDN and incubated with Dox (Fig. 10, third reduced growth to <5% over the same time course. We observed panel), thus illustrating the lack of effect of adenoviral infection on similarly dramatic inhibitory effects on longitudinal growth in the proportion of different cell types in the developing and growing metatarsals infected with Ad-AktDN at the beginning of ex vivo bone. By contrast, expression of AktDN not only prevented culture, whereas, by contrast, infection with Ad-EGFP or Ad-AktDN longitudinal metatarsal growth, but also completely inhibited in the presence of Dox, was completely ineffective (Fig. 9). Thus, lengthening of the zone of proliferating cartilage and expansion of in this model system, the PI3-kinase–Akt pathway appears to be the mineralized zone (Fig. 10, bottom panel). As a consequence, needed for normal bone growth. the histological profile after 10 days of AktDN expression resembled 722 Journal of Cell Science 122 (5)

Fig. 10. AktDN inhibits chondrocyte maturation and osteoblast development in cultured neonatal mouse metatarsal bones. Neonatal mouse metatarsals were infected with Ad-AktDN and Ad-tTa, and incubated in DMEM with 0.5% BSA with or without Dox for 10 days followed by histological analysis. Pictured on the left are hematoxylin and eosin stained sections of representative metatarsals at day 0 and day 10 of culture after control incubations (top two panels) or after infection with Ad-AktDN and Ad-tTa (bottom two panels); ϫ40 magnification. Zones of proliferating (PC) or hypertrophic chondrocytes (HC) are indicated, as is the central mineralized zone (MZ). The charts in the center represent graphical analysis of each component as a percentage of the total length of each bone. Images on the right show the boxed regions on the left panels at ϫ100 magnification.

that of control metatarsals at the onset of ex vivo incubation (Fig. within bone. Similarly, as measured by both calcein labeling of 10, compare top and bottom panels). Thus, Akt signaling appears living bones (Fig. 11C) and Alizarin red staining of histological to be required for both cartilage and bone growth in isolated sections (Fig. 11D), AktDN prevented the normal accumulation of

Journal of Cell Science metatarsal bones. mineralized matrix. Thus, based on these results, we conclude that We further examined the effects of inhibiting Akt signaling on continuous Akt signaling is necessary for full osteoblast maturation osteoblasts within the mid-diaphyseal mineralizing zone. Cell- and function in vivo, as well as in vitro. counting experiments demonstrated that the number of morphologically recognizable osteoblasts per ϫ400 microscopic Discussion field nearly doubled (from 16±3 to 28±7) during 10 days of organ BMPs have a central role in bone development and osteoblast culture, whereas total cell density did not change (Fig. 11A). As differentiation (Li and Cao, 2006), but require interactions with other the mineralizing zone also expanded twofold in absolute length (Fig. growth-factor-activated signals. Here, we demonstrate essential 10), it appears that the total number of osteoblasts increased by a crosstalk between BMP2 and the IGF-activated PI3-kinase–Akt factor of four during ex vivo bone development. We recorded nearly pathway to initiate osteogenic differentiation in uncommitted identical results in bones infected with Ad-AktDN and incubated mesenchymal precursor cells, and to promote maturation of with Dox. By contrast, when AktDN was expressed in the absence committed osteoblasts. We also show that IGF-stimulated and Akt- of Dox, both osteoblast numbers and total cell density were mediated signaling is crucial for longitudinal bone growth by unchanged at day 10 compared with control metatarsals at day 0 exerting facilitating effects on both chondrocyte and osteoblast (Fig. 11A). Our interpretation of these results is that Akt