FHL2 mediates dexamethasone‐induced mesenchymal cell differentiation into osteoblasts by activating Wnt/β‐catenin signaling‐dependent Runx2 expression

Abstract

The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step in bone formation. However, the mechanisms involved in the early stages of osteogenic differentiation are not well understood. In this study, we identified FHL2, a member of the LIM-only subclass of the LIM protein superfamily, that is up-regulated during early osteoblast differentiation induced by dexamethasone in murine and human MSCs. Gain-of-function studies showed that FHL2 promotes the expression of the osteoblast transcription factor Runx2, alkaline phosphatase, type I collagen, as well as in vitro extracellular matrix mineralization in murine and human mesenchymal cells. Knocking down FHL2 using sh-RNA reduces basal and dexamethasone-induced osteoblast marker gene expression in MSCs. We demonstrate that FHL2 interacts with beta-catenin, a key player involved in bone formation induced by Wnt signaling. FHL2-beta-catenin interaction potentiates beta-catenin nuclear translocation and TCF/LEF transcription, resulting in increased Runx2 and alkaline phosphatase expression, which was inhibited by the Wnt inhibitor DKK1. Reduction of Runx2 transcriptional activity using a mutant Runx2 results in inhibition of FHL2-induced alkaline phosphatase expression in MSCs. These findings reveal that FHL2 acts as an endogenous activator of mesenchymal cell differentiation into osteoblasts and mediates osteogenic differentiation induced by dexamethasone in MSCs through activation of Wnt/beta-catenin signaling- dependent Runx2 expression.

Priming integrin α5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis

“…Total RNAs were isolated and relative, mRNA levels were evaluated by quantitative PCR analysis using microfluidic cards (Applied Biosystems) or LightCycler (Roche Applied Science) and normalized to 18S rRNA as described in ref. 36.…”

“…Viral production was performed as described in ref. 36. For transduction, subconfluent cells were incubated with lentivirus and 4 g/mL polybrene (Sigma) for 48 h.…”

“…Cell lysates and immunoprecipitation analysis were prepared as described in ref. 36. Densitometric analysis was performed using ImageQuant software (Agfa).…”

“…The ALP activity, protein content, ALP cytochemical detection, and in vitro matrix mineralization assays were performed as described in ref. 36.…”

Non-destructive characterisation of mesenchymal stem cell differentiation using LC-MS-based metabolite footprinting

“…4 Numerous in vitro studies have demonstrated the effect of osteoinductive treatment combining dexamethasone, ascorbic acid, and betaglycerophosphate (βGP) on MSC osteogenesis. Dexamethasone induces osteogenesis in vitro through activating WNT/ β-catenin signalling-dependent Runx2 expression, 5 whereas ascorbic acid increases collagen type 1 secretion. 6,7 βGP provides inorganic phosphate (Pi) that facilitates intracellular signalling molecules.…”