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

Abstract

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.

Recent Advances of Osterix Transcription Factor in Osteoblast Differentiation and Bone Formation

“…CHIP, another novel post-translational regulator of Osx, plays an important role in the regulation of Osx in protein levels in osteoblast precursor cells upon treatment with tumor necrosis factor-α (TNF-α). Unlike the previous description, CHIP mediates the inhibition of Osx by TNF-α in K55 and K386 ubiquitination sites of Osx ( Xie and Gu, 2015 ).…”

Inhibition of PTGS1 promotes osteogenic differentiation of adipose-derived stem cells by suppressing NF-kB signaling

“…We found that PTGS1 expression is highly induced in response to TNFα treatment and that the inflammatory cytokine could inhibit osteogenesis and bone formation by downregulating two core transcription factors for osteogenic differentiation: RUNX2 and OSX [24, 25]. TNFα is the key stimulating factor for activating the classical NF-κB pathway; for this reason, we hypothesized that targeting the NF-κB pathway may be a novel mechanism through which PTGS1 regulates the osteogenic differentiation of MSCs.…”

Immunoporosis: Role of Innate Immune Cells in Osteoporosis

“…TNF-α acts as anti-osteogenic by inhibiting differentiation, proliferation, and activities of osteoblast. It upregulates CHIP-ubiquitin ligase protein, which results in the degradation of osterix (pro-osteoblastic transcription factor) ( 48 ). TNF-α also inhibits expression of BMP-induced ‘special AT-rich sequence binding protein 2’ (SATB2), which is another pro-osteoblastic transcription factor, by triggering NF-κB binding to SATB2 promoter ( 49 ).…”