MOLECULAR-BASIS FOR HEME-DEPENDENT INDUCTION OF HEME OXYGENASE IN PRIMARY CULTURES OF CHICK-EMBRYO HEPATOCYTES - DEMONSTRATION OF ACQUIRED REFRACTORINESS TO HEME

The effects of heme on the induction of mRNA and protein synthesis for heme oxygenase-1 have been studied in primary cultures of chick embry o liver cells. Heme increased the amount of mRNA and the rate of heme oxygenase-1-gene transcription in a dose-dependent fashion, with a max imal 20-fold increase occurring at 20 muM heme. The largest increase i n the rate of transcription, measured by nuclear run-on assays, occurr ed at 5 h, 2 h earlier than the maximum increase in the amount of mRNA , measured by densitometry of Northern blots. 7-15 h after heme additi on, the half-life of heme-oxygenase-1 mRNA was 3.5 h in the presence o r absence of actinomycin D. In contrast. addition of cycloheximide mar kedly increased the stability of the message (half-life - 18 h), sugge sting that a short-lived protein plays a key role in modulating heme o xygenase-1 mRNA levels. The half-life of heme-induced heme-oxygenase-1 protein, measured by [S-35]methionine labelling and immunoprecipitati on, was 15 h. This long half-life of the protein can largely account f or the additional finding that, following addition of heme, the amount of enzyme protein in the cells increased for 10 h, after which it rem ained essentially constant for 15 h. A striking finding was that, afte r an initial burst of heme-stimulated gene transcription, the cells be came refractory to further heme-mediated induction. This acquired resi stance could not be attributed to the following: a longer duration of culture time; cellular toxicity caused by heme; a lack of heme in the medium or the cells; secretion of heme-binding proteins into the mediu m, preventing further heme uptake; the induction of cellular heme cata bolism sufficient to deplete cellular heme. Instead, the results sugge st a down-regulation of the intracellular machinery required for heme- dependent induction of heme oxygenase-1.