Protease-activated receptor-2 promotes osteogenesis in skeletal mesenchymal stem cells at the expense of adipogenesis: Involvement of interleukin-6

Bone marrow mesenchymal stem cells (MSCs) give rise to osteoblasts and adipocytes, with an inverse relationship between the two. The MSCs from protease-activated receptor-2 knockout (PAR₂ KO) mice have a reduced capacity to generate osteoblasts. Here we describe the observation that PAR₂ KO osteoblastic cultures generate more adipocytes than wildtype (WT) cultures. Osteoblasts from PAR₂ KO mice expressed lower levels of osteoblastic genes (Runx2, Col1a1 and Bglap), and higher levels of the adipocytic gene Pparg than WT osteoblasts. Bone marrow stromal cells from PAR₂ KO mice generated fewer osteoblastic colonies (assessed by staining for alkaline phosphatase activity and mineral deposition) and more adipocytic (Oil Red-O positive) colonies than cultures from WT mice. Similarly, cultures of the bone marrow stromal cell line (Kusa 4b10) in which PAR₂ was knocked down (F2rl1 KD), were less osteoblastic and more adipocytic than vector control cells. Putative regulators of PAR₂-mediated osteogenesis and suppression of adipogenesis were identified in an RNA-sequencing (RNA-seq) investigation; these include C1qtnf3, Gpr35, Grem1, Snorc and Tcea3, which were more highly expressed, and Cnr1, Enpep, Hmgn5, Il6 and Ramp3 which were expressed at lower levels, in control than in F2rl1 KD cells. Interleukin-6 (IL-6) levels were higher in medium harvested from F2rl1 KD cells than from control cells, and a neutralising anti-IL-6 antibody reduced the number of adipocytes in F2rl1 KD cultures to that of control cultures. Thus, PAR₂ appears to be a mediator of the reciprocal relationship between osteogenesis and adipogenesis, with IL-6 having a regulatory role in these PAR₂-mediated effects.