Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism, a process which leads to the generation of equimolar amounts of biliverdin, free iron and carbon monoxide (CO).⁽¹⁾ Heme oxygenase-1 (HO-1) is highly inducible by a vast array of stimuli, including oxidative stress, heat shock, ultraviolet radiation, ischemia-reperfusion, heavy metals, bacterial lipopolysaccharide (LPS), cytokines, nitric oxide (NO), and its substrate, heme.⁽²⁾ Heme oxygenae-2 (HO-2) is a constitutive gene, expressed in neurons, endothelium and many other cell types. Although both HO-1 and HO-2 catalyze the identical biochemical reaction, there are some fundamental differences between the two in genetic origin, primary structure, and molecular weight. HO-1, once expressed under various pathological conditions, has an ability to metabolize high amounts free heme to produce high concentrations of its enzymatic by-products that can influence various biological events, and has recently been the focus of considerable medical interest.⁽³⁾ HO-1 expression can confer cytoprotection and anti-inflammation in gastric and intestinal disease models. The cytoprotective effects of HO-1 are related to end-products formation. The pharmacological application of CO and biliverdin/bilirubin can mimic the HO-1-dependent cytoprotection and anti-inflammation in many injury models. In this review, we provide a comprehensive overview on the molecular mechanisms underlying the regulation and function of HO-1 and its possible clinical implications, especially in gastrointestinal diseases.

The transcriptional upregulation of the ho-1 gene, and subsequent de novo synthesis of the corresponding protein, occurs in response to elevated levels of its natural substrate heme and to a multiplicity of endogenous factors including NO, cytokines, heavy metals, heat shock, ultraviolet radiation, ischemia-reperfusion, and growth factors.^(4,5) Many agents that induce HO-1 are associated with oxidative stress in that they (i) directly or indirectly promote the intracellular generation of reactive oxygen species (ROS), (ii) fall into a class of electrophilic antioxidant compounds that includes plant-derived polyphenolic substances, or (iii) form complexes with intracellular reduced glutathione and other thiols. Two enhancer regions located at approximately –4 and –10 kb relative to the ho-1 transcriptional start site have been identified in the mouse gene. The dominant sequence element of the enhancers is the stress-responsive elements (StRE), which is structurally and functionally similar to the Maf-response element (MARE) and the antioxidant-response element (ARE).⁽⁶⁾ Several transcriptional regulators bind these sequences, including nuclear factor erythroid 2-related factor-2 (Nrf2) and BTB and CNC homolog 1 (Bach1) (Fig. 1). Nrf2 contains a transcription-activation domain and positively regulates HO-1 transcription, whereas Bach1 competes with Nrf2 and represses transcription.^(7–9) Under normal conditions, Nrf2 localizes in the cytoplasm, where it interacts with the actin-binding protein, Kelch-like ECH associating protein 1 (Keap1), and is rapidly degraded by the ubiquitin-proteasome pathway, which results in a lower accumulation of Nrf2 in the nucleus and reduced transcription of the HO-1 gene.⁽¹⁰⁾ Namely, Keap1 acts as negative regulator of Nrf2. Various stimuli, including electrophiles and oxidative stress, liberate Nrf2 from Keap1, allowing Nrf2 to translocate into the nucleus and to bind to stress- or antioxidant-response elements (StRE/ARE). Nuclearly translocated Nrf2 provides immediate transactivation of regulated encoding genes. In this sequence of Nrf2 activation, the phosphorylation of Nrf2 is an important event in the dissociation of Nrf2 from Keap1.⁽¹¹⁾ Furthermore, it has been demonstrated that the oxidation of Keap1 cysteine residues causes a change in the affinity of Keap1 with Nrf2, easily releasing Nrf2.^(12,13) Thus the Nrf2-Keap1 system is considered a major defense mechanism that plays a key role in the induction of HO-1.

Bach1 under baseline condition forms a heterodimer with small maf proteins that represses transcription of the ho-1 gene by binding to MARE in the 5'-untranslated region of the ho-1 promoter. Under conditions of excess heme, increased heme binding to Bach1 causes a conformational change and a decrease in DNA-binding activity followed by nuclear export of Bach1, which in turn leads to transcriptional activation of the ho-1 gene through MARE. Heme also induced nuclear translocation of Nrf2, a partner molecule for the family, and promotes stabilization of Nrf2. Thus, an intracellular heme concentration displaces Bach1 from the MARE sequences by heme binding, which then permits Nrf2 binding to a member of small maf proteins, ultimately resulting in transcriptional activation of ho-1 genes.

