SUMMARYObjective: Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (Na V ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on Na V channels. Methods: Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of Na V . Whole-cell patch-clamp recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 lM) or phenytoin (50 lM). Results: Na V expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current amplitude for carbamazepine (31.6 AE 5.9%, n = 9; p < 0.001), and for phenytoin (35.5 AE 6.9%, n = 7; p < 0.001). Significance: Mouse osteoblasts express Na V , and native Na V currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength.

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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 (PAR 2 KO) mice have a reduced capacity to generate osteoblasts. Here we describe the observation that PAR 2 KO osteoblastic cultures generate more adipocytes than wildtype (WT) cultures. Osteoblasts from PAR 2 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 PAR 2 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 PAR 2 was knocked down ( F2rl1 KD), were less osteoblastic and more adipocytic than vector control cells. Putative regulators of PAR 2 -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, PAR 2 appears to be a mediator of the reciprocal relationship between osteogenesis and adipogenesis, with IL-6 having a regulatory role in these PAR 2 -mediated effects.

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