| Abstract |
Bone forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Gap junctional communication can be regulated by a variety of physical (mechanical) and chemical factors. We studied whether the application of mechanical stretch (using the Flexercell Strain Unit) or the presence of prostaglandin E2 (PGE2) and parathyroid hormone (PTH) modulates intercellular communication through gap junctions. Gap junctional communication was assessed using a new developed method, the parachute assay, which allows monitoring of the dye diffusion (i.e., dye coupling) without disruption of the plasma membrane. We also examined whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype (i.e., the transcriptional activity of certain, osteoblast-specific, genes). We found that both physical (cyclic stretch) and chemical (PTH, PGE2) factors enhance gap junctional communication in osteoblastic cell cultures through an early and rapid redistribution of pre-formed Cx43 from the cytoplasm to the cell membrane and formation of functional gap junctions. Although, there was no change in the transcriptional activity of the Cx43 gene after prolonged mechanical stimulus, we did observe a clear and significant stimulation of Cx43 gene expression and new Cx43 protein synthesis after longer incubation of osteoblastic cells with the aforementioned chemical agents. Finally, we demonstrated that two different connexins, Cx43 and Cx45, modulate the expression of specific osteoblastic gene products by regulating the transcriptional activity of their promoters in a reciprocal manner.
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