The present studies extend this homeostatic role of GUCY2C beyond proliferation, differentiation, migration, DNA damage sensing and repair, and metabolism to maintenance of macromolecular permeability and the intestinal barrier. Elimination of GUCY2C signaling produced basal macromolecular hyperpermeability and potentiated barrier disruption resulting in colitis induced by DSS. Conversely, genetic or pharmacological induction of GUCY2C signaling reduced basal permeability and DSS-induced hyperpermeability and inflammation. Results in mouse models were recapitulated in monolayers of human intestinal epithelial cells in vitro, in the absence of other confounding physiological mechanisms like inflammation,Epigoitrin demonstrating a primary mechanistic role for GUCY2C in regulating the epithelial barrier and macromolecular permeability. Moreover, protection of the epithelial barrier by GUCY2C in vivo and in vitro was associated with alterations in steady state concentrations of tight junction proteins, including occludin and claudin 4. Regulation of steady-state concentrations of these components are one established mechanism modulating the dynamic assembly and deployment of tight junction complexes, barrier integrity and macromolecular permeability. Integration of crypt-villus homeostatic programs mediated by GUCY2C is coordinated through AKT1. Ligand activation of GUCY2C and accumulation of cGMP produces dephosphorylation of AKT1, modulating downstream signaling circuits regulating proliferation and metabolism. In the present study,Isoforskolin homeostatic regulation of epithelial barrier function and permeability by GUCY2C was mediated by AKT1. In that context, specifically reducing AKT1, but not AKT2, blocked alterations in occludin and claudin 4, but not JAM-A and claudin 2, and macromolecular hyperpermeability induced by eliminating GUCY2C signaling in mice. Further, in monolayers of human intestinal epithelial cells in vitro, eliminating AKT1 expression induced steady state levels of occludin and claudin 4, but not JAMA and claudin 2, and barrier function to levels achieved by ST activation of GUCY2C. Moreover, a constitutively activated form of AKT1 produced resistance to, while eliminating AKT1 expression from these cells mimicked, the effects of GUCY2C activation on epithelial cell permeability. Together, these results demonstrate a key mechanistic role for suppression of AKT1 signaling in the regulation of epithelial cell barrier function by GUCY2C. These results reinforce the established role of AKT in regulating the expression of junctional proteins, disrupting epithelial cell membrane complexes, and inducing barrier dysfunction. Further, they identify occludin and claudin 4, but not JAM-A and claudin 2, as specific downstream targets of AKT1 contributing to the regulation of epithelial barrier permeability in vitro and in vivo. They underscore the integrated homeostatic role of GUCY2C signaling, centrally coordinated through AKT, in regulating intestinal epithelial structure and function. There is an established mechanistic relationship between disruption of the intestinal epithelial barrier, inflammation resulting from systemic immune exposure to normally compartmentalized lumenal antigens, and the generation of reactive oxygen species producing DNA damage and genotoxic stress. In turn, reactive oxygen species producing DNA damage comprise one mechanism contributing to tumorigenesis generally, and to the evolution of colon cancer in the context of inflammatory bowel disease, specifically. Importantly, there is emerging recognition that this oxidative stress extends beyond the primary site of barrier disruption and inflammation, and is disseminated systemically.