Grant: Discovery Grants Program - Individual

Bones fulfill three important functions: mechanical (protection and locomotion), metabolic (reservoir of calcium) and hematopoietic (niches for stem cells). Bone integrity is preserved by the balance between bone degradation mediated by osteoclasts and bone formation achieved by osteoblasts (OBs). The role for OBs in the maintenance of bones requires tuned regulation of their proliferation, migration, differentiation, secretory functions for matrix deposition and mineralization. Given the high incidence of bone diseases in the elderly population, there is a need for improved understanding of the regulation of bone metabolism. The long-term goal of my research program is to establish the contribution for ion channels of the transient receptor potential (TRP) family in OB physiology, through modulation of calcium influx and signaling as second messenger. Over the present funding term we established an expression inventory of TRP channels in OBs, focussing on the major subfamilies TRP Canonical (TRPC), TRP Melastatin (TRPM) and TRP Vanilloid (TRPV). We demonstrated that TRPM7 is critical for proliferation, migration and differentiation of OBs. We also evidenced that TRPM7 gene expression is enhanced during OB differentiation and others reported upregulation of TRPV4 expression with OB differentiation. We recently found that bone anabolic factors promote TRPV4-mediated calcium influx in OBs. SHORT-TERM OBJECTIVES: For the current application, we intend to focus on TRPM7 and TRPV4, and address 3 specific objectives to unravel the roles of these channels in OB function. 1) Study in vitro the mechanism responsible for TRPV4 activation by bone anabolic factors: We propose to detail the mechanisms by which the anabolic factors PTH and Wnt5a support TRPV4 activation, by focusing on the cAMP-dependent and tyrosine-dependent channel phosphorylation. Activation mechanisms will be investigated with selective activators/blockers, by detection of TRPV4 phosphorylation and molecular biology through targeted mutation. These approaches will be combined with measurements of intracellular calcium and functional assays, all techniques that are well established in the laboratory. 2) Determine the mechanism responsible for enhanced expression of TRPM7 and TRPV4 in OB differentiation: The enhanced TRPM7 and TRPV4 gene expressions during OB differentiation suggest sustained contribution for these channels in OB functions. We will investigate how up-regulation of TRPM7 and TRPV4 expression occurs, by evaluating the transcription rate of TRPM7 and TRPV4 genes during OB differentiation, and by determining the contribution of miRNA for the regulation of TRPV4 and TRPM7 expression. 3) Determine the roles of TRPM7 in bone metabolism in vivo: Since our in vitro data suggest that TRPM7 regulates OB proliferation, migration and differentiation, we will document in bone tissue the impact of Trpm7 gene inactivation in OBs. For this, tamoxifen-inducible transgenic mice (Cre-Col1a1/ERT2) will be crossed with the Trpm7tm1clph mouse line, which contains loxP sites flanking the Trpm7 exon 17. Deletion of TRPM7 specifically in OBs will be induced in the mutant mouse line with administration of tamoxifen at different time of bone development. Analysis of the bone status of tamoxifen-treated mice will allow identifying in vivo the input for TRPM7 in OB functions. EXPECTED IMPACT: Given the gradual loss of bone mass with aging, a main challenge in the bone field is to increase knowledge on the bone-forming process. Intracellular calcium levels constitute an underestimated limiting step in various signaling pathways and this research will allow identifying regulatory roles for transient receptor potential channels in osteoblast physiology.