REGULATION OF HEME OXYGENASE-2 BY GLUCOCORTICOIDS IN NEONATAL RAT-BRAIN - CHARACTERIZATION OF A FUNCTIONAL GLUCOCORTICOID RESPONSE ELEMENT

Heme oxygenase-2 (HO-2) is constitutively expressed in mammalian tissu es; together with HO-1 (HSP32) it catalyzes the cleavage of heme to pr oduce biliverdin IX alpha, CO and Fe. Detection of a consensus sequenc e of the glucocorticoid response element (GRE) in the promoter region of the HO-2 gene prompted the present study which has investigated the role of glucocorticoids (Gcs) in the regulation of HO-2 protein and t ranscript development in the newborn rat brain and has examined the pr omoter activity of the GRE in HeLa cells. Using in situ hybridization histochemistry, we noted a pronounced increase in signal for HO-2 mRNA in the brain of 14 day-old rats postnatally treated with corticostero ne (5 mu g/g, 4X, starting 24-36 h after birth). And, using immunohist ochemistry, a striking increase in neuronal HO-2 immunostaining in tre ated brains was detected. The HO-2 GRE was tested for responsiveness t o dexamethasone (DX) using both a promoterless CAT expression vector, and a heterologous promoter containing luciferase expression vector in HeLa cells. The HO-2 promoter containing the GRE and transcription st art site induced CAT reporter gene activity in response to DX, whereas mutation or deletion in the GRE abolished hormone responsiveness. Sim ilarly, constructs containing the GRE conferred responsiveness to DX i n an orientation-independent manner and increased relative luciferase activity. Further, specific binding of glucocorticoid receptor protein to the GRE was observed; binding could be competed out only by excess cold GRE and not by mutated HO-2 GRE, or AP1. HO-2 mRNAs(similar to 1 .3 and similar to 1.9 kb) increased in HeLa cells treated with DX (5 m u M), the level reached a maximum at 24 h. DX did not effect HO-1 mRNA level. The increase in the HO-2 transcript was accompanied by an incr ease in HO-2 protein, as assessed by Western blot analysis, and an inc rease in HO activity, as measured by bilirubin formation. Also, an inc rease in intensity of immunostaining was noted in DX-treated HeLa cell s. We conclude that the GRE present in the HO-2 gene promoter region i s functional, and propose the direct involvement of the adrenal glucoc orticoids in modulation of HO-2 gene expression. In the context of bio logical functions of heme degradation products, we suggest that this r egulation may be of significance, particularly to the neurons.