YTHDC1 inhibits osteoclast differentiation to alleviate osteoporosis by enhancing PTPN6 mRNA stability in an m6A-HUR dependent manner.

Abstract

YTH domain containing 1 (YTHDC1) has been confirmed to mediate osteoporosis (OP) progression by regulating osteogenic differentiation. However, whether YTHDC1 mediates osteoclast differentiation and its molecular mechanism remain unclear. Quantitative real-time PCR and western blot analysis were performed to detect the levels of YTHDC1, protein tyrosine phosphatase non-receptor type 6 (PTPN6), nuclear factor of activated T cells 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), runt-related transcription factor 2 (RUNX2), alkalinephosphatase (ALP) human antigen R (HUR). YTHDC1 knockout (KO) mice was constructed by CRISPR/Cas9 system, and OP mice model was established by ovariectomy (OVX). Hematoxylin and eosin (H&E) staining and micro-CT were used to evaluate bone formation and bone mass. Mouse primary bone marrow macrophages cells (BMMs) were isolated and induced into osteoclasts. TRAP positive cells were detected using TRAP staining. MeRIP-qPCR, RIP-qPCR assay, RNA affinity isolation assay and Co-IP assay were used to confirm the interactions among YTHDC1, PTPN6 and HUR. YTHDC1 expression was reduced and positively correlated with lumbar bone mineral density (BMD) in OP patients. In OVX model of YTHDC1-KO mice, bone formation was reduced, bone histomorphology was changed, and osteoclastic-related factors (NFATc1 and TRAP) levels were enhanced. Overexpression YTHDC1 inhibited osteoclast differentiation. YTHDC1 increased PTPN6 mRNA stability in an m6A dependent manner. Moreover, YTHDC1 interacted with HUR to positively regulate PTPN6 expression. PTPN6 knockdown promoted osteoclast differentiation, and this effect was reversed by overexpressing HUR or YTHDC1. YTHDC1 was involved in regulating OP progression through inhibiting osteoclast differentiation by enhancing PTPN6 mRNA stability in an m6A-HUR dependent manner.