appears development and function. Based on these results, we conclude that to be required for osteoblast recruitment and/or differentiation within the PI3-kinase–Akt pathway is a crucial component of an interactive bone during the early postnatal period in mice, although osteogenic signaling network. alternatively, Akt signaling might be necessary for osteoblast viability. An IGF-activated PI3-kinase–Akt pathway regulates BMP2- We next assessed osteoblast maturation and function in metatarsal mediated osteoblast differentiation cultures by examining bone-cell-specific gene expression and We previously found that IGFBP5 could block BMP2-regulated mineralization. AktDN reduced induction of mRNA encoding Ocn osteogenic differentiation of mesenchymal stem cells by by 70%, but had no effect on transcripts encoding Runx2 or Osx sequestering IGF-I and IGF-II from the IGF-I receptor (Mukherjee (Fig. 11B). Since Runx2 and Osx are produced by both chondrocytes and Rotwein, 2008), and postulated that through this mechanism it and osteoblasts, whereas Ocn is synthesized exclusively by also prevented osteoblast maturation and blocked longitudinal differentiated osteoblasts (Karsenty, 1998), we interpret these data growth of mouse metatarsal bones. We now show that both a to indicate that AktDN interfered with osteoblast development chemical PI3-kinase inhibitor and a dominant-negative version of Akt in bone development 723

Fig. 11. AktDN inhibits osteoblast development and function in cultured neonatal mouse metatarsal bones. Neonatal mouse metatarsals were uninfected (Con) or were infected with Ad-AktDN and Ad-tTa, and incubated in DMEM plus 0.5% BSA with or without Dox for 10 days. (A) Cell counts of osteoblasts per field (h.p.f.) in histological sections of the mineralized zone at ϫ400 magnification [mean ± s.d., n=4; *P<0.001, vs Con (day 0)]. (B) Results of RT-PCR experiments at day 10 for mRNA encoding Runx2, Osx, Ocn, and S17. (C) Representative fluorescent images of calcein-labeled mineralizing zone after incubation for 10 days; graph shows the relative difference in length of the calcein- labeled mineralizing zone at day 10 in metatarsals incubated with or without Dox (mean ± s.d., n=4; *P<0.001, vs +Dox). (D) Representative images showing Alizarin-red-stained histological sections after a incubation for 10 days; graph shows the difference in the Alizarin-red- stained area at day 10 in metatarsals incubated with or without Dox (mean ± s.d., n=4; *P<0.01).

Akt (AktDN), also can inhibit BMP2-initiated osteogenesis (Figs 2 Runx2 upregulated components of the PI3-kinase–Akt pathway, and 4). By contrast, blocking the Mek-Erk signaling pathway had which then enhanced the functions of Runx2 (Fujita et al., 2004). no effect on the onset or progression of BMP2-activated osteoblast Others have found that a dominant-negative Akt could reduce the differentiation (Fig. 2). As the inhibitory actions of IGFBP5 or activity of a BMP2-dependent promoter-reporter gene, possibly by LY294002 on BMP2-stimulated osteogenesis could be reversed by inhibiting the nuclear accumulation of activated Smad1 and Smad5 a recombinant adenovirus encoding an activated version of Akt (Figs (Ghosh-Choudhury et al., 2002), but these results have not been 5 and 6), our results document that Akt-regulated signaling is the replicated, and we see no inhibitory effects of dominant-negative key pathway of IGF action in promoting osteoblast differentiation Akt on Smad-mediated Sox9 and JunB gene expression (Fig. 4B). in collaboration with BMPs. In another mesenchymal derivative, skeletal muscle, Akt has been Several previous studies have supported facilitating roles for IGF- shown to collaborate with myogenic transcription factors to enhance

