Melatonin promotes the proliferation and differentiation of antler chondrocytes via RUNX2 dependent on the interaction between NOTCH1 and SHH signaling pathways

Abstract

Melatonin (MT), an endogenous hormone secreted by pineal gland, has the sedative, anti-inflammatory and antioxidant functions. However, there are few studies on whether MT affects the proliferation and differentiation of antler chondrocytes. The present study investigated the influences of MT on the proliferation and differentiation of antler chondrocytes, explored its regulation on runt-related transcription factor 2 (RUNX2), NOTCH1 and sonic hedgehog (SHH) signaling, and elucidated their interplays. The results showed that MT promoted the proliferation of antler chondrocytes and induced the differentiation of chondrocytes into hypertrophic chondrocytes as evidenced by the significant increase of collagen type X (COL X), alkaline phosphatase (ALP) and matrix metalloproteinase 13 (MMP13) expression and ALP activity, the well-established markers for hypertrophic chondrocytes, but this effectiveness was neutralized by the addition of MT receptor antagonist. Further analysis indicated that MT activated the NOTCH1 and SHH signaling whose blockage abrogated the inducement of MT on the proliferation and differentiation of antler chondrocytes. SHH was identified as a downstream target of recombination signal binding protein for immunoglobulin kappa J region (RBPJ), a transcription factor of NOTCH1 signaling. Meanwhile, MT stimulated the expression of RUNX2 through activating the SHH signaling whose downstream transcription factor glioma-associated oncogene 1 (GLI1) directly controlled the transcription of RUNX2 through binding to its promoter region. Moreover, repression of GLI1 counteracted the proliferative effect of MT on antler chondrocytes and attenuated the advancement of MT on chondrocyte differentiation, while supplementation of recombinant RUNX2 protein recued above effects. Collectively, MT induced the proliferation and differentiation of antler chondrocytes via RUNX2 dependent on the interaction between NOTCH1 and SHH signaling pathways.

Keywords: NOTCH1 and SHH signaling; RUNX2; antler chondrocytes; differentiation; melatonin; proliferation.