Regulation of COX-2 and PGE2-Dependent Recovery of Intestinal Barrier Function
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Date
2004-09-29
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Abstract
The regulation of intestinal barrier function is of importance in various clinical situations. For example, in cases of ischemia/reperfusion injury, there is a disruption in the gastrointestinal barrier. This may lead to release of pro-inflammatory cytokines and neutrophil (PMN) infiltration. It has been demonstrated that COX-2, which is upregulated during inflammation, and its downstream product PGE2 are important for recovery of barrier function, yet their regulation has not been fully characterized. Study 1: To study the effects of PMNs on acutely injured mucosa, we applied PMNs isolated from circulation to ischemic-injured porcine ileal mucosa. Since COX-2 is upregulated by inflammatory mediators such as IL-1beta, which is released by PMNs, we postulated that PMNs enhance recovery of ischemia-injured mucosa by a pathway involving IL-1beta and COX-2. Application of 5x10⁶ PMNs to ischemia-injured ileal mucosa significantly enhanced transepithelial resistance (TER), an effect inhibited by the selective COX-2 inhibitor NS-398 and an IL-1beta receptor antagonist. Western blots revealed up-regulation of COX-2 in response to PMNs, which was inhibited by an IL-1beta receptor antagonist. Real time PCR revealed increased mRNA COX-2 expression, which preceded increased COX-2 protein expression in response to IL-1beta. We concluded that PMNs augment recovery of TER in ischemia-injured ileal mucosa via IL-1beta-dependent upregulation of COX-2. Study 2: Mitogen activated protein kinase (MAPK) pathways transduce signals from a diverse array of extracellular stimuli, including IL-1beta. The three primary MAPK signaling pathways are the extracellular regulated kinases (ERK 1&2), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Because COX-2 expression is regulated in part by MAPK's, we postulated that MAPK pathways would play an integral role in recovery of porcine ischemia-injured ileal mucosa. Treatment of tissues with the p38 MAPK inhibitor SB-203580 or the ERK 1&2 inhibitor, PD-98059, abolished recovery. Western blots revealed that SB-203580 inhibited upregulation of COX-2 whereas PD-98059 had no effect on COX-2 expression. Inhibition of TER by SB-203580 or PD-98059 was overcome by exogenous PGE2. The JNK inhibitor, SP-600125, significantly increased TER and resulted in COX-2 upregulation. Thus, COX-2 expression appears to be positively and negatively regulated by the p38 MAPK and the JNK pathways respectively. Alternatively, ERK 1&2 appears to be involved in COX-2-independent reparative events. Study 3: In previous studies, we had shown that PGE2 restored barrier function via a signal transduction pathway involving Cl⁻ secretion and recovery of interepithelial tight junctions (TJs). To study these mechanisms, we utilized human colonic T84 cells. We postulated that PGE2 induced chloride secretion would precede increases in TER associated with re-distribution of critical proteins to TJs. T84 cells were grown to confluence, but utilized at lower TER values (200-500 ohms.cm²) to simulate our ileal mucosal model of 'leaky' restituted epithelium. Basolateral application of PGE2 induced transient increases in Isc, indicative of chloride secretion, followed by sustained increases in TER. Basolateral application of the Na/K/2Cl cotransporter inhibitor bumetanide, abolished the PGE2-induced rise in Isc and subsequent elevations in TER. PGE2 induced a shift in claudin-1 from the Triton-X soluble to insoluble fraction, beginning 4-hour after PGE2 administration, which was prevented by bumetanide. Alternatively, there were no changes in occludin or claudins-3 and -5. Immunoflourescence demonstrated that PGE2 increased accumulation of claudin-1 at the apical lateral membrane. Additionally, we showed that PGE2 increased tyrosine phosphorylation of claudin-1 within 30 min, an effect prevented by bumetanide. Therefore, PGE2-induced chloride secretion in T84 cells is directly linked with increases in TER, and these elevations in TER are associated with phosphorylation of claudin-1 and a shift in claudin-1 to the tight junction.
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tight junction, epithelium
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Degree
PhD
Discipline
Physiology
