Abstract
Background Managing healing of impaired bone fracture has long been a subject of concern. Prolonged or uncontrolled inflammation exerts detrimental effects on bone healing. Tumor necrosis factor (TNF)-α is a critical inflammatory factor, whose absence impairs normal bone formation. However, TNF-α exposure for longer duration inhibits bone regeneration. In this study, we aim to find a new therapeutic target for the impaired osteogenesis induced by long exposure of TNF-α.MethodsIn vitro, mRNA microarray analysis was used to identify differentially expressed genes. Cell proliferation assay was used to assess the proliferation of cells. qPCR and Western blotting analysis were applied to detect the expression of target genes and proteins respectively. ALP staining and Alizarin Red staining (ARS) were used to evaluate ALP activity and mineral deposition respectively. Co-immunoprecipitation was used to detect the interaction of proteins. In vivo, a murie tibial fracture model was established, histological evaluation and radiographic analysis was used to confirm bone regeneration in fracture healing. statistical significance between two groups was determined by Student’s t test, one-way ANOVA or Bonferroni’s post-hoc test according to the distribution of the tested population.ResultsIn this study, we show that heat shock protein family B (small) member 7 (HSPB7) mitigates negative impact of long-term TNF-α exposure on bone formation by binding heat shock protein family H (small) member 1 (HSPH1). HSPH1, in turn, inhibits the ATPase of heat shock cognate A8 (HSCA8), one of the key components involved in chaperone-mediated autophagy (CMA). Deletion of genes encoding for either HSCA8 or lysosome-associated membrane protein 2A (LAMP2A), another component of CMA, failed to reverse the osteogenic differentiation of hBMSCs induced by TNF-α by deleting HSPH1. Moreover, LAMP2A overexpression reverse the impaired osteogenesis induced by TNF-α, and this effect was attenuated by DKK1, a specific Wnt/β-catenin signaling pathway inhibitors. Thus, a heat shock protein family network composed by HAPB7, HSPH1 and HSCA8 rescued the impairment of bone healing by TNF-α through the CMA/β-catenin pathway, making it a potential therapeutic agent for bone regeneration in cases of prolonged or severe inflammation in the clinical settings. ConclusionsTaken together, these findings indicate that a heat shock protein family network including HSPB7, HSPH1 and HSCA8 protects the impaired osteogenesis induced by TNF-α via the CMA/β-catenin pathway. And in vivo, HSPB7 overexpression lentiviral particles effectively protects the impaired fracture healing induced by TNF-α in a mouse tibia fracture healing model.
Paracrine Effects of Recombinant Human Adiponectin Promote Bone Regeneration
