Activation of guanylate cyclase C signaling pathway protects intestinal epithelial cells from acute radiation-induced apoptosis.
ABSTRACT Uroguanylin (UGN) is a peptide hormone that binds to and activates the intestinal epithelial cell (IEC) transmembrane receptor guanylate cyclase C (GC-C), which in turn increases intracellular cGMP. Gene targeting of murine UGN or GC-C results in significantly lower levels of cGMP in IECs. On the basis of effects of cGMP in nonintestinal systems, we hypothesized that loss of GC-C activation would increase intestinal epithelial apoptosis following radiation-induced injury. We first compared apoptosis from the proximal jejunum of C57BL/6 wild-type (WT) and GC-C knockout (KO) mice 3 h after they received 5 Gy of gamma-irradiation. We then investigated whether supplementation via intraperitoneal injection of 1 mM 8BrcGMP would mitigate radiation-induced apoptosis in these experimental animals. Identical experiments were performed in BALB/c UGN WT and KO mice. Apoptosis was assessed by quantitating morphological indications of cell death, terminal dUTP nick-end labeling, and cleaved caspase 3 immunohistochemistry. Both UGN KO and GC-C KO mice were more susceptible than their WT littermates in this in vivo model of apoptotic injury. Furthermore, cGMP supplementation in both GC-C and UGN KO animals ameliorated radiation-induced apoptosis. Neither WT strain demonstrated significant alteration in apoptotic susceptibility as a result of cGMP supplementation before radiation injury. These in vivo findings demonstrate increased radiosensitivity of IECs in UGN and GC-C KO mice and a role for cGMP as a primary downstream mediator of GC-C activation in the protection of these IECs from radiation-induced apoptosis.
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ABSTRACT: In Crohn’s Disease (CD) and ulcerative colitis (UC), the major manifestations of inflammatory bowel disease (IBD), genetically predisposed individuals develop chronic intestinal inflammation in response to environmental stimuli, which are mainly derived from luminal flora. Intestinal responses to luminal flora breaching the intestinal barrier require cytokine-regulated activation of elements of innate and acquired immunity, leading to a targeted and contained inflammatory response. Recent population-based genetic analyses have identified polymorphisms in specific genes relevant to pathways critical for inflammatory signalling and cellular response to stress as carrying increased risk for the development of either CD or UC. Specifically, key mediators of apoptosis and autophagy are implicated in the genetic vulnerability to IBD. Patients with IBD have a compromise of their intestinal barrier integrity, as do their first-degree relatives even in the absence of clinical disease, underscoring the critical nature of barrier integrity in the prevention of aberrant immune responses to intestinal flora. Here we explore the relationships between two of the key proinflammatory cytokines mediating intestinal inflammation in IBD, TNF-α and IFNγ, and the mechanisms by which they regulate epithelial apoptosis and intestinal barrier. Specifically we review factors regulating the balance between pro- and antiapoptotic stimuli resulting from the activation of NF-κB and Aktdependent signalling by proinflammatory cytokines, as well as the influence of oxygen tension and nutritional factors on these pathways.Journal of Clinical and Cellular Immunology. 03/2011; S3(S3):003.
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ABSTRACT: Analysis of knockout animals indicates that 3',5'cyclic guanosine monophosphate (cGMP) has an important role in gut homeostasis but the signaling mechanism is not known. The goals of this study were to test whether increasing cGMP could affect colon homeostasis and determine the mechanism. We increased cGMP in the gut of Prkg2(+/+) and Prkg2(-/-) mice by treating with the PDE5 inhibitor Vardenafil (IP). Proliferation, differentiation and apoptosis in the colon mucosa were then quantitated. Vardenafil (Vard) treatment increased cGMP in colon mucosa of all mice, but reduced proliferation and apoptosis, and increased differentiation only in Prkg2(+/+) mice. Vard and cGMP treatment also increased dual specificity protein phosphatase 10 (DUSP10) expression and reduced phospho-c-Jun N-terminal kinase (JNK) levels in the colon mucosa of Prkg2(+/+) but not Prkg2(-/-) mice. Treatment of Prkg2(-/-) mice with the JNK inhibitor SP600125 reversed the defective homeostasis observed in these animals. Activation of protein kinase G2 (PKG2) in goblet-like LS174T cells increased DUSP10 expression and reduced JNK activity. PKG2 also increased goblet cell-specific MUC2 expression in LS174T cells, and this process was blocked by DUSP10-specific siRNA. The ability of cGMP signaling to inhibit JNK-induced apoptosis in vivo was demonstrated using dextran sodium sulfate (DSS) to stress the colon epithelium. Vard was a potent inhibitor of DSS-induced epithelial apoptosis, and significantly blocked pathological endpoints in this model of experimental colitis. In conclusion, Vard treatment activates cGMP signaling in the colon epithelium. Increased PKG2 activity alters homeostasis by suppressing proliferation and apoptosis while promoting differentiation. The PKG2-dependent mechanism was shown to involve increased DUSP10 and subsequent inhibition of JNK activity.Cell Death and Differentiation advance online publication, 22 November 2013; doi:10.1038/cdd.2013.163.Cell death and differentiation 11/2013; · 8.24 Impact Factor