TPA induced expression and function of human connexin 26 by post-translational mechanisms in stably transfected...

Connexin 26 (Cx26) has been proposed to be a tumor suppressor gene and its expression may modulate development, cell growth and differentiation in various tissues, including the brain. 12-O-tetradecanoylphorbol-13-acetate (TPA) may serve as either tumor promoter (in mammary gland amd skin) or as a differentiating agent (in neuroblastoma and leukemic cells) and may also modulate expression, function and phosphorylation of gap junctions. In this study, to determine the effects of TPA on Cx26 expression and its function in neuroblastoma, we transfected N2A mouse neuroblastoma cells (which are gap junction deficient) with the coding region of human Cx26 gene (which lacks TPA response elements) and examined the changes of expression and function of Cx26 following 10 nM TPA treatment. Individual clones of transfectants stably expressed distinct levels of exogenous Cx26 as judged by Northern and Western blots, immunocytochemistry and electrophysiological recordings. Cx26 channels displayed unitary conductances of about 140-155 pS. Increase of Cx26 expression following TPA treatment was markedly observed using immunocytochemistry and Western blots of membrane fractions although it was not detected in Northern or Western blots of whole cells. This increase in Cx26 expression in the plasma membrane was accompanied by an increase of function as evidenced in measurements of junctional conductance. These results suggest that induction of exogenous Cx26 in neuroblastoma cells by TPA treatment is controlled by post-translational mechanisms.