Journal of Cell Science mediated signaling in osteogenesis in cell culture models, but with the abundance of transcriptional co-activators on muscle gene disparate results regarding the intracellular pathways implicated promoters (Wilson and Rotwein, 2007), and to reduce co-repressors (Merriman et al., 1990; Niu and Rosen, 2005; Strong et al., 1991; (Serra et al., 2007). It thus might be reasonable to postulate an Strong et al., 1994). Fujita and colleagues found that either PI3- analogous role for Akt signaling in osteoblast differentiation, kinase or Mek inhibitors could reduce the amount of bone-specific although to date there is little experimental evidence for or against alkaline phosphatase produced by cells overexpressing Runx2 this idea. (Fujita et al., 2004). Raucci and co-workers also showed that these chemical inhibitors could decrease alkaline phosphatase activity in Akt signaling is required in all phases of osteoblast two additional osteogenic cell lines, and attributed the negative differentiation and maturation effects of the PI3-kinase inhibitor to enhanced cell death (Raucci Osteoblast differentiation can be divided into several phases, et al., 2008). These authors also found that a constitutively active including lineage commitment, characterized in part by expression Akt led to increased accumulation of several bone-specific mRNAs, of Runx2 and Osx (Komori, 2008), early differentiation, in which including those encoding Runx2 and Osx (Raucci et al., 2008). other bone-cell-specific mRNAs and proteins are produced (Deng Several other groups also showed that chemical inhibition of PI3- et al., 2008), and maturation, marked by accumulation of bone- kinase or Mek could reduce markers of osteoblast differentiation specific alkaline phosphatase, extracellular matrix deposition and (Ghosh-Choudhury et al., 2002; Ghosh-Choudhury et al., 2007; mineralization (Balcerzak et al., 2003; Hoshi et al., 2000; Zaidi, Hanai et al., 2006; Noda et al., 2005; Osyczka and Leboy, 2005). 2007). We now find that Akt activity appears to be required for Our observations thus appear to be one of the few studies to clearly each of these stages of differentiation, because AktDN can block discriminate between the PI3-kinase–Akt and Mek-Erk pathways progression from one step to the next (Fig. 7). Most remarkable in in terms of osteogenic outcomes. this regard is the inhibitory action of AktDN on mineralization, which What mechanisms might mediate Akt-regulated osteoblast was seen even when AktDN was added relatively late in the differentiation? A dominant-negative Akt decreased the differentiation process, at a time when alkaline phosphatase activity transcriptional actions of Runx2 in a model system in which Runx2 was already maximal (compare Fig. 7D with 7E). These latter was overexpressed (Fujita et al., 2004). These authors additionally results, which complement our previous data using IGFBP5 to block found that overexpression of Runx2 led to an increase in abundance osteogenesis (Mukherjee and Rotwein, 2008), are supported by the of Akt and of both regulatory and catalytic PI3-kinase subunits, inhibitory effects of AktDN on osteoblast maturation and function and postulated the existence of a positive-feedback loop in which in cultured metatarsal bones (Fig. 11), and also are consistent with 724 Journal of Cell Science 122 (5)

the defective mineralization phenotype seen in mice lacking the diminished osteocalcin gene expression and reduced mineralization IGF-I receptor in mature osteoblasts (Zhang et al., 2002). (Fig. 11). Thus, sustained Akt activity appears to be required for Mineralization represents the outcome of a complex series of steps the normal cartilage and bone cell development that leads to that include active transport of calcium and inorganic phosphate longitudinal bone growth during the early postnatal period, at least into osteoblasts, release of matrix vesicles into the extracellular in metatarsals. These observations are additionally supported by