While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.

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Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4 -/-bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4 -/-BMMs with WT OBLs or a high concentration of RANKL failed to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity.

Parathyroid hormone (PTH) is a potent anabolic agent for the treatment of osteoporosis. However, its mechanism of action in osteoblast and bone is not well understood. In this study, we show that the anabolic actions of PTH in bone are severely impaired in both growing and adult ovariectomized mice lacking bone-related activating transcription factor 4 (ATF4). Our study demonstrates that ATF4 deficiency suppresses PTH-stimulated osteoblast proliferation and survival and abolishes PTH-induced osteoblast differentiation, which, together, compromise the anabolic response. We further demonstrate that the PTH-dependent increase in osteoblast differentiation is correlated with ATF4-dependent up-regulation of Osterix. This regulation involves interactions of ATF4 with a specific enhancer sequence in the Osterix promoter. Furthermore, actions of PTH on Osterix require this same element and are associated with increased binding of ATF4 to chromatin. Taken together these experiments establish a fundamental role for ATF4 in the anabolic actions of PTH on the skeleton.

Background: Hypertriglyceridemia is the most common lipid disorder with incompletely understood mechanisms. Results: ATF4 deficiency attenuates lipogenesis in the liver and protects against high fructose-induced hypertriglyceridemia in mice. Conclusion: ATF4 plays a pivotal role in regulating hepatic lipid metabolism. Significance: ATF4 is a contributing factor for the pathogenesis of hypertriglyceridemia.

PTH is an important peptide hormone regulator of calcium homeostasis and osteoblast function. However, its mechanism of action in osteoblasts is poorly understood. Our previous study demonstrated that PTH activates mouse osteocalcin (Ocn) gene 2 promoter through the osteoblast-specific element 1 site, a recently identified activating transcription factor-4 (ATF4) -binding element. In the present study, we examined effects of PTH on ATF4 expression and activity as well as the requirement for ATF4 in the regulation of Ocn by PTH. Results show that PTH elevated levels of ATF4 mRNA and protein in a dose- and time-dependent manner. This PTH regulation requires transcriptional activity but not de novo protein synthesis. PTH also increased binding of nuclear extracts to osteoblast-specific element 1 DNA. PTH stimulated ATF4-dependent transcriptional activity mainly through protein kinase A with a lesser requirement for protein kinase C and MAPK/ERK pathways. Lastly, PTH stimulation of Ocn expression was lost by small interfering RNA down-regulation of ATF4 in MC-4 cells and Atf4(-/-) bone marrow stromal cells. Collectively, these studies for the first time demonstrate that PTH increases ATF4 expression and activity and that ATF4 is required for PTH induction of Ocn expression in osteoblasts.

In this study, we determined the molecular mechanisms whereby forkhead transcription factor Foxo1, a key downstream signaling molecule of insulin-like growth factor 1 (IGF1)/insulin actions, regulates Runx2 activity and expression of the mouse osteocalcin gene 2 (Bglap2) in osteoblasts in vitro. We showed that Foxo1 inhibited Runx2-dependent transcriptional activity and osteocalcin mRNA expression and Bglap2 promoter activity in MC-4 preosteoblasts. Co-immunoprecipitation assay showed that Foxo1 physically interacted with Runx2 via its C-terminal region in osteoblasts or when co-expressed in COS-7 cells. Electrophoretic mobility shift assay demonstrated that Foxo1 suppressed Runx2 binding to its cognate site within the Bglap2 promoter. IGF1 and insulin prevented Foxo1 from inhibiting Runx2 activity by promoting Foxo1 phosphorylation and nuclear exclusion. In contrast, a neutralizing anti-IGF1 antibody decreased Runx2 activity and osteocalcin expression in osteoblasts. Chromatin immunoprecipitation assay revealed that IGF1 increased Runx2 interaction with a chromatin fragment of the proximal Bglap2 promoter in a PI3K/ AKT-dependent manner. Conversely, knockdown of Foxo1 increased Runx2 interaction with the promoter. This study establishes that Foxo1 is a novel negative regulator of osteoblast-specific transcription factor Runx2 and modulates IGF1/insulin-dependent regulation of osteocalcin expression in osteoblasts.Foxo1 is a forkhead transcription factor that is defined by its amino forkhead DNA binding domain and carboxyl trans-activation domain. Foxo1 plays a pivotal role in mediating the effect of insulin or insulin-like growth factor 1 (IGF1) 2 on the expression of genes involved in cell growth, differentiation, metabolism, and longevity (1-5). Insulin or IGF1 exerts its inhibitory effect on gene expression via a highly conserved sequence (TG/ ATTTT/G), termed the insulin response element (IRE), in the promoter of genes that are negatively regulated by insulin/ IGF1. In the absence of insulin/IGF1, Foxo1 resides in the nucleus and binds as a trans-activator to the IRE, enhancing promoter activity. In response to insulin, Foxo1 is phosphorylated at three highly conserved phosphorylation sites (Thr-24, Ser-256, and Ser-319) through the PI3K-dependent pathway, resulting in its nuclear exclusion and inhibition of target gene expression (1, 2, 6). Except for Foxo6 (7), all members of the Foxo superfamily undergo insulin/IGF1-dependent phosphorylation and nuclear exclusion. Failure to phosphorylate Foxo1 results in its permanent nuclear localization and constitutive trans-activation of gene expression. This phosphorylation-dependent Foxo1 translocation has been viewed as an acute mechanism for insulin or growth factors to inhibit gene expression, as insulin-induced Foxo1 phosphorylation is kinetically coupled to its subsequent translocation to the cytoplasm (1, 8). Osteoblasts, the bone-forming cells, originate from multipotential mesenchymal cells. Osteoblast activity and function are regulated by a number of gro...

Background: Bone remodeling must be precisely controlled to maintain a healthy bone mass. Results: Knock-out of FBLP-1/migfilin causes a severe osteopenic phenotype in mice. Conclusion: FBLP-1/migfilin regulates bone remodeling through modulating both osteoblast behavior and osteoclast differentiation. Significance: Identifying molecules that regulate bone remodeling is critical for understanding the pathogenesis of bone diseases and developing novel therapeutic approaches.