Abstract

The down regulation of gap junctional communication (GJC) has been strongly implicated in carcinogenesis. This is the result of reduced expression of connexin proteins such as connexin 43 (Cx43). Both retinoids and carotenoids increase GJC through the stimulation of Cx43 expression; this increase has been proposed as a mechanism by which retinoids and carotenoids inhibit neoplastic transformation. Using three retinoic acid receptor (RAR) antagonists, (Ro41-5253, BMS453 and BMS493) we demonstrate for the first time that activation of RAR by retinoids (TTNPB) is necessary for Cx43 upregulation. Ro41-5253 was also able to significantly reduce, but not completely inhibit β-carotene, and was ineffective against the non-pro-vitamin A carotenoids astaxanthin or lycopene. Simultaneous treatment with both retinoids and carotenoids resulted in super-induction of Cx43, which suggests that these two compounds operate through separate mechanisms. A PPAR-γ agonist (GW1929) was able to induce Cx43, while GW9662, a potent PPAR-γ antagonist was able to significantly inhibit carotenoid induction of Cx43. These data demonstrate for the first time that PPAR-γ is both necessary and sufficient for carotenoid induction of Cx43. Due to the lack of a retinoic acid response element or a PPAR response element in the Cx43 promoter we examine how these compounds might upregulate Cx43. Using luciferase constructs we show that both retinoid and carotenoid treatment results in upregulation of the Cx43 promoter. All treatments demonstrated a fold-increase in promoter activity that was comparable to their protein induction for the same time frame. Computer analysis of human, mouse and rat Cx43 promoter sequences led to the identification of several evolutionally conserved elements previously shown to be modulated by retinoids or PPAR-γ. EMSA studies revealed no specific binding to TCF, AP-2, nuclear receptor half-sites or changes in binding with treatment to a GC-box by Sp1/Sp3. However, sit-directed mutagenesis of the GC-box resulted in both an increase in basal level activity, as well as a loss of responsiveness to TTNPB treatment and in part to β-carotene as well. We conclude therefore that the induction of Cx43 by retinoids (via RAR) and carotenoids (via PPAR-γ) while occurring at the transcriptional level, operate through separate mechanisms.