space, and nucleation and deposition of hydroxyapatite granules in previous studies, which have suggested that Akt signaling is the osteoid (Balcerzak et al., 2003; Hoshi et al., 2000). Among required for proteoglycan and collagen production in chondrocytes factors that control mineralization is the sodium-dependent mediated by Runx2 (Fujita et al., 2004), and that Akt1 is important phosphate transporter Pit-1, which appears to be regulated by IGF- for normal rates of bone formation and for preventing osteoblast I in osteoblasts (Kavanaugh and Kabat, 1996; Palmer et al., 1997; apoptosis (Kawamura et al., 2007). Selz et al., 1989). As mineralization is an important step in fracture In summary, we have shown that the IGF-activated PI3- healing (Schindeler et al., 2008), an understanding of the regulatory kinase–Akt signaling pathway is a potent facilitator of osteoblast mechanisms has the potential to lead to better treatment options. differentiation, bone growth, and mineralization. Our results point to a key role for IGF-mediated signaling in all phases of Akt signaling in bone growth and endochondral ossification osteogenesis, and provide an impetus to define the mechanisms of We showed previously that incubation with IGFBP5 prevented both interaction with BMPs and other regulators of cartilage and bone longitudinal growth and mineralization of cultured neonatal mouse development and function. metatarsal bones, and found that the inhibitory effects of IGFBP5 depended on its ability to bind IGFs with high affinity (Mukherjee Materials and Methods and Rotwein, 2008). We now show that a chemical PI3-kinase DN Reagents inhibitor and adenoviral-delivered Akt also block metatarsal Fetal calf serum, horse serum, Dulbecco’s modified Eagle’s medium (DMEM), and growth (Figs 8 and 9). In these experiments, the metatarsals were phosphate-buffered saline (PBS) were purchased from Mediatech-Cellgrow (Herndon, incubated in serum-free medium, and because IGFBP5, LY294002 VA). Okadaic acid was from Alexis Biochemicals (San Diego, CA), and NBT/BCIP DN tablets and protease inhibitor tablets were from Roche Applied Sciences (Indianapolis, and Akt all exerted similar inhibitory effects, we conclude that IN). Calcein, sodium orthovanadate, alizarin red, ascorbic acid and β-glycerol locally produced IGFs are responsible for activating the PI3- phosphate were purchased from Sigma (St Louis, MO). Trypsin-EDTA solution and kinase–Akt signaling pathway that is essential for longitudinal bone Superscript III first-strand synthesis kit were from Invitrogen (Carlsbad, CA). The growth. The defects seen in our metatarsal model resemble the bone BCA protein assay kit was from Pierce Biotechnologies (Rockford, IL) and Immobilon-FL was from Millipore Corporation (Billerico, MA). AquaBlock EIA/WIB phenotype in mice lacking the IGF-I receptor (Zhang et al., 2002) solution was from East Coast Biologicals (North Berwick, ME). IGF-I (Gropep) was or both Akt1 and Akt2 (Peng et al., 2003), in which ossification stored at –80°C at a concentration of 10 mM in 0.01 M HCl until use. Doxycycline was delayed and osteopenia resulted. Conversely, the opposite (Dox, Clontech, Palo Alto, CA) was used at a final concentration of 1 mg/ml. LY294002 (Biomol Research Laboratories, Plymouth Meeting, PA) was stored in phenotype was observed in transgenic mice lacking Pten in dimethyl sulfoxide at –20°C at a concentration of 20 mM until use; 4- osteoblasts, in which progressive increases in bone volume and hydroxytamoxifen (HT) was from Sigma, and was stored in ethanol at –20°C at a density were seen throughout life (Liu et al., 2007). As Pten concentration of 50 mM until use. UO126 (Promega, Madison, WI) was stored in ethanol at –80°C at a concentration of 20 mM until use. Other chemicals were dephosphorylates and inactivates phosphatidylinositol (3,4,5)- purchased from commercial vendors.

