Presence Of Heme Oxygenase-1 Research Articles

Physical Interactions between Heme Oxygenase‐1 and the Cytochromes P450 Lead to Complex Changes in Enzyme Activity

The cytochromes P450 (P450s) catalyze the oxygenation of endogenous and exogenous organic substrates, including most prescribed drugs. Heme oxygenase-1 (HO-1) is an inducible enzyme responsible for the rate-limiting step of heme catabolism. HO-1 and the P450s are membrane-bound proteins expressed on the cytosolic face of the endoplasmic reticulum where they must interact with NADPH-cytochrome P450 reductase (POR) for activity. Because POR levels are often limiting, HO-1 and the P450s must compete for POR binding in order to function. However, simple competition for POR is often complicated by homomeric and heteromeric P450•P450 interactions. We hypothesized that HO-1 might participate with the P450s in their competition for POR and that this competition might lead to changes in P450 and/or HO-1 activity. We were also interested in determining whether HO-1 could physically interact with P450s and measuring what functional effects any P450•HO-1 complex might have. Using bioluminescence resonance energy transfer (BRET), we were able to establish that HO-1 formed physical complexes with CYP1A1, CYP1A2, and CYP2D6 in transiently transfected HEK293T cells. The stability of these complexes was determined by measuring the BRET signal generated by GFP- and Renilla luciferase-tagged P450 and HO-1 in the presence and absence of untagged POR. The presence of POR did not have a significant effect on the interactions of HO-1 with CYP1A2 and CYP2D6 but caused an increase in the BRET signal generated by the HO-1/CYP1A1 pair. Further BRET studies of these three P450s showed that the presence of HO-1 inhibited the formation of POR•P450 complexes, while, conversely, the presence of untagged P450 caused marginal disruption of POR•HO-1 complex formation. The differences in the response of the HO-1•CYP1A1 complex and the HO-1•CYP1A2 complex to POR indicated the possibility of different functional consequences of these P450/HO-1 interactions. Activity measurements of purified enzymes in lipid reconstituted systems confirmed that this was the case. CYP1A1 and HO-1 had large inhibitory effects on each other over a large range of POR concentrations. HO-1 activity in the presence of CYP1A1 approached that of HO-1 alone only in the presence of a large excess of POR. The parallel experiments with HO-1 and CYP1A2 yielded results that differed both qualitatively and quantitatively from those seen with CYP1A1. Like CYP1A1, CYP1A2 caused inhibition of HO-1 activity, though the effect of CYP1A2 was much milder. The effect of HO-1 on CYP1A2 was also smaller in magnitude than was observed with CYP1A1, but CYP1A2 activity increased in the presence of HO-1 and subsaturating POR. In the presence of HO-1 and excess POR, CYP1A2 activity was slightly inhibited. Kinetic analysis of these results showed that they were inconsistent with a simple model of competition between HO-1 and P450 for POR.

Amelioration of Murine Macrophage Activation Syndrome by Monomethyl Fumarate in Both a Heme Oxygenase 1-Dependent and Heme Oxygenase 1-Independent Manner.

Macrophage activation syndrome (MAS) is characterized by increased serum levels of ferritin and heme oxygenase 1 (HO-1), and yet no known function is ascribed to these molecules in MAS. Because HO-1 is antiinflammatory, we hypothesized that pharmacologic activation of HO-1 could ameliorate MAS disease activity. Dimethyl fumarate (DMF), a treatment approved by the US Food and Drug Administration for multiple sclerosis, activates HO-1. Monomethyl fumarate (MMF) is the active metabolite of DMF. We therefore evaluated whether MMF could elicit HO-1-dependent therapeutic improvements in a murine model of MAS. We induced MAS by repeated activation of Toll-like receptor 9 (TLR-9) in wild-type and myeloid-specific HO-1-deficient mice. MMF was administered twice daily to test its efficacy. We assessed organ weights, serum cytokine levels, histologic features of the spleen and liver tissue, and complete blood cell counts to evaluate disease activity. Statistical testing was performed using Student's t-test or by 2-way analysis of variance as appropriate. The presence of HO-1 was required for the majority of TLR-9-induced interleukin-10 (IL-10). IL-10 production in TLR-9-induced MAS was found to correlate with the myeloid-HO-1 gene dose in myeloid cells (P < 0.001). MMF treatment increased the levels of HO-1 in splenic macrophages by ~2-fold (P < 0.01), increased serum levels of IL-10 in an HO-1-dependent manner in mice with TLR-9-induced MAS (P < 0.005), and improved multiple disease parameters in both an HO-1-dependent and HO-1-independent manner. TLR-9-induced production of IL-10 is regulated by HO-1 activity both in vitro and in vivo. Therapeutic enhancement of the HO-1/IL-10 axis in a murine model was able to significantly ameliorate MAS disease activity. These results suggest that HO-1 may be viable as a MAS therapeutic target, and treatment with DMF and MMF should be considered in future investigations of MAS therapy.

Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1

Heme oxygenase 1 (HO-1) and the cytochromes P450 (P450s) are endoplasmic reticulum–bound enzymes that rely on the same protein, NADPH-cytochrome P450 reductase (POR), to provide the electrons necessary for substrate metabolism. Although the HO-1 and P450 systems are interconnected owing to their common electron donor, they generally have been studied separately. As the expressions of both HO-1 and P450s are affected by xenobiotic exposure, changes in HO-1 expression can potentially affect P450 function and, conversely, changes in P450 expression can influence HO-1. The goal of this study was to examine interactions between the P450 and HO-1 systems. Using bioluminescence resonance energy transfer (BRET), HO-1 formed HO-1•P450 complexes with CYP1A2, CYP1A1, and CYP2D6, but not all P450s. Studies then focused on the HO-1–CYP1A2 interaction. CYP1A2 formed a physical complex with HO-1 that was stable in the presence of POR. As expected, both HO-1 and CYP1A2 formed BRET-detectable complexes with POR. The POR•CYP1A2 complex was readily disrupted by the addition of HO-1, whereas the POR•HO-1 complex was not significantly affected by the addition of CYP1A2. Interestingly, enzyme activities did not follow this pattern. BRET data suggested substantial inhibition of CYP1A2-mediated 7-ethoxyresorufin de-ethylation in the presence of HO-1, whereas its activity was actually stimulated at subsaturating POR. In contrast, HO-1–mediated heme metabolism was inhibited at subsaturating POR. These results indicate that HO-1 and CYP1A2 form a stable complex and have mutual effects on the catalytic behavior of both proteins that cannot be explained by a simple competition for POR.

The anti-tumor effects of calorie restriction are correlated with reduced oxidative stress in ENU-induced gliomas

The anti-tumor effects of calorie restriction (CR) and the possible underlying mechanisms were investigated using ethylnitrosourea (ENU)-induced glioma in rats. ENU was given transplacentally at gestational day 15, and male offspring were used in this experiment. The brain from 4-, 6-, and 8-month-old rats fed either ad libitum (AL) or calorie-restricted diets (40% restriction of total calories compared to AL rats) was studied. Tumor burden was assessed by comparing the number and size of gliomas present in sections of the brain. Immunohistochemical analysis was used to document lipid peroxidation [4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA)], protein oxidation (nitrotyrosine), glycation and AGE formation [methylglyoxal (MG) and carboxymethyllysine (CML)], cell proliferation activity [proliferating cell nuclear antigen (PCNA)], cell death [single-stranded DNA (ssDNA)], presence of thioredoxin 1 (Trx1), and presence of heme oxygenase-1 (HO-1) associated with the development of gliomas. The results showed that the number of gliomas did not change with age in the AL groups; however, the average size of the gliomas was significantly larger in the 8-month-old group compared to that of the younger groups. Immunopositivity was observed mainly in tumor cells and reactive astrocytes in all histological types of ENU-induced glioma. Immunopositive areas for HNE, MDA, nitrotyrosine, MG, CML, HO-1, and Trx1 increased with the growth of gliomas. The CR group showed both reduced number and size of gliomas, and tumors exhibited less accumulation of oxidative damage, decreased formation of glycated end products, and a decreased presence of HO-1 and Trx1 compared to the AL group. Furthermore, gliomas of the CR group showed less PCNA positive and more ssDNA positive cells, which are correlated to the retarded growth of tumors. Interestingly, we also discovered that the anti-tumor effects of CR were associated with decreased hypoxia-inducible factor-1α (HIF-1α) levels in normal brain tissue. Our results are very exciting because they not only demonstrate the anti-tumor effects of CR in gliomas, but also indicate the possible underlying mechanisms, i.e. anti-tumor effects of CR observed in this investigation are associated with reduced accumulation of oxidative damage, decreased formation of glycated end products, decreased presence of HO-1 and Trx1, reduced cell proliferation and increased apoptosis, and decreased levels of HIF-1α.

