The broad, long term objective for this research proposal is to investigate the function of the cGMP signaling pathway via guanylyl cyclase C (GC-C) activation within the gut to help preserve epithelial integrity. We propose to determine the effects of loss of GC-C activation on regulation of the mucosal epithelial barrier during inflammation. Our preliminary in vivo data show that loss of the GC-C receptor can increase intestinal epithelial cell (IEC) apoptosis in a gamma irradiation model of intestinal injury. Amelioration of apoptosis by cGMP administration, demonstrates the novel cGMP mediated potential to promote IEC survival. Moreover, our preliminary in vitro data has illustrated that ligand activators of GC-C have proliferative effects on lECs. Moreover, we have preliminary evidence by microarray that guanylin expression is suppressed in pediatric patients with colitis. Collectively, these findings illustrate the novel, yet to date, unexplored hypothesis that GC-C receptor activation via cGMP signaling, will decrease IEC monolayer apoptosis and increase proliferation during acute colitic injury and will promote monolayer restitution via cell migration during recovery from this injury. This proposal outlines three specific aims to accomplish this objective as follows:
Aim 1 : To characterize the effects of loss of GC-C activation in vivo on IEC survival during acute wounding injury and repair by a DSS model. Colitic severity will be determined by clinical and histologic criteria. Apoptosis will be evaluated by two modalities: morphologic changes by light microscopy and cleaved caspase 3 activation by immunostaining. Proliferation will be evaluated by BrdU incorporation. These criteria will be assessed in WT and GC-C KO mice during and after recovery from acute injury.
Aim 2 : To delineate the interactions between inflammatory cytokines, GC-C activation, and IEC survival in a modified in vitro model. Cultured murine colonic explants will be perturbed by IFNg or TNFa. Consequent alterations in GC-C expression (real time PCR), cGMP accumulation (ELISA), and apoptosis will be determined.
Aim 3 : To characterize the relationship between GC-C activation and cGMP accumulation during in vitro wounding and migration of intestinal epithelial cells. We will compare restitution after guanylin supplementation in IEC- 18 cells with a functional and dominant negative GC-C construct. Migration will be assessed using inverted phase microscopy. In conclusion, IBD is a term that refers to illnesses caused by chronic inflammation in the intestine. Maintaining gut epithelial integrity can have an important role in protecting the intestine from injury. We are investigating if specific activation of a receptor, guanylyl cyclase C, on these lining cells, during inflammatory injury can promote their survival and also help repair these barrier cells after injury. ? ? ?
| Garin-Laflam, M P; Steinbrecher, K A; Rudolph, J A et al. (2009) Activation of guanylate cyclase C signaling pathway protects intestinal epithelial cells from acute radiation-induced apoptosis. Am J Physiol Gastrointest Liver Physiol 296:G740-9 |
