Perfluorooctanoic acid and perfluorooctane sulfonate inhibit in vitro osteogenesis: possible role of connexin 43-mediated gap-junctional intercellular communication.

In the current study, we investigated the effects of two legacy per- and polyfluoroalkyl substances (PFASs) namely perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) on osteogenesis. The alterations of connexin 43 (Cx43)-mediated gap junctions (GJs) were further explored as a potential mechanism. The two cell models (C3H10T1/2 and MC3T3-E1 cells) differentiated into osteoblasts (OBs) were utilized, and treated with PFOA and PFOS at the doses of 0.25, 2.5, 25, and 75 μM. Real-time PCR and Western blot were applied to assess the mRNA and protein expression of osteogenic-specific markers and Cx43. ALP staining and ARS staining were used to evaluate the osteogenesis process. The scrape-loading dye transfer assay was performed to assess the GJ-mediated intercellular coupling. To investigate the role of gap-junctional intercellular communication (GJIC) in the PFAS-induced osteogenic inhibition, the Cx43-specific GJIC enhancer, rotigaptide (ZP123), was added into the differentiation medium of C3H10T1/2 cells. After the exposure of PFOA and PFOS, the osteogenic molecules were down-regulated and the calcium deposition was reduced in the two cell models, indicating the inhibitory effects of the legacy PFASs. The Cx43 expression and GJIC activity were significantly suppressed, and the usage of ZP123 rescued the adverse impact on osteogenesis, suggesting the remarkable role of GJIC herein.