Intestinal epithelial cells (IEC) form a barrier to intestinal contents and antigens by the formation of tight junctions between adjacent cells. Alterations in this barrier function, such as seen with inflammatory bowel disease (IBD), can induce or exacerbate the disease inflammation. Interleukin-1 (IL-1), a major inflammatory cytokine with a known role in IBD, can cause a breakdown of this epithelial barrier function. IL-1 can also induce IEC to secrete several potent pro-inflammatory cytokines that may be critical in initiating or amplifying local responses in IBD. Therefore, investigating mechanisms that regulate IL-1 intracellular signaling is an important focus in mucosal research. Our preliminary experiments with IEC cell lines suggest that inhibition of the Rho activated kinase, ROCK, suppresses IL-1 induced pro-inflammatory cytokine secretion and expression of mRNA for claudin-18, an epithelial tight junction protein whose expression is altered in IBD. Further, this action is through suppressing the signaling pathway leading to AP-1 activation, while the pathway leading to NF-:B is unaltered. This suggests that ROCK may play a significant role in IL-1 induced inflammatory responses by IEC. Therefore, the long-term goal of this proposal will be to determine the role of ROCK in the regulation of IL-1 stimulated inflammatory responses by IEC. This will be accomplished by the following Specific Aims: (1) To investigate the role of ROCK in regulating IL-1 induced JNK signaling in IEC. JNK, a critical signaling component of the IL-1 pathway leading to AP-1 activation, was found to be suppressed when ROCK activity was suppressed. These experiments will be done using the RNA interference (RNAi) technique to knockdown ROCK1 and/or 2 expression in IEC cell lines. (2) To determine the regulatory effect of ROCK1 on the expression of proteins important to epithelial tight junctions. Using RNAi knockdown of ROCK, the role of ROCK in the IL-1 induced expression of myosin light chain kinase and claudins important to epithelial tight junctions will be determined. Further, the IL-1 induced and ROCK1 mediated alterations in the localization of claudins to IEC tight junctions will be examined by immunofluorescence. Finally, (3) To examine the mechanism by which IL-1 induces ROCK1 activation. The Rho family small GTPase (RhoA, B, or C) which is responsible for activating ROCK will be determined by kinase activation assays and confirmed by determining the effect of RNAi knockdown of the identified Rho protein on IL-1 signaling and claudin expression. These studies will help to define a potentially important mechanism for regulating the IL-1 signaling pathway that may be essential for the function of IEC in mucosal infections, inflammation, and wound healing. These studies may also identify potential new targets for controlling IEC barrier function and cytokine responses in mucosal inflammations, and thereby limiting the contribution of IEC to harmful inflammations.

Public Health Relevance

The inflammatory bowel diseases result in an uncontrolled inflammation of the intestine and colon, which can be worsened by a decrease in the barrier function of the epithelial cells that line these organs. The project outlined in this proposal will examine a new component of a pathway that regulates the capacity of epithelial cells to act as a barrier against the intestinal contents and produce inflammation-inducing factors. Knowledge of this novel regulation of the pathway will provide insight into potential therapies that could control epithelial cell barrier function and participation in intestinal inflammatory responses.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Academic Research Enhancement Awards (AREA) (R15)
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Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
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Hamilton, Frank A
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State University of NY, Binghamton
Schools of Arts and Sciences
United States
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Young, Rebecca S; Wiles, Brody M; McGee, Dennis W (2017) IL-22 Enhances TNF-?- and IL-1-Induced CXCL8 Responses by Intestinal Epithelial Cell Lines. Inflammation 40:1726-1734
Perey, Aaron C; Weishaar, Isabelle M; McGee, Dennis W (2015) The effect of ROCK on TNF-?-induced CXCL8 secretion by intestinal epithelial cell lines is mediated through MKK4 and JNK signaling. Cell Immunol 293:80-6
Rafferty, Brian J; Unger, Benjamin L; Perey, Aaron C et al. (2012) A novel role for the Rho-associated kinase, ROCK, in IL-1-stimulated intestinal epithelial cell responses. Cell Immunol 280:148-55