Abstract
Bone metabolism is regulated by several mechanisms triggered by the interaction among local and systemic molecules such as hormones, cytokines and lipid mediators that act in autocrine, paracrine and endocrine manners. In this context, it is known that specific inflammatory mediators produced in the bone microenvironment can modulate osteoclastogenesis and so the bone metabolism. In turn, the leukotrienes (LTs) are inflammatory mediators synthesized by both osteoblasts and aosteoclasts that signal through G protein-coupled transmembrane receptors. There are four types of receptors named BLT1, BLT2 and cysLT1, cysLT2. Leukotriene B4 (LTB4) binds to BLT1/2 receptors while the cysteinyl-leukotrienes, named LTC4, LTD4, and LTE4, binds to cysLT1/2 receptors. Generally, these receptors are coupled to G±i and G±q subunits type receptors which binding to LTs lead to inhibition of adenylate cyclase and enhancement of calcium influx, respectively. However, it is unknown the impact of these mediators produced endogenously in the induction and /or inhibition of osteogenic differentiation, as well as the intracellular signaling and expression of microRNA triggered during this response. Thus, in MC3T3-E1 cells cultivated in osteogenic medium, the measurement of LTB4 and LTD4 and the expression of the 5-lipoxygenase (5-LO), enzyme responsible for LTs synthesis, alkaline phosphatase (ALP), Runx-2 and osterix in the presence or not of LT synthesis inhibitor (compound MK 886), of BLT1/2 or cysLT1/2 receptors antagonists will be evaluated by RT-PCR. Next, the ligand-receptor interaction and intracellular signaling induced by exogenous LTB4 and LTD4, in the presence or not of MK 886, or specific receptor antagonists will be determined by quantifying cAMP and intracellular calcium assessed by immunoassays. Also, the profile of microRNA expression induced by LTB4 or LTD4 during osteogenic differentiation will be assessed. Finally, we will investigate the contribution of LTs synthesized by preosteoblasts in the in vitro osteoclastogenesis from hematopoietic stem cells using osteoblast conditioned medium.
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