Journal of Cell Science trisphosphate (PIP3), the product of PI3-kinase, and because PIP3 is essential for membrane targeting and activation of Akt (Franke, Antibodies 2008; Hanada et al., 2004), these results predict enhanced Akt The following polyclonal antibodies were purchased from commercial suppliers: anti- Smad1, anti-Akt, anti-phospho-Akt (Ser473), anti-Erk1 and 2, anti-phospho-Erk1 and activity in Pten-deficient osteoblasts. In fact, cultured calvarial -Erk2, Cell Signaling Technology (Beverly, MA); anti-α-tubulin, Sigma; anti-IGFBP- osteoblasts engineered to lack Pten did show increased Akt 5, anti-phospho-Smad, Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal phosphorylation and phosphorylation of several Akt substrates, and anti-T7 antibody was from Novagen (San Diego, CA) and anti-HA was from Roche the cells differentiated more extensively than controls (Liu et al., Applied Sciences (Indianapolis, IN). Goat anti-rabbit IgG-IR800 and goat anti-mouse IgG-IR680 were from Rockland Immunochemical (Gilbertsville, PA). 2007). No results have been reported yet on mice in which a constitutively active Akt has been targeted to osteoblasts, but a Recombinant adenoviruses similarly high bone density phenotype might be anticipated. The following adenoviruses (Ad) have been described: Ad-EGFP (Tureckova et al., 2001), Ad-IGFBP-5 (Mukherjee et al., 2008), Ad-BMP2 (Mukherjee and Rotwein, 2008), Ad-tTA (tetracycline transactivator protein), Ad-iAkt (inducible Akt) Akt is essential for optimal chondrogenesis and osteogenesis (Tureckova et al., 2001) and Ad-AktDN [dominant negative Akt (Wilson et al., 2003)]. Histological analysis of growing mouse metatarsal bones revealed All viruses were purified by centrifugation through CsCl density gradients and titered a growth-associated proportional increase in the zone of proliferating as described (Wilson et al., 2003). chondrocytes, a decline in the extent of terminally differentiated Production of BMP2 and IGFBP5 hypertrophic chondrocytes and a more-than-proportional rise in the C3H10T1/2 mouse embryonic fibroblasts (ATCC: CCL226) were incubated at 37°C length of the central mineralized zone, which contained osteoblasts in humidified air with 5% CO2 in DMEM with 10% fetal calf serum. Cells were as well as other cell types (Fig. 10). These growth-related changes infected at ~50% of confluent density with Ad-BMP2 [multiplicity of infection (MOI) of 500]. The following day, medium was replaced with DMEM plus 2% horse serum; in the profile of cell types within the cultured metatarsals were 2 days later, conditioned medium was collected, clarified, and stored in aliquots at completely inhibited by AktDN (Fig. 10), thus demonstrating –80°C until use. The concentration of BMP2 was determined by immunoblotting negative effects on both chondrocyte and osteoblast development. with purified standards purchased from R&D systems (Minneapolis, MN) (see supplementary material Fig. S2). Mouse IGFBP-5 was produced in C3H10T1/2 cells In their analysis of isolated tibias from E15.5 mouse embryos, Ulici following infection with Ad-IGFBP-5, and was purified by heparin affinity and colleagues also found that LY294002 could impair chondrocyte chromatography (Mukherjee et al., 2008). differentiation and inhibit longitudinal bone growth (Ulici et al., 2008). In addition, AktDN blocked the normal twofold increase in Osteogenic differentiation Confluent C3H10T1/2 cells were incubated in osteogenic medium (DMEM, 10% osteoblast number seen in the central mineralizing zone, and also fetal calf serum, 50 μg/ml ascorbic acid, 10 mM β-glycerol phosphate and 200 ng/ml impaired both osteoblast maturation and function, as measured by BMP2) in the absence or presence of LY294002 (20 μM), UO126 (10 μM), or IGFBP- Akt in bone development 725

