β-glycyrrhetinic Acid Alters Phosphorylation States of the Gap Junction Protein Connexin-43 in Enterocyte-like Cells
Becker, Russell E.
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Necrotizing enterocolitis (NEC) is an intestinal inflammatory disease representing a leading cause of morbidity and mortality among premature neonates. NEC may be associated with an impaired capability for intestinal epithelial restitution, the coordinated cellular migration process by which damage to the intestinal mucosa heals in healthy individuals. The mechanisms governing this restitution process are poorly understood; however, the level of coordination involved strongly suggests that intercellular communication plays a role. Our laboratory hypothesizes that gap junctions may be an important mediator of this communication, and we have recently demonstrated that treatment with β-glycyrrhetinic acid (β-GA), a well-described chemical gap junction communication inhibitor, also inhibits coordinated restitution in an in vitro assay. In an attempt to elucidate the molecular mechanism for this migration inhibition, the present study utilizes immunoblotting to analyze biochemical effects of β-GA on the gap junction protein connexin-43 in IEC-6 enterocyte-like cells originally isolated from the rat intestine. Connexin-43 is expressed in these cells, and β-GA treatment up to 10 µM was shown to cause dramatic increases in overall connexin-43 protein levels. Additionally, treatment with β-GA caused a marked shift in the phosphorylation state of connexin-43, from more phosphorylated to less phosphorylated forms. Importantly, these effects were observed at dosages similar to those required for inhibition of communication and restitution in previous studies. Taken together, these data indicate that the blockage of gap junctions by β-GA, leading to decreased intercellular communication and impaired restitution capability, may occur through an increase in total connexin-43 protein levels, or a shift from more phosphorylated to less phosphorylated forms of connexin-43.