Heme Oxygenase-1 Protein Localizes to the Nucleus and Activates Transcription Factors Important in Oxidative Stress

Abstract

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is an integral membrane protein of the smooth endoplasmic reticulum. However, we detected an HO-1 immunoreactive signal in the nucleus of cultured cells after exposure to hypoxia and heme or heme/hemopexin. Under these conditions, a faster migrating HO-1 immunoreactive band was enriched in nuclear extracts, suggesting that HO-1 was cleaved to allow nuclear entry. This was confirmed by the absence of immunoreactive signal with an antibody against the C terminus and the lack of a C-terminal sequence by gas chromatographymass spectrometry. Incubation with leptomycin B prior to hypoxia abolished nuclear HO-1 and the faster migrating band on Western analysis, suggesting that this process was facilitated by CRM1. Furthermore, preincubation with a cysteine protease inhibitor prevented nuclear entry of green fluorescent proteinlabeled HO-1, demonstrating that protease-mediated C-terminal cleavage was also necessary for nuclear transport of HO-1. Nuclear localization was also associated with reduction of HO activity. HO-1 protein, whether it was enzymatically active or not, mediated activation of oxidant-responsive transcription factors, including activator protein-1. Nevertheless, nuclear HO-1 protected cells against hydrogen peroxide-mediated injury equally as well as cytoplasmic HO-1. We speculate that nuclear localization of HO-1 protein may serve to up-regulate genes that promote cytoprotection against oxidative stress.Heme oxygenase (HO) 3 catalyzes the degradation of heme and the formation of biliverdin and carbon monoxide. It is highly inducible in response to various stimuli, including oxidative stress, heavy metals, UV radiation, and inflammation (1-4). Cytoprotective roles for HO have been demonstrated in many models; however, the mechanisms by which this occurs are still under intensive study. Many have speculated that either heme catabolites, such as biliverdin, or its derivative, bilirubin, and carbon monoxide or the degradation of the pro-oxidant heme results in cytoprotection against oxidative stress (5-7). Nevertheless, all of the by-products of the HO reaction, despite being potentially cytoprotective, are also cytotoxic. Bilirubin is a potent neurotoxin (8), as is carbon monoxide (9). Furthermore, the HO reaction releases iron, which could interact with cellular oxidants to generate the hydroxyl radical (10). Transfection with an inactive HO-1 mutant protein results in cytoprotection against chemically induced oxidative stress (11). Because this effect of the mutant HO-1 could not be attributable to changes in heme catabolites, it alludes to a role for the HO-1 protein itself. Furthermore, the inactive form of HO-1 increased catalase and glutathione content (11). This suggests that the HO-1 protein itself may play a role in cellular signaling. If this were true, HO-1 would need to migrate to the nucleus or produce nuclear changes that affect transcription. There are several examples of cytoplasmic enzymes serving in nuclear functi...

Heme Oxygenase-1 Contributes to an Alternative Macrophage Activation Profile Induced by Apoptotic Cell Supernatants

“…This might also apply to Adora A 2A , because overexpression of HO-1 as well as exposure of RAW264.7 macrophages to CO augmented Adora A 2A mRNA as well as protein level (Haschemi et al, 2007). The rather marginal effect seen with CO and bilirubin in our experiments may open a further possibility that HO-1 translocates to the nucleus to bind to a transcription factor or a protein complex, resulting in enhanced transcription of Adora A 2A (Lin et al, 2007). Adora A 2A agonists are capable of not only blocking the inflammatory potential of human macrophages, such as pathogenstimulated NO, TNF-␣, or IL-12 production (Hasko et al, 2007) but also promoting wound healing in disease states such as diabetes (Montesinos et al, 1997).…”

Differential hepatoprotective mechanisms of rutin and quercetin in CCl4-intoxicated BALB/cN mice

“…However, rutin acted as a strong HO-1 inducer in a rat model of liver ischemia-reperfusion injury [46] , suggesting a cell typedependent activation of this enzyme. Additionally, the nuclear translocation of HO-1 could be involved in the regulation of genes responsible for cytoprotection against oxidative stress [47] .…”

Zinc Protoporphyrin Regulates Cyclin D1 Expression Independent of Heme Oxygenase Inhibition

“…Increased ZnPP under certain pathological circumstances might modulate the activity of these transcription factors by replacing heme. Furthermore, HO-1 protein, which can be dramatically up-regulated by ZnPP, translocates into the nucleus and acts as a self-regulator under oxidative stress (50,51). Thus, ZnPP-enhanced HO-1 protein may also play a role in ZnPP-mediated transcriptional regulation although preliminary observations do not yet confirm this.…”