Conditional inactivation of the CXCR4 receptor in osteoprecursors reduces postnatal bone formation due to impaired osteoblast development

Cysteine (C)-X-C motif chemokine receptor 4 (CXCR4), the primary receptor for stromal cell-derived factor-1 (SDF-1), is involved in bone morphogenic protein 2 (BMP2)-induced osteogenic differentiation of mesenchymal progenitors. To target the in vivo function of CXCR4 in bone and explore the underlying mechanisms, we conditionally inactivated CXCR4 in osteoprecursors by crossing osterix (Osx)-Cre mice with floxed CXCR4 (CXCR4(fl/fl)) mice to generate knock-outs with CXCR4 deletion driven by the Osx promoter (Osx::CXCR4(fl/fl)). The Cre-mediated excision of CXCR4 occurred exclusively in bone of Osx::CXCR4(fl/fl) mice. When compared with littermate controls, Osx::CXCR4(fl/fl) mice developed smaller osteopenic skeletons as evidenced by reduced trabecular and cortical bone mass, lower bone mineral density, and a slower mineral apposition rate. In addition, Osx::CXCR4(fl/fl) mice displayed chondrocyte disorganization in the epiphyseal growth plate associated with decreased proliferation and collagen matrix syntheses. Moreover, mature osteoblast-related expression of type I collagen α1 and osteocalcin was reduced in bone of Osx::CXCR4(fl/fl) mice versus controls, suggesting that CXCR4 deficiency results in arrested osteoblast progression. Primary cultures for osteoblastic cells derived from Osx::CXCR4(fl/fl) mice also showed decreased proliferation and impaired osteoblast differentiation in response to BMP2 or BMP6 stimulation, and suppressed activation of intracellular BMP receptor-regulated Smads (R-Smads) and Erk1/2 was identified in CXCR4-deficient cells and bone tissues. These findings provide the first in vivo evidence that CXCR4 functions in postnatal bone development by regulating osteoblast development in cooperation with BMP signaling. Thus, CXCR4 acts as an endogenous signaling component necessary for bone formation.     

Identification of C‐terminal Hsp70‐interacting protein as a mediator of tumour necrosis factor action in osteoblast differentiation by targeting osterix for degradation

In patients with inflammatory arthritis, tumour necrosis factor (TNF)‐α are overproduced in inflamed joints. This leads to local erosion of cartilage and bone, periarticular osteopenia, as well as osteoporosis. But less is known regarding the molecular mechanisms that mediate the effect of TNF‐α on osteoblast function. The purpose of this study was to test that C terminus of Hsc70‐interacting protein (CHIP) has a specific role in suppressing the osteogenic activity of osteoblasts under inflammatory conditions. C2C12, MC3T3‐E1 and HEK293T cell lines were cultured and cotransfected with related plasmids. After transfection, the cells were cultured further in the presence or absence of murine TNF‐α and subjected to real time RT‐PCR, Western blot, Ubiquitination assay, Co‐immunoprecipitation, Luciferase reporter assay, Small interfering RNAs and Mineralization assay. The expression levels of TNF‐α‐induced CHIP and Osx were examined by RT‐PCR and Western blot analysis. Co‐immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that CHIP indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering RNAs examined whether TNF‐α target the bone morphogenetic protein signalling through CHIP. We established stable cell lines with the overexpression of HA‐CHIP, Mineralization assay and CHIP siRNA demonstrated the important roles of CHIP on osteoblast function in conditions in which TNF‐α is overexpressed. We found that the K55 and K386 residues are ubiquitination site(s) in Osx, and that TNF‐α inhibits osteoblast differentiation by promoting Osx degradation through up‐regulation of E3 ubiquitin ligase CHIP in osteoblast. Thus, CHIP targets Osx for ubiquitination and degradation in osteoblasts after chronic exposure to TNF‐α, and inhibition of CHIP expression in osteoblasts may be a new mechanism to limit inflammation‐mediated osteoporosis by promoting their differentiation into osteoblasts.     

转录因子Osterix对成骨细胞Col1a1基因的反式激活作用

Osterix（Osx）是一种具有锌指结构的转录因子,对骨形成十分重要. 但到目前为止,直接接受Osterix调控的靶基因尚不清楚.用骨形态发生蛋白2（bone morphogenetic protein 2,BMP2）诱导原代培养小鼠成骨细胞的骨分化,定量RT PCR检测Ⅰ型胶原蛋白(collagen Ⅰ a 1, Col1a1)与Osx的转录水平.结果发现,二者的转录时相具有相同的变化模式.为了确定二者之间的关系,采用腺病毒表达系统在原代成骨细胞中过表达Osx. 数据表明,Osx能够明显上调Col1a1的转录水平.用EMSA（electromobility shift assay）检测这些过表达Osx基因的成骨细胞核抽提物,迁移条带的出现表明,Osx能够直接与Col1a1的启动子相结合.结果提示,在原代培养的成骨细胞中,Osterix通过直接与启动子相结合,调控Col1a1基因的转录.