The role of Cx43 in mammary epithelial cell and osteoblast differentiation and disease

Gap junctions mediate direct intercellular communication and are essential to the development, differentiation and homeostasis of many tissues. The fundamental subunits of gap junctions are connexin (Cx) proteins. There are 21 human connexins that each form channels with unique selectivity, permeability, gating and regulation. It is also now recognized that connexins have intrinsic functions that extend beyond gap junctional intercellular communication (GJIC). We sought to elucidate mechanisms by which Cx43 regulates cell differentiation and disease progression using breast cancer and bone as model tissues. We over-expressed Cx26 or Cx43 in GJIC-deficient MDA-MB-231 human breast cancer cells that aberrantly localize these connexins to intracellular compartments. Nevertheless, the cells were able to form partially differentiated organoids in 3-dimensional culture whereas control cells remained incapable of differentiation. We determined that the tumor suppressive mechanisms of Cx26 and Cx43 included promotion of an epithelial versus mesenchymal phenotype and an inhibition of angiogenesis in vitro and in vivo. Importantly, these properties were achieved independent of notable increases in GJIC but rather by the regulation of important molecules by connexin expression. Moreover, connexin expression in MDA-MB-231 cells had little effect on their behavior in an osteoblast-influenced microenvironment and did not affect the growth or differentiation of the MC3T3-E1 osteoblast cell line. To examine the role of Cx43 in bone differentiation and disease, we expressed dominant-negative Cx43 mutants linked to the human disease oculodentodigital dysplasia (ODDD) in GJIC-competent osteoblasts and examined their differentiation potential in comparison to the differentiation of osteoblasts from the Gja1Jrt /+ mouse model of ODDD harboring a germline Cx43 mutation. We found that when Cx43 channel function was disrupted at the onset of development, late stage osteoblast differentiation was impaired; however, introduction of the mutant to the cells after they were committed to the osteoblast lineage had minimal effect. Collectively, these studies determined that Cx43 is critical to proper mammary epithelial cell and osteoblast differentiation and our data also supports a role for both GJIC-dependent and -independent mechanisms.;Keywords. Connexin, Cx43, Cx26, differentiation, breast cancer, osteoblast, oculodentodigital dysplasia, gap junctional intercellular communication.