The role of connexin 43 gap junction protein in adrenal cell proliferation and steroidogenesis

To test the hypothesis that connexin 43 (α₁-Cx43) gap junctions play a significant role in adrenocortical cell proliferation and adrenocorticotropin (ACTH) stimulated steroidogenesis, adrenocortical primary cells in culture as well as various adrenocortical cell lines were used. The cells were examined for their ability to proliferate and produce steroids after modulation of α₁-Cx43 gap junction expression and function by chemical treatments as well as transfection of sense and antisense α ₁-Cx43 cDNAs. In addition, α₁-Cx43 gap junctions were characterized in an adrenocortical cell line isolated from a tumor to evaluate whether α₁-Cx43 expression is different in transformed adrenocortical cells. Finally the results obtained from the adrenocortical cells were extrapolated to a non-adrenal tissue. The role of α₁-Cx43 gap junctions in proliferation of cancerous endocrine cells, specifically prostate cancer cell lines (ML, MLL and AT-1), was characterized.; To analyze the role of Cx43 in proliferation and steroidogenesis of the cells, a bovine adrenocortical cell line (SBAC) was treated with 18α-glycerrhetinic acid (GA), a potent inhibitor of gap junction function, or transfected with α ₁-Cx43 antisense cDNA to reduce connexin expression in the cells. Measurement of cell growth in the GA treated and antisense transfected SBAC populations revealed that these cells at a higher rate than the control populations. Both GA treated and anti-α₁-Cx43 transfected cells also showed a decrease in steroidogenesis in response to treatment with ACTH compared to control cell populations. A human adrenocortical cell line (H295) that does not express endogenous α₁-Cx43 exhibited a decrease in cell population growth in comparison to control cell populations after transfection with α₁-Cx43.; Further study using a human adrenocortical cell line originating from an adrenal carcinoma patient (SW13) revealed that α₁-Cx43 expression can be modulated with dibutyryl cyclic adenosine monophosphate (DbcAMP). In the SW13 cells, total α₁-Cx43 protein was not altered following DbcAMP treatment, yet more surface gap junctions were formed and increased gap junctional communication was exhibited. Finally, using prostate cancer cell lines, α₁-Cx43 expression was modulated with DbcAMP treatment and an inverse relationship between cell metastatic capacity and gap junction expression was exhibited; such that a decrease in cell proliferation was noted following increased α₁-Cx43 gap junction expression.; Together these analyses showed an inverse relationship between α ₁-Cx43 gap junction expression and adrenal cortical cell proliferation. Suppression of α₁-Cx43 gap junction function via decreased expression or chemical inhibition resulted in reduced hormone responsiveness in the adrenal cortical cells. In contrast, when α₁-Cx43 gap junction expression was increased, cell population growth was decreased. Therefore, α₁-Cx43 gap junction expression is critical to normal adrenal cortical cell function in culture. These observations support the hypothesis that gap junctions play a significant role in adrenocortical cell proliferation and steroidogenesis and are a necessary component for hormone-mediated response.