Circadian transcription factor Dbp promotes rat calvarial osteoprogenitors osteogenic differentiation through Kiss1/GnRH/E2 signaling pathway loop.

Abstract

To determine the mechanism by which D-site-binding protein (Dbp) regulates rat calvarial osteoprogenitors (OPCs) osteogenic differentiation. α-Smooth muscle actin (α-SMA) + rat calvarial OPCs were extracted and purified using immunomagnetic beads. Cells were transduced with Dbp-lentivirus and divided into Dbp knockdown, Dbp overexpression and vehicle groups. After osteogenic induction for 21 days, Alizarin red staining and alkaline phosphatase (ALP) activity were examined. Expression levels of Runx2, Ocn, Osterix, Bmp4, Kiss1, and GnRH were determined using a quantitative real-time polymerase chain reaction. The observed changes in Kisspeptin, GnRH, ERα, and Runx2 were further validated via Western blot analysis. Furthermore, E2 and GnRH secretion levels were detected via an enzyme-linked immunosorbent assay (ELISA). Chromatin immunoprecipitation (ChIP) and luciferase assay were used to assess the effects of Dbp on the Kiss1 gene promoter. The coexpression of Dbp and Kisspeptin or GnRH was also evaluated via immunofluorescence. Following osteogenic induction, Dbp overexpression significantly increased calcium nodule formation and ALP activity, as well as Runx2, Ocn, Osterix, Bmp4, Kiss1, and GnRH messenger RNA expression, while Dbp knockdown presented the opposite results. Western blot analysis and ELISA results showed that Dbp significantly promotes Runx2, E2/ERα, Kisspeptin, and GnRH expression. These findings were confirmed by the ChIP assay, which indicated that the estrogen receptor promotes Kisspeptin expression after binding to the Kiss1 gene promoter, which is regulated by Dbp. Immunofluorescence assay showed that Dbp coexpression with Kisspeptin or GnRH varied depending on Dbp expression levels. Collectively, the circadian transcription factor Dbp promotes α-SMA + rat calvarial OPCs osteoblastic differentiation through Kiss1/GnRH/E2 signaling pathway loop.