Role of connexin43 in osteoblast response to physical load.

Gap junctions are hexameric transmembrane channels formed by connexins, and are responsible for direct cell-to-cell communication. The most abundant gap junction protein in bone is connexin43 (Cx43), although connexin45 (Cx45) is also expressed. In the present study, we tested the hypothesis that bone cell responses to mechanical stimulation are dependent on the type of gap junction communication provided by Cx43 in vitro and in an in vivo model of physical load. Application of cyclic stretch to calvaria osteoblasts results in a modest but detectable increase in PGE2 levels, and the amount of PGE2 produced was lower in cells isolated from Cx43 null mice. Mice with an osteoblast-specific deletion of the Cx43 gene were subjected to an in vivo four-point bending protocol on the tibia. This resulted in fast and exuberant formation of woven bone at the region directly below the loading fulcrum in both osteoblast Cx43-deleted and wild-type mice. However, indirect measurement of endosteal bone apposition suggested a less pronounced effect of physical load in Cx43-deficient than in wild-type mice. Taken together, these results indicate that deficiency of Cx43 in osteoblasts attenuates but does not abolish anabolic responses to mechanical strain.

Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters.

Loss-of-function mutations of gap junction proteins, connexins, represent a mechanism of disease in a variety of tissues. We have shown that recessive (gene deletion) or dominant (connexin45 overexpression) disruption of connexin43 function results in osteoblast dysfunction and abnormal expression of osteoblast genes, including down-regulation of osteocalcin transcription. To elucidate the molecular mechanisms of gap junction-sensitive transcriptional regulation, we systematically analyzed the rat osteocalcin promoter for sensitivity to gap junctional intercellular communication. We identified an Sp1/Sp3 containing complex that assembles on a minimal element in the -70 to -57 region of the osteocalcin promoter in a gap junction-dependent manner. This CT-rich connexin-response element is necessary and sufficient to confer gap junction sensitivity to the osteocalcin proximal promoter. Repression of osteocalcin transcription occurs as a result of displacement of the stimulatory Sp1 by the inhibitory Sp3 on the promoter when gap junctional communication is perturbed. Modulation of Sp1/Sp3 recruitment also occurs on the collagen Ialpha1 promoter and translates into gap junction-sensitive transcriptional control of collagen Ialpha1 gene expression. Thus, regulation of Sp1/Sp3 recruitment to the promoter may represent a potential general mechanism for transcriptional control of target genes by signals passing through gap junctions.