Connexin 43 Contributes to Estrogen Protection against Oxidative Stress in Cortical Astrocytes




Kubelka, Nicholas K.


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Age-related brain disorders are associated with the decline in the ability of brain cells to cope with homeostatic challenge. Although all major brain cell types have the capacity to respond to homeostatic challenges, astrocytes are particularly well-equipped to counteract these challenges. Here, we focused on Connexin 43 (Cx43) as a protein that is not only highly expressed in astrocytes, but whose expression is critical to inter-cellular communication that in turn, can influence cell viability. Most studies to date have focused on the expression (i.e., abundance) of Cx43. However, a critical limitation of these studies is that they did not thoroughly examine functionality of the Cx43 channels. In particular, there is a paucity of data describing the differential contributions of Cx43-containing hemichannels versus Cx43-containing gap junctions to cellular functions. We hypothesized the astrocyte Cx43 hemichannel as a yet unreported target of androgens and estrogens based on three notions. First, our laboratory has determined that astrocytes are a relevant and important target of such gonadal steroid hormones as estrogens (e.g., 17[beta]-estradiol (abbreviated herein as estradiol or E2)) and androgens (such as DHT), through which these hormones promote healthy brain cell function. Second, oxidative stress is associated with an increase in Cx43 opening. Finally, the Cx43 gene promoter contains functional estrogen response element (ERE) half sites, and estradiol, as well as other estrogenic compounds, decrease Cx43 channel opening in peripheral (non-CNS) tissue. Based on these notions, we hypothesized that gonadal androgens and estrogens will inhibit Cx43 hemichannel opening in cortical astrocytes as well. My data revealed that while E2, dihydrotestosterone (DHT), and the estrogenic metabolite of DHT (3[beta]diol) all protect primary cortical astrocytes from the mixed metabolic/oxidative insult, iodoacetic acid (IAA), only DHT decreased astrocyte Cx43 mRNA expression. Consistent with their cytoprotective effects, however, all three steroids decrease astrocyte Cx43 hemichannel opening, and antagonized the increased opening of Cx43 hemichannels induced by IAA. In an effort to pursue the mechanism by which these steroids reduced Cx43 hemichannel opening, we evaluated the phosphorylation of Cx43 at two key residues, Ser 368 and Tyr 265. Phosphorylation at these residues is associated with channel closing, and as such, we predicted that the three hormones would increase the phosphorylation of Cx43 at one or both of these residues. Whereas Tyr265 phosphorylation was unaffected any of the three hormones, DHT significantly reduced the phosphorylation of Cx43 at Ser368. These observations may indicate that while all three steroids contribute to astrocyte protection through a mechanism that involves blocking astrocyte Cx43 hemichannel opening, DHT may induce molecular changes in the astrocytes that are distinct from those induced by estradiol or 3[beta]diol. The knowledge gained through the experiments conducted not only enhance our understanding of how Cx43 hemichannels and Cx43 gap junctions influence astrocyte function and viability but also define Cx43 hemichannels as relevant targets of gonadal steroid hormone induced regulation of cell viability. Such knowledge may facilitate the development of more precise therapeutics (i.e., selectively targeting Cx43 hemichannels without activity at Cx43 gap junctions in the same cells or tissue), the benefit of which would be to better treat age-associated neurodegenerative disorders as well as disorders of peripheral tissue