Connexin 43 as a Mediator of Estrogen-induced Protection against Oxidative Stress

Hypothesis: 17-β estradiol (E2) and the estrogenic metabolite of dihydrotestosterone (DHT), 5-α-androstane-3,17-β-diol (3βdiol), protect against oxidative stress by increasing the expression and function of Connexin 43 (Cx43) – containing gap junctions in cortical astrocytes. Methods: In order to assess the expression of Cx43 and cell viability, real time RTPCR and the MTT assay was used, respectively. Cerebral cortical astrocytes derived from postnatal day 2 female C57/Bl6 mice were treated with physiologically relevant concentrations of E2, DHT, 3βdiol, or the vehicle control, DMSO, and evaluated for Cx43 mRNA expression. For the cell viability assays, astrocytes were pre-treated with either E2, DHT or DMSO vehicle control and then exposed to iodoacetic acid (IAA) oxidative insult. To further determine the role of Cx43 gap junctions, either Gap19 (inhibitor of the Cx43 hemichannel) or Gap26 (inhibitor of the dimeric Cx43 containing gap junction) were co-applied with the insult. Results: E2 treatment (3 hr) significantly increased Cx43 mRNA expression relative to DMSO control, while both DHT and 3βdiol (also applied for 3 hr) did not. Longer treatment with E2 (18 hr) yielded a non-significant trend to increase Cx43 mRNA expression. In the viability assays, neither E2 or DHT alone (18 hours pre-treatment) nor the Cx43 hemichannel selective inhibitor peptide (Gap19) alone protected against IAA toxicity. However, Gap19 did increase the protective efficacy of not only E2, but DHT as well. Interestingly, inhibition of both Cx43 hemichannels and the dimeric Cx43-containing gap junctions using Gap26, in and of itself, conferred protection against IAA toxicity. Like Gap19, Gap26 also significantly enhanced the protective efficacy of E2. Conclusions: Both E2 and DHT showed some regulatory interaction with astrocyte Cx43 that impacted protection against oxidative stress. This is the first evidence that E2 or DHT regulate Cx43 in the brain and the mechanisms underlying these interactions remain to be further characterized.