5 (150 nM). osteogenic medium was replaced every 48 hours for up to 10 days. Cell Results were visualized and images captured using the LiCoR Odyssey and version counting was performed as described (Wilson and Rotwein, 2007). Alternatively, 1.2 analysis software. C3H10T1/2 cells were infected at ~50% of confluent density with Ad-tTA at an MOI of 125, and either Ad-EGFP, Ad-iAkt, or Ad-AktDN at MOIs of 500. One day later, We thank Svetlana Lutsenko for reagents and Ronen Schweitzer for cells were washed and osteogenic medium was added, along with other chemicals use of his microscope and imaging system, and appreciate the assistance as described in individual figure legends. of the histology core of the Department of Pathology at OHSU. The Mouse metatarsal bone culture studies presented in this manuscript were supported by National Metatarsal bones were isolated from newborn C57BL6 mice (days 0-3 after birth), Institutes of Health R01 grants DK42748 and DK63073 (to P. R). as described (Krishnan et al., 2001; Mukherjee and Rotwein, 2008) and were incubated Deposited in PMC for release after 12 months. in DMEM containing 0.5% bovine serum albumin, 50 μg/ml ascorbic acid, 1 mM β μ -glycerol phosphate and 100 g/ml penicillin-streptomycin solution at 37°C in References humidified air with 5% CO2 for up to 10 days. In some experiments LY294002 (20 μ Akune, T., Ogata, N., Hoshi, K., Kubota, N., Terauchi, Y., Tobe, K., Takagi, H., Azuma, M) was added the next day. In others, bones were infected the next day with Ad- Y., Kadowaki, T., Nakamura, K. et al. (2002). Insulin receptor substrate-2 maintains ϫ 7 DN ϫ 7 tTA (1 10 PFU/ml) plus either Ad-EGFP or Ad-Akt (6 10 PFU/ml). Images predominance of anabolic function over catabolic function of osteoblasts. J. Cell Biol. were captured at days 1, 4, 7 and 10 with a Nikon DXL1200 camera attached to a 159, 147-156. Lieca MZ FLIII microscope. Mineralization was assessed by addition of medium Bach, L. A., Headey, S. J. and Norton, R. S. (2005). IGF-binding proteins-the pieces are containing calcein (500 ng/ml) for 2 hours. After rinsing three times with PBS, falling into place. Trends Endocrinol. Metab. 16, 228-234. fluorescent images were captured with a Roper Scientific Cool Snap FX CCD camera Balcerzak, M., Hamade, E., Zhang, L., Pikula, S., Azzar, G., Radisson, J., Bandorowicz- attached to a Nikon Eclipse T300 microscope using an Apple PowerPC computer Pikula, J. and Buchet, R. (2003). The roles of annexins and alkaline phosphatase in running IP Labs Scientific Image Processing software v3.9.4r2 (Scanalytics, Rockville, mineralization process. Acta Biochim. Pol. 50, 1019-1038. MD). Chalaux, E., Lopez-Rovira, T., Rosa, J. L., Bartrons, R. and Ventura, F. (1998). JunB is involved in the inhibition of myogenic differentiation by bone morphogenetic protein- Bone histology 2. J. Biol. Chem. 273, 537-543. Deng, Z. L., Sharff, K. A., Tang, N., Song, W. X., Luo, J., Luo, X., Chen, J., Bennett, Metatarsals were fixed in 4% paraformaldehyde for 18 hours at 4°C and stored in E., Reid, R., Manning, D. et al. (2008). Regulation of osteogenic differentiation during 70% ethanol. Bones were embedded in paraffin blocks and sectioned. Staining with skeletal development. Front. Biosci. 13, 2001-2021. hematoxylin and eosin or with alizarin red was performed after hydrating slides in Duan, C. and Xu, Q. (2005). Roles of insulin-like growth factor (IGF) binding proteins graded concentrations of ethanol and water, as described (Mukherjee et al., 2005; in regulating IGF actions. Gen. Comp. Endocrinol. 