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. 2012 Oct 19;287(43):35975-84.
doi: 10.1074/jbc.M112.372458. Epub 2012 Sep 4.

Transforming growth factor β suppresses osteoblast differentiation via the vimentin activating transcription factor 4 (ATF4) axis

Na Lian  1 , Tonghui Lin, Wenguang Liu, Weiguang Wang, Lingzhen Li, Stephanie Sun, Jeffry S Nyman, Xiangli Yang
Affiliations

Transforming growth factor β suppresses osteoblast differentiation via the vimentin activating transcription factor 4 (ATF4) axis

Na Lian et al. J Biol Chem. .

Abstract

ATF4 is an osteoblast-enriched transcription factor of the leucine zipper family. We recently identified that vimentin, a leucine zipper-containing intermediate filament protein, suppresses ATF4-dependent osteocalcin (Ocn) transcription and osteoblast differentiation. Here we show that TGFβ inhibits ATF4-dependent activation of Ocn by up-regulation of vimentin expression. Osteoblasts lacking Atf4 (Atf4(-/-)) were less sensitive than wild-type (WT) cells to the inhibition by TGFβ on alkaline phosphatase activity, Ocn transcription and mineralization. Importantly, the anabolic effect of a monoclonal antibody neutralizing active TGFβ ligands on bone in WT mice was blunted in Atf4(-/-) mice. These data establish that ATF4 is required for TGFβ-related suppression of Ocn transcription and osteoblast differentiation in vitro and in vivo. Interestingly, TGFβ did not directly regulate the expression of ATF4; instead, it enhanced the expression of vimentin, a negative regulator of ATF4, at the post-transcriptional level. Accordingly, knockdown of endogenous vimentin in 2T3 osteoblasts abolished the inhibition of Ocn transcription by TGFβ, confirming an indirect mechanism by which TGFβ acts through vimentin to suppress ATF4-dependent Ocn activation. Furthermore, inhibition of PI3K/Akt/mTOR signaling, but not canonical Smad signaling, downstream of TGFβ, blocked TGFβ-induced synthesis of vimentin, and inhibited ATF4-dependent Ocn transcription in osteoblasts. Thus, our study identifies that TGFβ stimulates vimentin production via PI3K-Akt-mTOR signaling, which leads to suppression of ATF4-dependent Ocn transcription and osteoblast differentiation.

