ISOLATION OF A NOVEL INDUCIBLE RAT HEAT-SHOCK PROTEIN (HSP70) GENE AND ITS EXPRESSION DURING ISCHEMIA HYPOXIA AND HEAT-SHOCK

Most of the members of the mammalian heat-shock protein (HSP) gene fam ily have been studied and isolated from human and mouse cells. Few stu dies have concentrated on the HSPs of rat, a commonly used experimenta l animal. We have isolated and characterized a novel inducible rat HSP 70 gene using an HSP70 cDNA sequence obtained from an ischaemic rat he art cDNA library. The isolated rat HSP70 gene was found to be a functi onal gene, as indicated by RNAase-protection and Northern-blot analysi s. The deduced amino acid sequence of the inducible rat HSP70 exhibits a high degree of similarity to previously isolated mammalian inducibl e HSP70 gene products. Expression of the inducible HSP70 gene in rat m yogenic cells (H9c2) is markedly increased after relatively short peri ods of hypoxia as well as by heat shock. Two heat-shock elements (HSE) are present in the rat HSP70 promoter. Transient transfection of rat HSP70 promoter/chloramphenicol acetyltransferase constructs into H9c2 cells shows that the presence of either of the two HSEs is sufficient for heat-shock inducibility. In contrast, induction of the rat HSP70/c hloramphenicol acetyltransferase constructs by hypoxia is only detecta ble when both HSEs are present. This leads us to conclude that the ind uction of HSP70 by hypoxia and heat shock occurs through the same regu latory HSEs but the activation of the inducible HSP70 gene by heat sho ck is several-fold higher than by hypoxia.