β-glycyrrhetinic Acid Alters Phosphorylation States of the Gap Junction Protein Connexin-43 in Enterocyte-like Cells
Abstract
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.