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Heme oxygenase (HO) is the rate-limiting enzyme in heme catabois 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. (11,12). Two HO isozymes, the products of two distinct genes, have been described (13,14). HO-1 is the inducible form which is ubiquitously distributed in mammalian tissues, whereas HO-2 is believed to be constitutively expressed, is not inducible by HO-1 inducers, and is present in tissues such as the brain and testis (14).One of the mechanisms by which hormones, growth factors, and other stimuli induce the expression of genes is by activating various transcription factors. This is a rapid process which frequently involves transcriptional or structural activation of the factor and allows its presence or transfer to the nucleus. These processes may be part of the mechanism by which various agents, including heme, increase HO expression and activity. Previous studies have shown the presence of AP-1-binding sequences and interleukin 6-, metal-, and heat-responsive elements in the HO-1 promoter region and have suggested the involvement of these nuclear factors in the regulation of several genes, including that encoding [13][14][15][16][17][18]. Erythropoietic cells are endowed with HO activity which is inducible by heme (6,7). We therefore used a human-derived erythroleukemic cell line, K562, to examine the presence of transcription factors which might be involved in heme-induced HO-1 expression and to determine whether binding sequences for these factors were present in the promoter region of the human HO-1 gene. tTo whom reprint requests should be addressed. MATERIALS AND METHODS 5987The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The effects of various metalloporphyrins on hepatic heme oxygenase (EC 1. 14.99.3) activity were examined in order to identify compounds that could inhibit heme degradation to bile pigment and might therefore be utilized to suppress the development of hyperbilirubinemia in the newborn. Among nine metal-protoporhyrin IX chelates (i.e., metal-hemes) studied, Snheme, Mn-heme, and Zn-heme substantially diminished heme oxygenase activity in vivo in the rat. These metalloporphyrins act as competitive inhibitory.substrates in the heme oxygenase reaction but are not themselves oxidatively. degraded. Sn.cheme was the most potent enzyme inhibitor (1I = 0.911 FAM) in liver, spleen, kidney, and .skin. Sn-heme administered to newborn animals within the first 72 hr after birth blocked the postnatal increase in heme oxygenase activity that occurs in various tissues. Its effect on the enzyme levels was prompt and protracted. Sn-heme administration also entirely prevented the development of hyperbilirubinemia that normally occurs postnatally. The effect of the metalloporphyrin in lowering the increased concentrations of serum bilirubin in neonates was prompt (within 1 day) and persisted throughout the 42 days after birth. No deleterious effects of Snheme treatment of the newborn were observed. This demonstrates that a synthetic metalloporphyrin that is a potent competitive inhibitor of heme oxidation can, when administered to the newborn, also prevent the hyperbilirubinemia that normally develops postnatally. The potential clinical implications ofthese findings are evident, and it is suggested that the pharmacological properties of Sn-heme and related synthetic metalloporphyrins merit further study. Chemical blockade of the postnatal induction of heme oxygenase activity [heme,hydrogen-donor In this study we examined the ability of nine metalloporphyrins to alter heme oxygenase synthesis or function in liver, spleen, and other tissues of the rat. Of these compounds three markedly inhibited heme degradation by the enzyme; three substantially enhanced this process,. presumably by inducing heme oxygenase; and three had intermediate effects on the enzyme. Two of these. metal-protoporphyrin IX complexes (Snheme and Mn-heme) were employed in whole animal. studies to determine whether the ability to block heme oxygenase activity demonstrated in vitro could be shown to have physiological expression by also blocking the development of postnatal hyperbilirubinemia in the neonate during the 6-week period after birth.We report here that a metalloporphyrin, Sn-heme, which in vitro acts as a potent competitive substrate for heme in the heme oxygenase reaction can also, when administered to newborn animals, entirely prevent the hyperbilirubinemia that develops in the postnatal period.MATERIALS AND METHODS Materials. Male (160-200 g) and 15-day-pregnant female Sprague-Dawley rats purchased from Holtzman (Madison, WI) were used. To the extent possible pregnancy was synchronized in the latter group of animals so that large numbers of newb...

these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Heme oxygenase-1 (HO-1) is emerging as an important cytoprotective enzyme system in a variety of injury models. To optimize future therapeutic applications of HO-1, it is necessary to delineate the precise functions and mechanisms as well as modes of externally regulating HO-1 expression. Investigations have been limited by difficulties with the generation of HO-1 null mice and the lack of specific HO-1 inhibitors. Lung ischemiareperfusion (I-R) injury is the inciting event in acute lung failure following transplantation, surgery, and shock. To study the function of HO-1 in I-R-induced lung injury, we designed small interfering RNA (siRNA) sequences that effectively suppress HO-1 expression both in vitro and in vivo in an organ-specific manner. In this study we show that there is enhanced apoptosis, via increased Fas expression and caspase 3 activity, in the presence of HO-1 siRNA in endothelial cells and mouse lung during I-R injury, whereas HO-1 overexpression attenuates apoptosis. To the best of our knowledge, we are the first to demonstrate that lung-specific siRNA delivery can be achieved by intranasal administration without the need for viral vectors or transfection agents in vivo, thereby obviating potential concerns for toxicity if siRNA technology is to have clinical application in the future.

Obesity-associated inflammation causes insulin resistance. Obese adipose tissue displays hypertrophied adipocytes and increased expression of the cannabinoid-1 receptor. Cobalt protoporphyrin (CoPP) increases heme oxygenase-1 (HO-1) activity, increasing adiponectin and reducing inflammatory cytokines. We hypothesize that CoPP administration to Zucker diabetic fat (ZDF) rats would improve insulin sensitivity and remodel adipose tissue. Twelve-week-old Zucker lean and ZDF rats were divided into 4 groups: Zucker lean, Zucker lean–CoPP, ZDF, and ZDF–CoPP. Control groups received vehicle and treatment groups received CoPP (2 mg/kg body weight) once weekly for 6 weeks. Serum insulin levels and glucose response to insulin injection were measured. At 18 weeks of age, rats were euthanized, and aorta, kidney, and subcutaneous and visceral adipose tissues were harvested. HO-1 expression was measured by Western blot analysis and HO-1 activity by serum carbon monoxide content. Adipocyte size and cannabinoid-1 expression were measured. Adipose tissue volumes were determined using MRI. CoPP significantly increased HO-1 activity, phosphorylated AKT and phosphorylated AMP kinase, and serum adiponectin in ZDF rats. HO-1 induction improved hyperinsulinemia and insulin sensitivity in ZDF rats. Subcutaneous and visceral adipose tissue volumes were significantly decreased in ZDF rats. Adipocyte size and cannabinoid-1 expression were both significantly reduced in ZDF–CoPP rats in subcutaneous and visceral adipose tissues. This study demonstrates that HO-1 induction improves insulin sensitivity, downregulates the peripheral endocannabinoid system, reduces adipose tissue volume, and causes adipose tissue remodeling in a model of obesity-induced insulin resistance. These findings suggest HO-1 as a potential therapeutic target for obesity and its associated health risks.