142, 44-52. Serra et al., 1999). After staining, sections were dehydrated, coverslips added and Franke, T. F. (2008). Intracellular signaling by Akt: bound to be specific. Sci. Signal. 1, images were captured using a MicroPublisher cooled CCD camera (QImaging, Surrey, e29. British Columbia) attached to a Nikon Eclipse E800 compound microscope. For cell Fujio, Y., Mitsuuchi, Y., Testa, J. R. and Walsh, K. (2001). Activation of Akt2 Inhibits counting, osteoblasts were identified in the central mineralized zone by the cuboidal anoikis and apoptosis induced by myogenic differentiation. Cell Death Differ. 8, 1207- morphology of their nuclei, and were counted at ϫ400 magnification in histological 1212. sections stained with hematoxylin and eosin. Fujita, T., Azuma, Y., Fukuyama, R., Hattori, Y., Yoshida, C., Koida, M., Ogita, K. and Komori, T. (2004). Runx2 induces osteoblast and chondrocyte differentiation and Alkaline phosphatase staining enhances their migration by coupling with PI3K-Akt signaling. J. Cell Biol. 166, 85- 95. Cells were washed with PBS, fixed with 70% ethanol for 10 minutes, and incubated Ghosh-Choudhury, N., Abboud, S. L., Nishimura, R., Celeste, A., Mahimainathan, L. with 500 ml NBT/BCIP solution (1 tablet in 10 ml distilled water) for 20 minutes at and Choudhury, G. G. (2002). Requirement of BMP-2-induced phosphatidylinositol 20°C (Mukherjee and Rotwein, 2008). After three washes with distilled water, images 3-kinase and Akt serine/threonine kinase in osteoblast differentiation and Smad- were captured and analyzed with the LiCoR Odyssey Infrared Imaging System, using dependent BMP-2 gene transcription. J. Biol. Chem. 277, 33361-33368. software version 1.2 (LiCoR, Lincoln, NE). Alkaline phosphatase activity was Ghosh-Choudhury, N., Mandal, C. C. and Choudhury, G. G. (2007). Statin-induced determined spectrophotometrically at 405 nM after incubating cell lysates (10 μg) Ras activation integrates the phosphatidylinositol 3-kinase signal to Akt and MAPK for in a 96-well format for 20 minutes at 20°C in 50 μl of a 1 mg/ml solution of p- bone morphogenetic protein-2 expression in osteoblast differentiation. J. Biol. Chem.

Journal of Cell Science nitrophenyl phosphate (Mukherjee and Rotwein, 2008). 282, 4983-4993. Giustina, A., Mazziotti, G. and Canalis, E. (2008). Growth hormone, insulin-like growth Alizarin red staining factors, and the skeleton. Endocr. Rev. 29, 535-559. Cells were fixed in 70% ethanol for 10 minutes, and stained with 2% alizarin red Hadjidakis, D. J. and Androulakis, I. I. (2006). Bone remodeling. Ann. NY Acad. Sci. 1092, 385-396. solution (pH 4.1-4.5) for 1 minute at 20°C (Mukherjee and Rotwein, 2008). Images Hanada, M., Feng, J. and Hemmings, B. A. (2004). Structure, regulation and function were obtained by scanning plates on a Canon flat-bed scanner or with the LiCoR of PKB/AKT-a major therapeutic target. Biochim. Biophys. Acta 1697, 3-16. Odyssey and results were quantified as described (Mukherjee and Rotwein, 2008). Hanai, Y., Tokuda, H., Ishisaki, A., Matsushima-Nishiwaki, R., Nakamura, N., Yoshida, M., Takai, S., Ohta, T. and Kozawa, O. (2006). Involvement of p44/p42 Analysis of gene expression MAP kinase in insulin-like growth factor-I-induced alkaline phosphatase activity in Whole-cell RNA (2 μg), isolated as described (Mukherjee and Rotwein, 2008), was osteoblast-like-MC3T3-E1 cells. Mol. Cell Endocrinol. 