Abstract: Objective To determine the effect of ostecytic TGF-β/Smad4 signaling on osteoblastic and osteoclastic differentiation in bone marrow stromal cells (BMSCs). Methods Mice with osteocytic TGF-β/Smad4 conditional knock down (Smad4ot CKD) were generated as previously by crossing DMP1-8kb-Cre mice with Smad4lox(ex8)/lox(ex8) mice. The osteocytes were isolated from tibial and femoral diaphysis and co-cultured with wild-type BMSCs. ALP staining, Alizarin red staining and TRAP staining were performed to show osteoblastic and osteoclastic differentiation. Then, their marker genes were detected by qPCR and proteins measured by Western blot. Results The expression of Runx2 and Osterix were reduced in smad4 CKDot co-cultured with BMSCs compared with controls (P<0.01). Similarly, the specific markers of osteoblastic differentiation were decreased (P<0.01). Additionally, the expression of RANKL was not obviously changed in with BMSCs. However, OPG was highly expressed in control group compared with smad4 CKD ot co-cultured group (P<0.05). Thus, the radio of RANKL/OPG was significantly reduced (P<0.05). Furthermore, the expression of RANK was inhibited. Conclusions The terminally-differentiated osteocytes are the cells regulating bone metabolism, while down-regulation of osteocytic-TGF-β/Smad4 inhibits BMSC osteoblastic and osteoclastic differentiation.

Key words: osteocyte, TGF-β/Smad4, BMSCs, osteoblastic differentiation, osteoclastic differentiation