The mitogen-activated protein kinase (MAPK)-activated signaling pathway was also recognized as able to mediate the induction of HO-1 by extracellular stimuli. The phosphatidylinositol 3 kianse (PI3K)/Akt signaling pathway is also involved in HO-1 regulation.⁽¹⁴⁾ Akt can directly phosphorylate HO-1 protein at Ser-188 and modulate its activity. In addition to the transcriptional regulation, a recent study has shown that HO-1 is subjected to post-translational regulation by the ubiquitin-proteasome system through an ER-associated degradation pathway.⁽¹⁵⁾

It is most likely that the many properties including anti-inflammation and cytoprotection afforded by HO-1 may be attributed not only its own action but also to other actions of three by-products of HO-1 activity. Especially in anti-inflammation, the degradation of the pro-oxidant heme by HO-1 itself, the signaling action of CO, the antioxidant properties of biliverdin/bilirubin, and the sequestration of free iron by ferritin could all concertedly contribute to the anti-inflammatory effects observed with HO-1.

In 1999, Yachie et al.^(36,37) firstly reported the first case of human HO-1 deficiency. This patient suffered persistent hemolytic anemia and an abnormal coagulation/fibrinolysis system, which were associated with elevated thrombomodulin and von Willebrand factor, indicating persistent endothelial damage. Mice with a HO-1 null mutation have been shown to develop anemia associated with hepatic and renal iron overload⁽³⁸⁾ and right ventricular infarction after chronic hypoxia exposure.⁽³⁹⁾ Absence of HO-1 exacerbates ischemia and reperfusion injury,^(40,41) atherosclerotic lesion and vascular remoldeling,⁽⁴²⁾ chronic renovascular hypertension and acute renal failure,⁽⁴³⁾ and end-organ damage and mortality after lipopolysaccharide injection.⁽⁴⁴⁾ These findings provide strong evidence to support that HO-1 has important functions in normal physiology and pathophysiology, especially associated with oxidative stress.

Nrf2 regulates the inducible expression of a group of detoxication enzymes, such as glutathione S-transferase and NAD(P)H: quinone oxidoreductase, via antioxidant response elements. In addition, Ishii et al.⁽⁴⁵⁾ have shown that Nrf2 also controls the expression of a group of electrophile- and oxidative stress-inducible proteins and activities, which includes HO-1, A170, and peroxiredoxin using peritoneal macrophages from Nrf2-deficient mice. Nrf2-deficient mice have an increased susceptibility to dextran sulfate sodium-induced colitis.⁽⁴⁶⁾ It has been reported that anti-inflammatory and anti-oxidative properties with the HO-1 induction by transforming growth factor-b1 or 15-deoxy-D^(12,14)-prostaglandin J₂ are clearly cancelled in Nrf2-deficient mice.^(47,48)

Mice lacking the gene for Bach1 have dramatic increases in HO-1 expression in the heart, lung, liver, and gastro-intestinal tract, indicating a role for Bach1 in tonic suppression of HO-1 transcription. Bach1 deficiency ameliorates lipopolysaccharide-induced hepatic injury,⁽⁴⁹⁾ hyperoxic lung injury,⁽⁵⁰⁾ myocardial injury induced by ischemia-reperfusion,⁽⁵¹⁾ hypertensive cardiopathy,⁽⁵²⁾ spinal cord injury,⁽⁵³⁾ indomethacin-induced intestinal injury,⁽⁵⁴⁾ and atherosclerosis in apolipoprotein E.⁽⁵⁵⁾ Recently, we have investigated the role of Bach1 in the pathogenesis of indomethacin-induced intestinal injury using Bach1-deficient mice, which are kindly presented from Prof. Igarashi (Tohoku University, Japan).⁽⁵⁶⁾ We have shown that the indomethacin-induced intestinal injury is remarkably improved in Bach1-deficient mice (Fig. 2), and that the increased expression of inflammatory chemokines and myeloperoxidase activity in the intestinal mucosa is suppressed in Bach1-deficient mice, respectively.⁽⁵⁴⁾ In addition, these beneficial effects observed in Bach1-deficient mice are reversed by the cotreatment with an HO-1 inhibitor, SnPP, indicating that these effects are mediated by the HO-1 activity.

