Activating Transcription Factor4Regulates Osteoclast Differentiation

Bone is a dynamic organ that exhibits multiple functions that include the regulation of calcium levels, providing mechanical support to soft tissues, housing the central nervous system, and supporting hematopoiesis. These functions are accomplished by continuous tissue renewal, called bone remodeling. The osteoclast is a unique bone-resorbing cell which contains multiple nuclei, derived from committed myeloid progenitors. Their differentiation and function are regulated by a number of other cells and their products, especially by RANKL and M-CSF, which are secreted by bone marrow stromal cells and osteoblasts. Excessive osteolysis is an important clinical problem in many common lesions, including cancer metastases in bone, rheumatoid arthritis, and implanted joint prosthesis failure, as well as metabolic diseases such as Paget’s disease of bone and osteoporosis. Therefore, new therapeutic strategies are urgently needed to efficiently inhibit excessive osteolysis or promote bone formation, furthermore to prevent or alleviate osteoporosis formation and to improve the quality of patients’ life. During the past decade, many major breakthroughs have been made in understanding osteoclast differentiation; the process of osteoclast differentiation and its function have become clearer. Many factors have important roles during this process including M-CSF, RANKL, OPG and MITF etc. Activating transcription factor4(ATF4) is an important transcription factor, which is firstly found to be crucial in eye lens fiber formation through site-specific mutagenesis. Later on, more and more evidence showed that it is critical for osteoblast differentiation and bone formation. It has been reported that ATF4regulates osteoclast differentiation and ultimately bone resorption through its expression in osteoblasts and regulation of the production of RANKL. But no one shows that ATF4can directly affect osteoclast differentiation.Our hypothesis is that ATF4can directly regulate osteoclast differentiation.First, we confirmed that ATF4protein was expressed and phosphorylated in primary mouse bone marrow monocytes (BMMs). Then loss-and gain-of-function approaches were used to establish a direct role of this factor in osteoclast differentiation. In the bone of Atf4-/-mice, the percentage of TRAP positive area is drastically decreased and the signal is weaker. When cultured in vitro differentiation system, the number of TRAP positive multiple nucleated cells (MNCs)(>3nuclei) in Atf4-/-group is dramatically decreased than in WT group. Furthermore, we found the bone resorption ability is not impaired in At/4-/-osteoclast in vitro. Conversely, transgenic over-expression of ATF4driven by an OCL-specific Trap promoter dramatically increased osteoclastogenesis in vitro and in vivo, transgenic mice had a severe osteopenic phenotype, serum level of CTX, an indicator of in vivo osteoclast activity, was dramatically elevated and the expression of most of genes related to osteoclast differentiation are up-regulated both at mRNA level and protein level.What’s more, the formation of the granulocyte-macrophage colony forming units (GM-CFUs) colony is also severely compromised in At/4-/-bone marrow cell cultures. GM-CFU is the earliest identifiable hematopoietic precursor able to form osteoclasts. Coculture with wt osteoblasts or high concentration of RANKL failed to restore the OCL differentiation defect in Atf4-/-BMM cultures. As the cellular effects of RANKL are mediated by its receptor RANK, we then determined the level of RANK in ATF4WT and KO BMM by IHC and Western blot, and we found the RANK expression is dramatically reduced in the absence of ATF4, and its mRNA level couldn’t be upregulated by M-CSF. ATF4is critical for RANKL to activate multiple MAPK pathways in OCL progenitors. Lack of ATF4compromised RANKL-induced activation of MAPK pathways. In contrast, no difference was seen in RANKL activation of NF-kB and PI3K/Akt pathways. ATF4deficiency did not markedly affect M-CSF signaling in BMMs.Till now, NFATcl is the most important master regulator of osteoclast differentiation. The expression of NFATcl is dramatically reduced in the deficiency of ATF4both in vitro and in vivo. Over expression of NFATcl by retrovirus vector in wt and Atf4-/-BMMs can dose-dependently increase the number of TRAP-positive MNCs in Atf4-/-BMM cultures. Over expression of ATF4by adenovirus vector in BMMs can increase the expression of NFATcl dose-dependently. And we then found that ATF4activate the Nfatcl promoter in a dose-dependent manner in vitro; also, ATF4can interact with a chromatin fragment of the proximal Nfatcl promoter in RAW264.7osteoclast-like cells by chromatin immunoprecipitation (ChIP) assays and this interaction was greatly stimulated by RANKL.ATF4is regulated by M-CSF and PI3K/Akt pathway in BMMs. The level of ATF4protein was dramatically edueed-in the absence of M-CSF in a time-dependent manner. However, this reduction was completely blocked by M-CSF. LY294002, a specific inhibitor of the PI3K/AKT pathway, dramatically reduced total and phospho-ATF4in the presence of M-CSF. Importantly, exposure to LY294002for only24h prior to the addition of differentiation media inhibited in vitro OCL differentiation in a dose-dependent manner. The lack of ATF4causes a lineage shift between OCLs and macrophages, resulting in an increase in macrophages.Our results demonstrate that ATF4plays an intrinsic role in regulating osteoclast differentiation and may provide therapeutic target for treating osteoclast-based or involved bone diseases.