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First published online 10 February 2009
doi: 10.1242/jcs.042770

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Akt promotes BMP2-mediated osteoblast differentiation and bone development

Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA

View larger version (35K): [in this window] [in a new window]   Fig. 1. BMP2 promotes osteoblast differentiation. Results are shown of experiments in which C3H10T1/2 cells were incubated in osteogenic media (OM) without or with BMP2 (200 ng/ml) for up to 7 days. (A) Immunoblots of whole-cell protein lysates for serine-phosphorylated Smad1, Smad5 and Smad8 (pSmad1,5,8), total Smads, Akt phosphorylated at Ser473 (pAktS473) and total Akt. (B) Results of RT-PCR assays showing expression of osteoblast-specific genes Dlx5, Runx2, osterix (Osx) and osteocalcin (Ocn), and control gene S17 after incubation for up to 7 days in osteogenic medium with or without BMP2. (C) Representative images of qualitative alkaline phosphatase (AP) staining in cells after incubation in osteogenic medium with or without BMP2 for 7 days. (D) Measurement of mineralization assessed by Alizarin red staining 7 days after incubation in osteogenic medium with or without BMP2.

View larger version (60K): [in this window] [in a new window]   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 -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).

View larger version (58K): [in this window] [in a new window]   Fig. 3. Acute effects of BMP2 on signaling and gene expression. (A) BMP2 activates Smads but not Akt. Immunoblots of whole-cell protein lysates for serine phosphorylated Smad1, Smad5 and Smad8 (pSmad1,5,8), total Smads, Akt phosphorylated at Ser473 (pAktS473), and total Akt after incubation of C3H10T1/2 cells in serum-free medium with BMP2 (200 ng/ml), 10 nM IGF-I or both growth factors for 0, 15, 30 or 60 minutes. (B) Results of RT-PCR experiments for mRNA encoding Sox9, JunB, Dlx-5, Runx2 and S17 after incubation for 0, 6, 12, or 24 hours in osteogenic medium without BMP2, with BMP2 or with BMP2 plus 20 µM LY294002.

View larger version (45K): [in this window] [in a new window]   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 -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.

View larger version (35K): [in this window] [in a new window]   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.

View larger version (34K): [in this window] [in a new window]   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.

View larger version (34K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   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).

View larger version (26K): [in this window] [in a new window]   Fig. 9. AktDN inhibits growth of cultured neonatal mouse metatarsal bones. Neonatal mouse metatarsals were infected with Ad-EGFP and Ad-tTa, or Ad-AktDN and Ad-tTa, and were incubated in DMEM containing 0.5% BSA with or without Dox for up to 10 days. (A) Representative image of metatarsal bone for EGFP expression at 10 days after infection with Ad-EGFP and Ad-tTa 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 expression of endogenous Akt (lower band) and -tubulin in tissue lysates from metatarsals after 10 days of culture. (C) Representative images of metatarsal bones after incubation with Ad-EGFP or Ad-AktDN with or without Dox, or no adenovirus (Con) for 10 days. (D) Relative change in metatarsal bone length after infection with Ad-AktDN for 4, 7, or 10 days with or without Dox treatment compared with day 0 (mean ± s.d., n=3 experiments; *P<0.001, **P<0.01 vs +Dox).

View larger version (67K): [in this window] [in a new window]   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); x40 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 x100 magnification.

View larger version (29K): [in this window] [in a new window]   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 x400 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).

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