Lysyl oxidase inhibits BMP9-induced osteoblastic differentiation through reducing Wnt/β-catenin via HIF-1a repression in 3T3-L1 cells

BackgroundBone morphogenetic protein 9 (BMP9) is a promising growth factor in bone tissue engineering, while the detailed molecular mechanism underlying BMP9-oriented osteogenesis remains unclear. In this study, we investigated the effect of lysyl oxidase (Lox) on the BMP9 osteogenic potential via in vivo and in vitro experiments, as well as the underlying mechanism.MethodsPCR assay, western blot analysis, histochemical staining, and immunofluorescence assay were used to quantify the osteogenic markers level, as well as the possible mechanism. The mouse ectopic osteogenesis assay was used to assess the impact of Lox on BMP9-induced bone formation.ResultsOur findings suggested that Lox was obviously upregulated by BMP9 in 3T3-L1 cells. BMP9-induced Runx2, OPN, and mineralization were all enhanced by Lox inhibition or knockdown, while Lox overexpression reduced their expression. Additionally, the BMP9-induced adipogenic makers were repressed by Lox inhibition. Inhibition of Lox resulted in an increase in c-Myc mRNA and beta-catenin protein levels. However, the increase in BMP9-induced osteoblastic biomarkers caused by Lox inhibition was obviously reduced when beta-catenin knockdown. BMP9 upregulated HIF-1 alpha expression, which was further enhanced by Lox inhibition or knockdown, but reversed by Lox overexpression. Lox knockdown or HIF-1 alpha overexpression increased BMP9-induced bone formation, although the enhancement caused by Lox knockdown was largely diminished when HIF-1 alpha was knocked down. Lox inhibition increased beta-catenin levels and decreased SOST levels, which were almost reversed by HIF-1 alpha knockdown.ConclusionLox may reduce the BMP9 osteoblastic potential by inhibiting Wnt/beta-catenin signaling via repressing the expression HIF-1 alpha partially.