Asymmetric Expression/ Distribution of Connexins is Essential for Contractile Function in the Mammary Gland

Intercellular communication is essential for glandular functions and homeostasis. In the mammary gland, heterotypic interactions between the luminal epithelial cells specialized for milk production and the underlying contractile myoepithelial cells (myoeps) are necessary for the ejection of milk from the alveoli through the ducts and into the nipple at parturition. Recently, the role of the myoepithelial compartment in the development and differentiation of the mammary tissue has gained increasing attention. A growing body of evidence suggests that perturbation of the normal expression pattern of molecules specific to the myoepithelium alters the growth and differentiation of the entire mammary tissue. This is not surprising, since the mammary myoeps, with their location in the gland, are able to integrate multiple signals from neighboring cells and the underlying basement membrane (BM), and in turn, relay these signals to the luminal compartment to control cell growth, differentiation, and to maintain mammary architecture. Thus, studying the signaling pathways and interactions between the luminal and myoepithelial compartments is of major importance for understanding normal mammary development and function. This is highlighted by the important finding from the Bissell laboratory showing that myoeps mediate the polarity of the acinar structures in three-dimension by the production of the BM protein Laminin111, concomitant with another finding from Runswick et al., that desmosomal cadherins between the luminal epithelial cells and myoeps play an essential role in cellular positioning and tissue morphogenesis. Gap junctions couple cells homotypically and heterotypically and coordinate reciprocal responses between the different cell types. Connexins (Cxs) are the main mammalian gap junction proteins, and their distribution in the heterotypic gap junctions is not always symmetrical; in the mammary gland, Cx26, Cx30 and Cx32 are expressed in the luminal epithelial cells and Cx43 in myoepithelial cells. Expression of all four Cxs peaks during late pregnancy until late lactation suggesting essential roles for these proteins in functional differentiation of the gland. In this study, I addressed the pertinent question of how altering cell- specific expression of junctional proteins leads to defective tissue architecture and function. In particular, I investigated the importance of connexin-mediated intercellular interactions, and of cell-type specific expression of connexins in maintaining mammary function. To that end, I used a transgenic mouse where the luminal Cx26 is expressed under the regulation of a Keratin-5 promoter, thus targeting the expression of the protein to the myoep compartment otherwise lacking Cx26. K5-Cx26 dams have an unexpected mammary phenotype; transgenic dams are unable to feed their pups to weaning age, leading to litter starvation and demise in early to mid-lactation. I thus asked why ectopic expression of the luminal Cx26 to mammary myoepithelial cells impairs mammary function, and by what mechanism. The mammary glands of K5-Cx26 female mice develop normally but pups are unable to receive milk. This is despite the normal levels of beta casein and whey acidic protein present in the mammary glands of transgenic mice, and suggesting a defect in delivery rather than milk production. Primary mammary organoids, isolated from wild-type FVB females, contracted in culture upon treatment with oxytocin; however, primary mammary organoids from the transgenic mice failed to respond to oxytocin. Interestingly, I found that ectopic expression of Cx26 to myoepithelial cells alters the expression of endogenous Cx43 and inhibits gap junction-mediated dye coupling in myoepithelial cells expressing high levels of Cx26. Inhibition of gap junctional communication or knock-down of Cx43 in wild-type organoids similarly impairs contraction in response to oxytocin in culture, and suggests that the contractile defect in K5-Cx26 dams is caused by Cx26 acting as a dominant negative to Cx43 function in the mammary myoepithelial cells. This is the first report of a trans-dominant-negative effect of ectopic Cx26 on Cx43 expression in vivo and highlights the importance of tissue- and cell-type- specific expression of Cxs for normal mammary gland development and function.