L-carnitine affects osteoblast differentiation in NIH3T3 fibroblasts by the IGF-1/PI3K/Akt signalling pathway.

Fibroblasts in soft tissues are one of the progenitors of ectopic calcification. Our previous experiment found that the serum concentrations of small metabolite L-carnitine (LC) decreased in an ectopic calcification animal model, indicating LC is a potential calcification or mineralization inhibitor. In this study, we investigated the effect of LC on NIH3T3 fibroblast osteoblast differentiation, and explored its possible molecular mechanisms. Two concentrations of LC (10 µM and 100 µM) were added in Pi-induced NIH3T3 fibroblasts, cell proliferation was compared by MTT assays, osteoblast differentiation was evaluated by ALP activity, mineralized nodules formation, calcium deposition, and expressions of the osteogenic marker genes. Our results indicated that 10 µM LC increased the proliferation of NIH3T3 cells, but 100 µM LC slightly inhibited cell proliferation. 100 µM LC inhibits NIH3T3 differentiation as evidenced by decreases in ALP activity, mineralized nodule formation, calcium deposition, and down-regulation of the osteogenic marker genes ALP, Runx2 and OCN, meanwhile 10 µM of LC exerts an opposite effect that promotes NIH3T3 osteogenesis. Mechanistically, 100 µM LC significantly inhibits IGF-1/PI3K/Akt signalling, while 10 µM LC slightly activates this pathway. Our study suggests that a decease in LC level might contribute to the development of ectopic calcification in fibroblasts by affecting IGF-1/PI3K/Akt, and addition of LC may benefit patients with ectopic calcification.

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

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.