Original descriptions of GCC null mice were marked by a paradoxical lack of an obvious phenotype and we and others suggested that the function of GCC would only be revealed by systemic study and perturbation of gastrointestinal function 
. This led to the establishment of roles for GCC in regulation of IEC proliferation, apoptosis, and migration 
. Epithelial barrier function is a crucial component of gut homeostasis, and dysregulation contributes to the pathogenesis of many intestinal diseases. In this paper, we have shown that jejunal permeability was increased in GCC−/− and UGN−/− mice compared to WT. GCC−/− mice exhibited ileal hyperpermeability and greater bacterial translocation after LPS challenge, accompanied by increased IFNγ levels. The level of phosphorylated MLC in IEC was significantly increased in GCC−/− and UGN−/− mice compared to WT; Claudin-2 and JAM-A expression in TJs were reduced in GCC deficient IEC. GCC knockdown in IEC monolayers was associated with increased permeability under basal conditions and enhanced IFNγ induced hyperpermeability in IEC monolayers. Our data strongly suggest that GCC signaling plays a role in the integrity of the intestinal mucosal barrier by regulating epithelial MLC phosphorylation and TJ assembly.
Functional TJP strands are located between polarized epithelial cells and characterize highly-differentiated gastrointestinal epithelial cells 
. GCC is highly expressed in differentiated enterocytes. Waldman and co-workers have shown that loss of GCC is associated with changes in IEC homeostasis, including increased proliferation in the crypt, increased migration along the crypt-villus axis, and increased apoptosis 
. The magnitude of these differences decreased from duodenum to colon and parallels the level of hyperpermeability that we saw in different segments of the intestinal tract, with highest disruption of barrier function in the jejunum. It is possible that the changes in barrier function and TJPs that we observed may contribute at least partially to the alteration of homeostatic processes in GCC−/− intestine. Dedifferentiated IECs can lead to immature production of TJPs, and loss of contact inhibition 
. Our results show that loss of GCC signaling reduced JAM-A and Claudin-2 in vivo
, which have been determined to be associated with tumor progression 
. Most recently, GCC activation was found a polar pattern of the effects on ion transport; GCC mucosal activation resulted in a potent cGMP-chloride secretion, which may add to its role in the intestinal barrier 
GN and UGN are expressed in the highly-differentiated compartment along the crypt-villus axis, associated with the transition from proliferation to differentiation 
. They exhibit a gradient of expression along the length of the gastrointestinal tract with UGN levels highest in the proximal intestine and GN levels highest in the colon 
. Consistently, we found that UGN−/− mice, which also exhibit a significant decrease in intestinal GN protein 
, had a dysfunctional jejunal barrier function at baseline. Together, this suggests that intestinal UGN and GN as well as their signaling via GCC are required for the maintenance of small bowel barrier function. Levels of cGMP are reduced by 50–75% in the intestines of both GC-C−/− and UGN−/− mice 
and this reduction may provide a basis for loss of TJ function that remains to be investigated.
IFN-γ selectively increases epithelial permeability to large molecules by direct alteration of TJP assembly 
. STAT-1 is an important signaling molecule for IFN-γ; and STAT1 activation in IECs leads to downstream proinflammatory gene expression, predisposing IECs to injury 
. In our studies, we found that GCC signaling is involved in a complicated modulation of gut mucosal immunity. An increased level of cytokines (IFNγ and IL12p70) was detected in GCC knock out mice at baseline accompanied by significantly elevated jejunal permeability, MLCK expression and STAT1 activation in IECs. An important mechanism through which IFNγ drives barrier dysfunction is by increasing expression of TNF and LIGHT receptors on epithelial cells and sensitizing the IEC monolayer to cytokine stimulation 
. Low dose LPS challenge resulted in a further disruption of barrier function in the ileum of GCC null mice along with significantly elevated luminal bacterial translocation. The barrier dysfunction predisposed GCC null mice to LPS induced sepsis and organ dysfunction, and subsequently resulted in increased mortality upon high dose LPS challenge. Our data also indicated that IFNγ mRNA expression and IELs was elevated in LCM-captured jejunal IEC compartment, suggesting that intestinal barrier dysfunction in GCC deficient mice is maintained by a continuous immune activation. These data indicates that loss of GCC signaling may lead to a dysregulation of the mucosal immune system, and triggers intestinal barrier dysfunction and immune activation.
Primary pathophysiologically relevant intestinal epithelial barrier dysfunction can broadly activate mucosal immune responses and accelerate the onset and severity of immune-mediated colitis, but is not sufficient for intestinal disease 
. Cytokine-induced epithelial barrier dysfunction can be mediated by increased MLCK expression and subsequent myosin II regulatory light chain (MLC) phosphorylation; TNFα, IFNγ, and LIGHT (a member of the TNFα superfamily) can cause MLCK-dependent barrier dysfunction 
. Furthermore, MLCK upregulation is correlated with IBD disease activity, also suggesting that it may contribute to barrier dysfunction in intestinal disease 
. Our data confirmed that loss of GCC signaling led to increased MLC phosphorylation, MLCK mRNA expression and IEC barrier dysfunction in mice. In comparison, our GCC knockdown studies in IEC monolayers highlight an increase in permeability accompanied by increased phosphorylation
of MLC due only to decreased levels of GCC. However, the manner in which GCC signaling mediates the regulation of MLCK activity needs to be explored in the future. Together, loss of GCC signaling leads to the activation of IFN-γ:MLCK pathway in IECs and may be an important initiating event that leads to barrier dysfunction, followed by pro-inflammatory factor production and a predisposition to LPS-induced injury.
We found that reduced JAM-A and Claudin-2 abundance was consistently associated with loss of GCC in both GCC deficient mice and in GCC knock down IEC monolayers. JAM-A has been demonstrated to regulate junctional assembly through recruiting and binding these proteins to its intracellular C-terminus in order to colocalize junctional proteins with the nascent junctions 
. JAM-A null mice exhibit increased intestinal mucosal permeability, and JAM-A has been determined to regulate epithelial permeability, inflammation, and proliferation 
. Aberrant expression of Claudin-2 has been linked to SAMP1/YitFc (SAMP) mice, that develop chronic ileitis 
. Claudin-2 can convert “tight” tight junctions into leaky ones, and it was identified as a cation-selective paracellular channel 
. Upregulation of pore-forming claudin 2 leads to altered tight junction structure and pronounced barrier dysfunction in mild to moderately active Crohn's disease 
. Conversely, reduced levels of claudin-2 or JAM-A may also lead to disrupted barrier function 
. Therefore, GCC signaling may be relevant to regulation of intestinal barrier function directly through interacting with TJPs. The precise mechanisms elucidating how GCC signaling is involved in the regulation of TJPs is the basis for ongoing investigation.
Disruption of intestinal barrier function leading to mucosal inflammation and immune activation may be a key factor in the pathogenesis of several diseases, including sepsis, IBD and IBS 
. IBS is characterized by an increased small bowel paracellular permeability and an increased load of luminal bacteria 
. Linaclotide (MD-1100), a GCC agonist, has been shown in animal studies to stimulate intestinal fluid secretion and transit, but not in GCC null mice 
. Linaclotide improved bowel habits and symptoms of IBS patients with chronic constipation although the mechanisms of action downstream of cGMP are uncertain 
. Our studies for the first time identify a novel GCC
TJP pathway that regulates intestinal barrier function. Therefore, augmenting intestinal GCC activation may represent a novel approach for restoring mucosal barrier function in intestinal disorders.