Abstract
The imbalance between osteogenic and adipogenic differentiation of Bone marrow-derived mesenchymal stem cells (BMSCs) is involved in the occurrence and development of osteoporosis (OP). Previous studies have indicated the potential of phosphatase and actin regulator 1 (Phactr1) in regulating osteogenic and adipogenic differentiation of BMSCs. The present study aims to investigate the function and mechanism of Phactr1 in regulating osteogenic and adipogenic differentiation of BMSCs. Herein, the expression of Phactr1 in bone and adipose tissue of OP rats was determined by immunohistochemical. BMSCs were subjected to osteogenic and adipogenic differentiation, and transfected with Phactr1 overexpression lentivirus, small interference RNA (siRNA) and KD025 (selective ROCK2 inhibitor). The relationship between Phactr1 and ROCK2 was detected by Co-IP experiment. The expression of Phactr1, Runx2, C/EBPα, RhoA and ROCK2 was detected by Western blot. Calcium nodule and lipid droplets were determined by alizarin red and Oil red O staining. Interestingly, Phactr1 increased in both bone and adipose tissue of OP rats. During osteogenic differentiation, Phactr1 decreased and active RhoA, ROCK2 increased, while overexpression Phactr1 inhibits the increase of Runx2. Phactr1 increased and active RhoA decreased, ROCK2 did not changed during adipogenic differentiation. While, Knockdown Phactr1 inhibits the increase of C/EBPα. Phactr1 and ROCK2 were combined in osteogenic differentiation, but not in adipogenic differentiation. By using KD025, the decrease of Phactr1 and increase of Runx2 were inhibited respectively in osteogenic differentiation. Meanwhile, when ROCK2 was inhibited, Phactr1, C/EBPα were significantly increased in adipogenic differentiation. These findings indicated that Phactr1 negatively regulates bone mass by inhibiting osteogenesis and promoting adipogenesis of BMSCs by activating RhoA/ROCK2.