Modulation of a cardiogenic shock inducible RNA by chemical stress: adapt73/PigHep3.

BACKGROUND

Understanding how mammalian cells respond to stress is important in the study, detection, and therapy of stress-related disorders. We have been studying cellular stress response in hamster HA-1 cells by using an adaptive response model. HA-1 cells respond to a minimally toxic "pretreatment" dose of hydrogen peroxide by synthesizing RNAs and proteins that protect them against subsequent exposure to a higher cytotoxic concentration of peroxide. The purpose of our studies is to identify and partially characterize any mRNA whose steady state level is significantly modulated during adaptation.

METHODS

HA-1 cells were exposed to a pretreatment dose of hydrogen peroxide and RNA extracted. The differential display technique was used to identify modulated mRNAs. The effects of calcium ionophore A23187 and cis (II)-platinum on the modulation of mRNA from HA-1 cells and A23187 on the modulation of mRNAs from human IMR-90 cells were also determined.

RESULTS

One of the RNAs induced by a pretreatment concentration of hydrogen peroxide was designated adapt73. The size of the induced adapt73 RNA was determined to be 2.1 kb. Induction of adapt73 was maximal 5 hours after peroxide treatment, but elevated levels were still obvious at 10 hours. This induction was not specific to oxidative stress, because other stress agents including as (II)-platinum and especially calcium ionophore A23187 also induced adapt73 mRNA levels. Partial sequencing of adapt73 and a subsequent GenBank homology search revealed extensive homology to a novel RNA from pig, designated PigHep3, that was identified as a cardiogenic shock response gene from liver in pigs that were undergoing resuscitation after circulatory shock. Homology to a completely sequenced but uncharacterized human homolog was also found. Using a partially sequenced expressed sequence tag (EST) human clone to adapt73, we probed Northern blots containing RNA from IMR-90 human fibroblasts treated with A23187. A strongly induced human adapt73 mRNA homolog was observed, almost identical in size to its hamster homolog. In vitro transcription and translation of the human EST clone revealed a translatable Adapt73 protein product.

CONCLUSIONS

These data indicate that adapt73/PigHep3 RNA can be induced by multiple chemical stress, that these inductions occur under protective or adaptive response conditions, that there is an inducible human homolog to adapt73, and suggest that adapt73 may be an important physiologic mediator of organ and cellular shock response in mammals.