Regulation of heme oxygenase-2 by glucocorticoids in neonatal rat brain: characterization of a functional glucocorticoid response element

Abstract

Heme oxygenase-2 (HO-2) is constitutively expressed in mammalian tissues; together with HO-1 (HSP32) it catalyzes the cleavage of heme to produce biliverdin IXα, CO and Fe. Detection of a consensus sequence 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 transcript development in the newborn rat brain and has examined the promoter 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 corticosterone (5 μg/g, 4×, starting 24–36 h after birth). And, using immunohistochemistry, a striking increase in neuronal HO-2 immunostaining in treated brains was detected. The HO-2 GRE was tested for responsiveness to 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 start site induced CAT reporter gene activity in response to DX, whereas mutation or deletion in the GRE abolished hormone responsiveness. Similarly, constructs containing the GRE conferred responsiveness to DX in 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 (∼1.3 and ∼1.9 kb) increased in HeLa cells treated with DX (5 μM), 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 increase in HO-2 protein, as assessed by Western blot analysis, and an increase in HO activity, as measured by bilirubin formation. Also, an increase in intensity of immunostaining was noted in DX-treated HeLa cells. We conclude that the GRE present in the HO-2 gene promoter region is functional, and propose the direct involvement of the adrenal glucocorticoids in modulation of HO-2 gene expression. In the context of biological functions of heme degradation products, we suggest that this regulation may be of significance, particularly to the neurons.