251, 42-48. reverse-transcribed with the Superscript III first-strand synthesis kit, using oligo (dT) Herpin, A. and Cunningham, C. (2007). Cross-talk between the bone morphogenetic primers in a final volume of 20 μl. PCR reactions were performed with 1 μl of cDNA protein pathway and other major signaling pathways results in tightly regulated cell- per reaction and previously published primer pairs for mouse Dlx5, Runx2, osterix, specific outcomes. FEBS J. 274, 2977-2985. osteocalcin and S17 (Mukherjee and Rotwein, 2008). Other oligonucleotide primers Hoshi, K., Ejiri, S. and Ozawa, H. (2000). Ultrastructural, cytochemical, and biophysical are as follows: mouse Sox9: top strand, 5Ј-AGGAAGCTGGCAGACCAGTA-3Ј; aspects of mechanisms of bone matrix calcification. Kaibogaku Zasshi 75, 457-465. Jiang, J., Lichtler, A. C., Gronowicz, G. A., Adams, D. J., Clark, S. H., Rosen, C. J. bottom strand, 5Ј-CGTTCTTCACCGACTTCCTC-3Ј; mouse JunB: top strand, 5Ј- and Kream, B. E. (2006). Transgenic mice with osteoblast-targeted insulin-like growth ACGGAGGGAGAGAAAAGCTC-3Ј; bottom strand, 5Ј-AAGGCTGT TC - Ј factor-I show increased bone remodeling. Bone 39, 494-504. CATTTTCGTG-3 . Cycle numbers ranged from 20-30 and results were visualized Kadowaki, T., Tobe, K., Honda-Yamamoto, R., Tamemoto, H., Kaburagi, Y., after agarose gel electrophoresis. Momomura, K., Ueki, K., Takahashi, Y., Yamauchi, T., Akanuma, Y. et al. (1996). Signal transduction mechanism of insulin and insulin-like growth factor-1. Endocr. J. Protein extraction and immunoblotting 43 Suppl., S33-S41. Whole-cell protein lysates and conditioned cultured medium were prepared from Karsenty, G. (1998). Transcriptional regulation of osteoblast differentiation during C3H10T1/2 cells as described (Mukherjee et al., 2008; Mukherjee and Rotwein, 2008), development. Front Biosci. 3, d834-d837. and aliquots were stored at –80°C until use. Metatarsals were homogenized in cell Kavanaugh, M. P. and Kabat, D. (1996). Identification and characterization of a widely lysis buffer with protease inhibitors using a hand-held Teflon homogenizer. After expressed phosphate transporter/retrovirus receptor family. Kidney Int. 49, 959-963. centrifugation at 14,000 r.p.m. for 10 minutes at 4°C in a microcentrifuge, supernatants Kawamura, N., Kugimiya, F., Oshima, Y., Ohba, S., Ikeda, T., Saito, T., Shinoda, Y., were collected and stored at –80°C until use. Protein samples (30 μg/lane) or medium Kawasaki, Y., Ogata, N., Hoshi, K. et al. (2007). Akt1 in osteoblasts and osteoclasts controls bone remodeling. PLoS. ONE. 2, e1058. (25 μl/lane) were resolved by SDS-PAGE and transferred to Immobilon-FL Khosla, S., Westendorf, J. J. and Oursler, M. J. (2008). Building bone to reverse membranes. After blocking with 25% AquaBlock solution for 1 hour at 20°C, osteoporosis and repair fractures. J. Clin. Invest 118, 421-428. membranes were incubated sequentially with primary and secondary antibodies Komori, T. (2008). Regulation of bone development and maintenance by Runx2. Front (Mukherjee and Rotwein, 2008). The following primary antibodies were used at a Biosci. 13, 898-903. 473 dilution of 1:1000: anti-Akt, anti-phospho-Akt (Ser ), anti-IGFBP-5, anti-phospho- Krishnan, V., Ma, Y., Moseley, J., Geiser, A., Friant, S. and Frolik, C. (2001). Bone Smad, anti-Smad1, anti-HA, anti-Erk1/2 and anti-phospho-Erk1/2. Anti-T7 was used anabolic effects of sonic/indian hedgehog are mediated by bmp-2/4-dependent pathways at 1:5000 and anti-α-tubulin at 1:15,000. Secondary antibodies were used at 1:5000. in the neonatal rat metatarsal model. Endocrinology 142, 940-947. 726 Journal of Cell Science 122 (5)

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