Abstract
BackgroundGuanylyl Cyclase C (GCC) signaling via uroguanylin (UGN) and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function.Methodology/Principal FindingsParacellular permeability of intestinal segments was assessed in wild type (WT) and GCC deficient (GCC−/−) mice with and without lipopolysaccharide (LPS) challenge, as well as in UGN deficient (UGN−/−) mice. IFNγ and myosin light chain kinase (MLCK) levels were determined by real time PCR. Expression of tight junction proteins (TJPs), phosphorylation of myosin II regulatory light chain (MLC), and STAT1 activation were examined in intestinal epithelial cells (IECs) and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi). We found that intestinal permeability was increased in GCC−/− and UGN−/− mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC−/− mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC−/− and UGN−/− mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability.Conclusions/SignificanceGCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury.
Highlights
Guanylyl cyclase C (GCC) is a transmembrane receptor for the endogenous peptides guanylin (GN) and uroguanylin (UGN) and for bacterial heat stable enterotoxin (ST) [1,2]
We found that jejunal paracellular permeability to FD4 was significantly higher in GCC2/2 mice under basal conditions; this was not seen in ileum and colon (Fig 1 B, C and D)
We found that JAM-A and Claudin-2, membrane-associated tight junction proteins (TJPs), were significantly reduced in intestinal epithelial cells (IECs) monolayers with Guanylyl Cyclase C (GCC) knock down; this was potentiated by interferon c (IFNc) and tumor necrosis factor a (TNFa) administration (Fig 7B)
Summary
Guanylyl cyclase C (GCC) is a transmembrane receptor for the endogenous peptides guanylin (GN) and uroguanylin (UGN) and for bacterial heat stable enterotoxin (ST) [1,2]. Our group has reported that activation of GCC signaling pathway protects intestinal epithelial cells from acute radiation-induced apoptosis [11]. It remains unknown whether GCC directly mediates the regulation of intestinal epithelial barrier function. Barrier function is highly regulated by tight junction proteins (TJPs), allowing the epithelium to control transmucosal permeability to solutes, water, and electrolytes [12,13]. Increased MLC kinase (MLCK) activity has been demonstrated to mediate intestinal epithelial barrier dysfunction induced by tumor necrosis factor a (TNFa) and interferon c (IFNc) [16,17,18]. In our current studies, employing GCC and GCC ligand deficient mice, we demonstrate that GCC signaling is required for the maintenance of homeostatic intestinal barrier function, identifying a novel pathway as well as a new potential therapeutic target for intestinal barrier dysfunction
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