Identification of binding sites for transcription factors NF-kappa B and AP-2 in the promoter region of the human heme oxygenase 1 gene.

Abstract

Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and its activity is induced by many agents, including its substrate heme, heavy metals, UV radiation, and other injurious oxidant conditions. We examined the presence of several regulatory elements in the promoter region of the human HO-1 gene which could possibly account for its induction in response to diverse agents or influences. Heme treatment increased both HO activity and HO-1 mRNA in the human erythroleukemic cell line K562. Electrophoretic mobility-shift assays of nuclear protein extracts from heme-treated and control cells with specific oligonucleotide probes containing binding sites for known transcription factors, including AP-1, AP-2, Sp1, NF-kappa B, CTF/NF1, TFIID, OKT1, and CREB, and oligonucleotides containing serum-, metal-, and glucocorticoid-responsive elements demonstrated a specific and marked increase in the NF-kappa B and AP-2 transcription factors and, to a lesser extent, an increase in AP-1. No significant increase in other transcription factors over the control, untreated cells was observed. DNase I footprint assays using purified transcription factors revealed the presence of NF-kappa B and AP-2 binding sites in the proximal part of the promoter region of the human HO-1 gene. Moreover, nucleotide sequence analysis of the HO-1 promoter region showed that the protected regions encompassed NF-kappa B and AP-2 consensus binding sites. The presence of regulatory sequences for the binding of transcription factors such as NF-kappa B and AP-2, whose activation is associated with the immediate response of the cell to an injury, may be an indication of the important role which HO-1 may play in defense mechanisms against tissue injury.