ECM-induced gap junctional communication enhances mammary epithelial cell differentiation

The relationship between gap junctional intercellular communication (GJIC) and mammary cell (CID-9) differentiation in vitro was explored. CID-9 cells differentiate and express beta-casein in an extracellular matrix (ECM)- and hormone-dependent manner. In response to interaction with the ECM, cells in culture modulated the expression of their gap junction proteins at the transcriptional and post-translational levels. In the presence of EHS-matrix, connexins (Cx)26, 32 and 43 localized predominantly to the plasma membrane, and enhanced GJIC [as measured by Lucifer Yellow (LY) dye transfer assays] was noted. Inhibition of GJIC of cells on EHS-matrix with 18 alpha glycyrrhetinic acid (GA) resulted in reversible downregulation of beta-casein expression. In the presence of cAMP, cells cultured on plastic expressed beta-casein, upregulated Cx43 and Cx26 protein levels and enhanced GJIC. This was reversed in the presence of 18 alpha GA. cAMP-treated cells plated either on a non-adhesive PolyHEMA substratum or on plastic supplemented with function-blocking anti-beta 1 integrin antibodies, maintained beta-casein expression. These studies suggest that cell-ECM interaction alone may induce differentiation through changes in cAMP levels and formation of functional gap junctions. That these events are downstream of ECM signalling was underscored by the fact that enhanced GJIC induced partial differentiation in mammary epithelial cells in the absence of an exogenously provided basement membrane and in a beta 1-integrin- and adhesion-independent manner.