Tauroursodeoxycholic acid attenuates inorganic phosphate-induced osteoblastic differentiation and mineralization in NIH3T3 fibroblasts by inhibiting the ER stress response PERK-eIF2α-ATF4 pathway

Abstract

Ectopic ossification occurs in a wide range of common and genetic diseases, but its molecular mechanisms and effective therapeutic targets remain to be clarified. The aim of the study is to investigate whether endoplasmic reticulum (ER) stress is involved in ectopic ossification and ER stress inhibitor tauroursodeoxycholic acid (TUDCA) has potential to treat the pathological conditions. In this study, inorganic phosphate (Pi)-induced NIH3T3 fibroblasts induced osteogenesis and mineralization was used as an in vitro model for ectopic ossification. Various concentrations of TUDCA (0.1, 1, 5, 10 μM) were added during osteogenic induction. Osteoblast differentiation and minerlization were determined by RT-qPCR, alkaline phosphatase (ALP) activity assay, Alizarin Red-S (AR-S) staining, and calcium deposition. ER stress signalling components were detected by Western-blot analysis. We found ER stress was activated by inorganic phosphate in NIH3T3 cells. During osteogenic induction, TUDCA inhibited NIH3T3 cells ALP activity and mineral nodule formation. In addition, TUDCA caused decreased expression of osteoblastic markers Runx2, Col1a1, ALP, OCN. Mechanistically, TUDCA inhibited the ER stress response PERK-eIF2α-ATF4 pathway during osteogenesis. In conclusion, TUDCA could inhibit fibroblasts mineralization via supressing the ER stress response PERK-eIF2α-ATF4 pathway, and has potential pharmacologic and therapeutic applications for treating ectopic ossification associated diseases.