Stepwise control of osteogenic differentiation by 5-HT2B receptor signaling:: Nitric oxide production and phospholipase A2 activation

During development, antagonists of 5-HT2 receptor subtypes cause morphological defects of mesodermal and neural crest derivatives including the craniofacial skeleton. We used an inducible mesoblastic cell line, Cl, able to fully convert into osteocytes within 12 days, to assess the involvement of 5-HT2 receptors during osteogenic differentiation. On day 5 of the osteogenic program, immediately before matrix mineralization, the cells selectively implement 5-HT2B receptors (5-HT2BR) which remain functional until terminal differentiation. In 5-HT-depleted medium, the receptor exhibits a constitutive activity leading to basal nitric oxide (NO) release and phospholipase A2 (PLA2)dependent arachidonic acid (AA) production. Blockade of this intrinsic activity affects the efficiency of mineralization by decreasing calcium incorporation within the matrix by 40%. Optimal bone matrix mineralization involves both NO and PLA2 signaling pathways. Moreover, between day 5 and day 10, at the beginning of mineral deposition, the 5-HT2BR promotes prostaglandin E2 production through AA-dependent cyclooxygenase (COX) activation. From day 10 onwards, when Cl osteoblasts undergo conversion into osteocyte-like cells, COX activity is quenched. Altogether these observations indicate that the 5-HT2BR contributes in an autocrine manner to osteogenic differentiation and highlight a switch in the downstream targets of the receptor at the terminal stage of the program. Finally, in addition to its autocrine function, the 5-HT2BR responds to 5-HT by increasing NO production and AA release. These findings raise concern regarding the use of 5HT(2B)R-related drugs that may interfere with bone metabolism in physiological or pathological situations. (c) 2005 Elsevier Inc. All rights reserved.