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

Abstract

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 (<i>Runx2</i>, <i>Col1a1</i> and <i>Bglap</i>), and higher levels of the adipocytic gene <i>Pparg</i> 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 (<i>F2rl1</i> 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 <i>C1qtnf3</i>, <i>Gpr35</i>, <i>Grem1</i>, <i>Snorc</i> and <i>Tcea3</i>, which were more highly expressed, and <i>Cnr1</i>, <i>Enpep</i>, <i>Hmgn5</i>, <i>Il6</i> and <i>Ramp3</i> which were expressed at lower levels, in control than in <i>F2rl1</i> KD cells. Interleukin-6 (IL-6) levels were higher in medium harvested from <i>F2rl1</i> KD cells than from control cells, and a neutralising anti-IL-6 antibody reduced the number of adipocytes in <i>F2rl1</i> 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.