The role of the small heat shock protein in tissue responses to injuries from tumor necrosis factor and epilepsy

Tolerance is an interesting phenomenon observed in tissues responding to injurious stress. For example, an initial sublethal heat shock treatment may induce cells to become thermotolerant; sensitive tumor cells may become resistant to tumor necrosis factor (TNF) after exposure to a sublethal dose; and animal studies revealed that prior neural seizures conferred a time dependent protective effect against subsequent seizures. However, synthesis of heat shock proteins (hsp), a ubiquitous response to various injurious stresses, may play a common protective role in response to these stressors. Previous studies in our laboratory showed pretreatment of tumor cells with heat (which induces hsp) can reduce their sensitivity to TNF cytotoxicity. Of the major hsp families, the small heat shock protein (hsp27) is the most elusive in function and is reported to be upregulated by TNF and oxidative stress. We hypothesize that hsp27 may play a role in the tolerance response to TNF cytotoxicity and may be also expressed to respond to seizure attacks.; We successfully expressed human hsp27 in TNF sensitive murine cell lines, L929 and WEHI-164 and studied TNF cytotoxicity using neutral red, clonogenic, and chromium-release cytotoxicity assays. The results revealed that expression of hsp27 in TNF sensitive cells conferred significant resistance to TNF cytotoxicity and this protection is accompanied by the continued presence of hsp27 in the cells. Some sensitization is seen in ActD-treated hsp27-expressing cells compared with the non-ActD treated cells, suggesting other short-lived protective proteins may also exist to achieve the maximum protective function.; Phospholipase A2 (PLA2) activation induced by TNF causes increased arachidonic acid release and then consequently leads to increase intracellular oxyradical levels in sensitive cells. Our ({dollar}sp3{dollar}H) arachidonic acid release assay revealed hsp27 overexpressing cells exposed to TNF had a much lower release rate of radioactivity than the controls. Prolonging treatment time and increasing TNF doses increased release in controls but had little effect on the hsp27 overexpressing cells. Morphometric assays of cell death induced by oxyradicals revealed hsp27 overexpressing cells were strongly resistant to oxidative stress generated by menadione. These results suggest that hsp27 is involved in inhibiting PLA2 activation mechanisms, indirectly and directly protecting cells from reactive oxyradicals.; Seizures also may be related to inappropriate reactive oxyradical releases. Epilepsy is a very common chronic disease with the feature of recurrent seizures. In the central nervous system, astrocytes are uniquely able to secrete TNF and have TNF receptors implying active hsp27 induction pathways exist. Yet, in the normal mammalian brain, hsp27 is not detectable by immunoblots. We also hypothesized that hsp27 is to protect cells ubiquitously from reactive oxyradicals, and should play a role in response to seizure attacks. Immunohistochemical studies revealed hsp27 was present in all the 15 surgical specimens tested from epilepsy patients and absent in all normal autopsy brain tissues. Double immunofluorescent staining showed hsp27 is expressed in some astrocyte specific protein (glial fibrillary acidic protein, GFAP) positive cells. Electron microscopic studies revealed hsp27 is localized in the cytoplasm of these cells. In situ hybridization of fresh surgically removed brain tissue from epilepsy patients also showed increased hsp27 mRNA in the astrocytes. (Abstract shortened by UMI.)...