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Figures

FIGURE 1.
FIGURE 1.
ATF4 is required for TGFβ to inhibit osteocalcin (Ocn) transcription. A–C, TGFβ inhibits ATF4-dependent reporter activity. Luciferase activity is decreased by treatment of rhTGFβ1 at indicated concentrations in stable ROS17/2.8 reporter cells. p6xOSE1-Luc, luciferase gene driven by 6 repeats of ATF4-binding elements reporter (A); p6xmOSE1-Luc, 6 repeats of mutant ATF4-binding element driving luciferase reporter (B); p3xAP1-Luc, 3 repeats of unrelated leucine-zipper protein AP1-binding site- driven reporter (C). D, inhibition of endogenous Ocn expression by TGFβ is attenuated in Atf4−/− calvarial osteoblasts. Quantitative RT-PCR (qRT-PCR) analysis of total RNA isolated from WT and Atf4−/− calvalrial osteoblasts treated with vehicle (−) or rhTGFβ1 (+, 0.5 ng/ml) in osteogenic medium for 10 days. Note that TGFβ decreased endogenous Ocn mRNA level in WT calvarial osteoblasts, which was attenuated (48% inhibition) in Atf4−/− mutant osteoblasts. E, TGFβ reduced alkaline phosphatase (ALP) activity in WT but not in Atf4−/− osteoblasts. ALP assay of WT and Atf4−/− calvarail osteoblasts treated with vehicle (−) or rhTGFβ (+, 0.5 ng/ml) in osteogenic medium for 2 days. F, TGFβ inhibits mineralized nodule formation in WT but not in Atf4−/− osteoblasts. von Kossa staining of calvarial osteoblasts treated with rhTGFβ1 (0.5 ng/ml) in osteogenic medium for 10 days. Note that TGFβ reduced the number of mineralized nodules (black colonies) dramatically (>90%) in the WT cultures but only slightly in Atf4−/− osteoblasts (<20%). G, ATF4 is required for the anabolic effect of anti-TGFβ antibody in vivo. μCT analysis of trabecular bones of WT and Atf4−/− femurs treated with control IgG antibody or anti-TGFβ monoclonal antibody (2G7, αTGFβ) neutralizing three forms of TGFβ ligand for 4 weeks. H, quantification of data shown in G. Note that 2G7 treatment increased trabecular bone volume (BV) versus total tissue volume (BV/TV) in WT femurs by 30% but failed to rescue the low BV/TV in Atf4−/− femur. n = 6.
FIGURE 2.
FIGURE 2.
TGFβ inhibits Ocn expression and stimulates vimentin expression post-transcriptionally. A, Northern blot analysis of total RNA from primary calvarial osteoblasts showing that TGFβ inhibits endogenous Ocn expression dose-dependently. B, Western blot analysis of total protein from primary calvarial osteoblasts. C, Northern blot analysis with indicated cDNA probes showing that TGFβ does not affect endogenous vimemtin mRNA level in calvarial osteoblasts. D, Western blot analysis showing that TGFβ stimulates vimenitn protein in calvarial osteoblasts. E, Western blot analyses showing that rhTGFβ1 (0.2 ng/ml) stimulates vimentin protein expression in the indicated primary osteoblasts and osteoblastic cell lines. F, Northern blot analyses showing that rhTGFβ1 (0.2 ng/ml) does not affect endogenous vimentin mRNA level in the indicated primary osteoblasts and osteoblastic cell lines.
FIGURE 3.
FIGURE 3.
Vimentin is required for the suppression of Ocn expression by TGFβ in osteoblasts. A, transient DNA transfection in 2T3 preosteoblastic cells demonstrating that rhTGFβ (0.5 ng/ml) decreased luciferase activity (lanes 1 and 2). Note that luciferase activity increased when siRNA-Vim was co-transfected (lanes 3 and 4). N.S., not statistically significant. B, Northern blot analysis of RNA from 2T3 osteoblastic cells transfected with siRNA vector or siRNA-Vim using indicated cDNA probes. Note that knockdown of endogenous vimentin by siRNA-Vim (top panel) blunted the inhibition of Ocn transcription by TGFβ (middle panel). C, qRT-PCR results confirming that vimentin knockdown by siRNA-Vim abolished the suppression of endogenous Ocn expression by rhTGFβ (0.5 ng/ml). D, Western blot analysis of long bone total protein extracts demonstrating that neutralizing the activity of TGFβ in vivo by anti-TGFβ decreases vimentin protein abundance. 10-week-old mice were treated with control antibody (IgG) or anti-TGFβ monoclonal antibody (αTGFβ) neutralizing three forms of TGFβ ligand for 4 weeks. E, proteasomal degradation inhibitor MG115 (MG) stabilizes ATF4 but not vimentin. Western blot analysis of nuclear extracts of COS1 cells transfected with expression plasmids of HA-Vim or Flag-ATF4 using anti-HA or anti-Flag antibodies. Sp1, loading control of nuclear extracts. F, protein synthesis is required for TGFβ to induce vimentin expression. ROS17/2.8 cells were pretreated with indicated inhibitors prior to the treatment of rhTGFβ1 (0.2 ng/ml). Note that TGFβ-induced vimentin is not affected by proteasomal inhibitor, MG115 (MG), or lysosomal inhibitors, chloroquine (ChlQ, 100 mm) and ammonium chloride (AC, 50 mm), but is diminished by protein translation inhibitor cycloheximide (CHX).
FIGURE 4.
FIGURE 4.
TGFβ stimulates vimentin protein synthesis via PI3K-mTOR-Akt signaling but not Smad signaling. A, schematic presentation of inhibitors that block canonical and noncanonical signaling pathways downstream of TGFβ. B and C, Western blot of ROS17/2.8 cells indicating that wortmannin (B) and rapamycin (C) dose-dependently blunted rhTGFβ1 (0.2 ng/ml) induced vimentin and phosphorylation of downstream targets Akt or S6K. D, Western blot analysis of ROS17/2.8 cells showing that SB505124 did not inhibit rhTGFβ1 (0.2 ng/ml) to stimulate vimentin expression. Note that SB505124 effectively inhibited Smad2 phosphorylation. E, Western blot analysis showing that overexpression of dominant negative form of Akt (Akt-AA) inhibited rhTGFβ1 (0.2 ng/ml)-induced vimentin protein level in ROS17/2.8. F, ATF4-dependent activation of Ocn transcription requires PI3K-Akt-mTOR signaling. Luciferase activity in ROS17/2.8 reporter cells containing p6xOSE1-Luc was effectively (30%) inhibited by rhTGFβ (1 ng/ml) and SB505124 (0.4 μm), which was blunted by wortmannin (5 nm) or rapamycin (5 nm). *, p < 0.01.
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