104: Heme oxygenase in human myometrial cells and tissue

MARTIN SLODZINSKI, Johns Hopkins University, Anesthesiology and Critical Care Medicine, Baltimore, Maryland OBJECTIVE: Heme oxygenase 1 (HO-1) has been extensively studied as an inducible stress response protein essential to heme metabolism. HO-1 may also be present in other systemic tissues and may in fact be inducible by non-heme substrates such as heavy metals, endotoxin, heat shock, inflammatory cytokines, and prostaglandins. Little is known regarding HO-1 and human myometrium. We postulate that HO-1 is critical for myometrial homeostasis and is instrumental in maintaining gravid uterine quiescence. STUDY DESIGN: With IRB approval, human myometrial tissue was obtained from the lower uterine segment of pregnant women undergoing cesarean delivery for pre-term and term labor. A western blot analysis was performed using polyclonal rabbit antibody (1:2000) to identify the presence of HO-1. Ratiometric imaging of intracellular calcium and isometric myometrial tissue force were measured in response to oxytocin and the competitive HO-1 inhibitor zinc protoporphyrin IX (PPN-9) and bilirubin. RESULTS: HO-1 is expressed in term and preterm human myometrial tissue. In primary cultured human myocytes, PPN-9 (10uM, 5minutes) inhibition of HO-1 irreversibly increased intracellular calcium. This increase was prevented if bilirubin (100uM) was applied with the PPN-9. In isometric myometrial contractions, PPN-9 (1nM)increased the amplitude and rate of contraction compared with controls. H202 (10uM-1mM) increased the amplitude of contraction in the PPN-9 more than the control tissue. Bilirubin partially abolished the H202 increase in amplitude. CONCLUSION: While extensive research has been performed regarding the function of HO-1 in heme degradation, HO-1 physiology is unexplored in human myometrium. Our investigation demonstrates that HO-1 is present within gravid myometrium and influences myometrial intracellular calcium homeostasis and myometrial tissue contractibility.

A clinical assessment of the effects of 10% carbamide peroxide gel on human pulp tissue

Bleaching vital teeth with 10% carbamide peroxide gel is a routine procedure in which there has been no evidence of associated permanent pulpal damage. Synthesis of the enzyme heme oxygenase-1 (HO-1) is increased after exposure of eukaryotic cells to conditions of oxidative stress (including H2O2) as a defense against the damaging effects of free radicals. Dental pulps were evaluated for HO-1 (aka Heat Shock Protein 32) presence in teeth treated with 10% carbamide peroxide. Seventeen intact first premolars scheduled for orthodontic extraction were bleached for 4 h immediately preceding extraction. Fourteen additional premolars from the same individuals were not bleached. All 31 teeth were extracted, fixed, demineralized, frozen, sectioned, and immunostained with anti-HO-1 antibody using a standard ABC protocol. There was no significant difference in the presence of HO-1 between total bleached versus total unbleached teeth using the Fisher's Exact Test (p < or = 0.05). However, the histological findings could be interpreted to suggest that coronal odontoblasts and endothelial cells in the underlying pulp proper may have the potential to respond to oxidative stress by increasing the synthesis of HO-1 (HSP32). This could represent a component of an initial defensive response by specific cells in strategic locations in the pulp that precedes classical inflammatory pathways.