Interferon gamma inhibits inter-enterocyte communication by reducing connexin 43 phosphorylation

Abstract

Introduction. Restitution of the intestinal barrier requires inter-enterocyte communication via the phosphorylated gap junction protein connexin 43. Experimental necrotizing enterocolitis (NEC) is characterized by elevated interferon gamma (IFN) and impaired restitution. We therefore hypothesized that IFN impairs gap junction (GJ) function and restitution by inhibiting phosphorylation of connexin43. We further sought to determine whether connexin 43 phosphorylation was altered in human NEC. Methods. Connexin 43 expression and phosphorylation was assessed in IEC-6 cells ± IFN (1000 u/ml) by SDS--PAGE and confocal microscopy. GJ function was detected by microinjecting IEC-6 cells ± IFN with the GJ tracer lucifer yellow (LY) and impermeant rhodamine dextran. GJ function was inhibited with oleamide (10 μM). In vitro restitution was assessed using time lapsed video microscopy of IEC-6 cells ± IFN migrating across a scraped wound. Small bowel mucosal scrapings were acquired from infants undergoing bowel resection for NEC and compared to specimens taken from infants without NEC. Data are mean ± SEM. Results. IFN significantly reduced phosphorylation of connexin 43 (band density relative to actin, Ctrl: 0.86 ± 0.3 versus IFNγ 0.37 ± 0.1, P < 0.05) and decreased membrane localization of the phosphorylated gap junctions. This correlated with decreased gap junction function as detected by loss of LY transfer (levels of intercellular transfer: Ctrl: 3.2 ± 0.2, IFN 2.4 ± 0.3, oleamide 0, P < 0.05). Oleamide and IFN significantly reduced enterocyte restitution (ctrl: 5 μm/h, oleam: 1.5 μm/h, IFN: 1.6 μm/h, P < 0.05), correlating with a lack of intercellular communication. Strikingly, phosphorylation of connexin 43 in the mucosa of infants with NEC was significantly reduced as compared to control bowel, which correlated with the extent of mucosal disease. Conclusion. IFN inhibits phosphorylation of connexin 43, leading to impaired inter-enterocyte communication and reduced restitution. The finding of decreased phosphoconnexin 43 in human NEC suggests that impaired inter-enterocyte communication may underlie the barrier dysfunction in this disease.