Heat shock proteins as physiological and clinically-relevant mediators of intestinal epithelial protection

Current therapies for inflammatory bowel diseases have largely been directed towards inhibiting the production or actions of immune and inflammatory mediators. In contrast, the possibility of enhancing the ability of enterocytes to withstand stress or injury caused by these agents has been essentially unexplored. Of the agents that are believed to have cytoprotective actions, little is known about their cellular mechanisms of action. Heat shock proteins (HSPs), a highly conserved family of inducible and constitutively-expressed stress proteins, are known mediators of cellular protection. Once synthesized, these stress proteins are believed to bind to specific cellular targets to preserve their structure and function. To investigate the role of inducible HSPs in protecting intestinal epithelial cells from inflammation-associated injury and to explore the possible cellular mechanisms of HSP action, we performed studies in the IEC 18 cell line, a normal, diploid enterocyte cell line derived from rat small intestine. Heat shock (42 degrees C x 23 minutes) stimulates the production of HSP72, as well as smaller amounts of HSP90. To specifically investigate the role of HSP72, an inducible transfection system (lac switch) was used. When HSP72 was induced under nonstress conditions, the cells demonstrated significant thermotolerance as well as resistance to the thiol-oxidant monochloramine. Various presumed cytoprotective agents were tested for their ability to induce HSP72. The amino acid glutamine, which is a major energy source for small intestinal enterocytes, stimulated the production of HSP72 and conferred protection against heat and oxidant injury in a concentration-dependent fashion. Some of the effect was due to metabolism, but a non-metabolizable analog, DON, was able to confer partial protection to the cells, suggesting that additional mechanisms are involved. Several pharmacological agents also modulated the production of HSP72. Salicylates are an important therapy for maintenance of IBD and have been shown to regulate the 'thermal threshold' for the induction of HSP72 production in HeLa cells. However, in IEC Is cells, a different effect of 5-ASA was observed. 5-ASA did not by itself induce HSP72, nor did it lower the temperature for induction by heat. In contrast, 5-ASA potentiated and accelerated the early response to thermal stress as determined by Western blots. HSP72 was induced earlier in the presence of 1-3 mM 5-ASA, an effect which may relate to its ability to alter heat shock factor-heat shock element binding during transcription. Incubation of IEC 18 cells with dexamethasone and prostaglandins (E and A series), but not short chain fatty acids, also induced the formation of HSP72 and conferred cytoprotection. In contrast, no responses were observed in non-epithelial HeLa cells. Thus, intestinal heat shock protein response appears to be unique and distinct from non-epithelial cells. In other studies, we have also determined that the mechanisms of HSP cytoprotection in IEC 18 cells appear to include mediated by (1) protection of protein synthesis and mRNA translation. (2) binding to and protection of critical cytoskeletal proteins, and (3) stabilization of cytosolic proteins such as dihydrofolate reductase. Through a better understanding of the processes involved in the induction and actions of mucosal heat shock proteins. new strategies may be envisioned for the treatment of inflammatory bowel diseases.