Heme Oxygenase‐1 (HO‐1) localizes to the nucleus in hyperoxia

Heme oxygenase‐1, an integral membrane protein of the smooth endoplasmic reticulum and the rate limiting enzyme in heme degradation, is inducible in response to oxidative stress. However, it may have detrimental effects if overexpressed beyond a certain threshold (Suttner et al, FASEB, 1999), therefore tight regulation of HO‐1 levels may need to be maintained. We further investigated the mechanisms of HO‐1 regulation in hyperoxia using in vivo and in vitro models. Cultured fibroblasts, RAW 264.7 cells and newborn and adult mice were exposed hyperoxia for 24, 48 and 72 h. Intact nuclei were isolated on sucrose gradient. Nuclear extracts were obtained from the intact nuclei using a commercially available kit (Pierce). HO‐1 antibody (SPA‐896, Stressgen) was used to detect immunoreactive proteins using Western blot as well as for fluorescent immunostaining. After hyperoxic exposure, two immunoreactive bands were observed in cultured cells and in the mouse lung: the native HO‐1 (32 kDa) and another band with molecular weight of ~28kDa. Immunohistochemistry and Western blot analysis using nuclear extracts confirmed nuclear localization of the HO‐1 signal. In addition, in vivo, the truncated nuclear HO‐1 was predominant in the newborn lungs after hyperoxic exposure as compared to similarly exposed adult mice, suggesting maturational differences. To understand whether nuclear localization of HO‐1 may be related to oxidative stress, cultured cells were incubated with N‐acetyl cysteine (NAC) prior to hyperoxic exposure. This prevented the nuclear localization of HO‐1. These data suggest that there may be an oxidant sensitive cleavage of HO‐1 protein with resultant nuclear migration. Our further efforts will be directed toward understanding the role of the nuclear form of HO‐1 in oxidative signaling.