We have investigated the expression of ho-1 mRNA and HO-1 protein in colon specimens obtained from patients with ulcerative colitis.⁽⁵⁷⁾ The expression of ho-1 mRNA in inflamed colonic mucosa is remarkably increased compared with normal controls. Furthermore, in the inflamed mucosa of active ulcerative colitis, HO-1 protein expression was also increased. These results suggest that the increased expression of HO-1 protein is mainly derived from the increase in transcription of ho-1 in the inflamed intestine. In the histological study, we have confirmed the expression of HO-1 in inflamed intestinal mucosa and that it was localized in the inflammatory cells, mainly mononuclear cells in the colonic submucosal layer, but not in epithelial cells. Some of the HO-1 positively stained cells were positively stained CD68 cells. Maestrelli et al.⁽⁵⁸⁾ have reported that the majority of HO-1-positive cells in the alveolar spaces were CD68-positive cells, and Yoshiki et al.⁽⁵⁹⁾ have reported that HO-1 expression localized in CD68-positive macrophages. Thus, the expression of HO-1 has been observed mainly in macrophages in various organs. However, other reports regarding the localization of HO-1 in human colonic mucosa have described HO-1 expression not only in inflammatory cells but also in the epithelial cells.^(60,61)

Recent studies showed that glutamine, the major fuel for enterocytes, induces HO-1 in intestinal mucosa of rats⁽⁶²⁾ as well as humans.⁽⁶³⁾ Substantial expression of HO-1 after glutamine administration is observed in villous epithelial cells, crypts and muscular layers. In rats, the protective effect of glutamine on the intestine is associated with HO-1 induction in a model of ischemia-reperfusion injury.⁽⁶²⁾ In human duodenal mucosa, HO-1 is constitutively expressed in nearly all types of intestinal epithelial cells and approximately 10% of lamina propria cells from the villi core, whereas its expression is minimal in deep mucosa. Glutamine increases intestinal HO-1 expression in both intestinal epithelial cells and lamina propria cells, and this histological finding is correlated with an increase in mRNA levels for HO-1. These data suggest that the modulation of HO-1 expression by glutamine may contribute to its protective effect on intestinal injury, together with the previously reported reduction of proinflammatory cytokines production.⁽⁶⁴⁾ Further investigation is required as to whether glutamine may affect HO-1 expression under conditions of intestinal inflammation, including inflammatory bowel disease. As one example, HO-1 mRNA expression was reported to be not affected in pouchitis.⁽⁶⁵⁾

It has been demonstrated that gastric cytoprotection induced by polaprezinc,⁽⁶⁶⁾ eupatilin,⁽⁶⁷⁾ and ketamine⁽⁶⁸⁾ against noxious agents is mediated by HO-1 induction (Table 1). Recent our study shows that lansoprazole, a gastric H⁺/K⁺ ATPase inhibitor, up-regulates HO-1 expression in rat gastric epithelial cells, and the up-regulated HO-1 has anti-inflammatory effects, and that lansoprazole-induced HO-1 induction is mediated by the activation, phosphorylation and nuclear translocation of Nrf2 in accompaniment with the dissection of oxidized Keap1.^(69,70) In this study, we firstly demonstrated that oxidation of Keap1 protein is crucial in the induction of HO-1 by lansoprazole.

In addition to cytoprotection by HO-1, it has been shown that HO-1 exerts a modulatory role on gastric smooth muscle excitability via CO production.^(71,72) Using a diabetic gastroparesis model, Choi et al.^(72,73) have demonstrated that Kit expression in interstitial cells of Cajal is lost during diabetic gastroparesis due to increased levels of oxidative stress caused by low levels of HO-1, and that CD206(+) M2 macrophages that express HO1 appear to be required for prevention of diabetes-induced delayed gastric emptying. Because lansoprazole induces HO-1 in macrophages,⁽⁷⁴⁾ it should be investigated whether lansoprazole could reverse delayed gastric emptying in diabetic mice via the induction of HO-1.

Three reports are available on the use of HO-1 inducers (lansoprazole, sulforafane, and Bach1-deficiency) in indomethacin-induced gastric mucosal injury^(54,75,76) (Table 1). Higuchi et al.⁽⁷⁵⁾ have shown that lansoprazole inhibits indomethacin-induced intestinal injury in rats and that the inhibition is reversed by SnPP, an HO-1 inhibitor. Because CORM, a CO donor, also ameliorates these injury, cytoprotective effects of HO-1, in part, exerts via CO-dependent manner. Many reports have confirmed the anti-inflammatory and cytoprotective effects of HO-1 inducers on small intestinal injuries induced by ischemia-reperfusion,^(77–83) lipopolysaccharide,^(84–86) radiation,^(87–89) and burn shock.^(90–93) Pang et al.^(86,94) have used live Lactococcus lactis secreting bioactive HO-1 to treat intestinal mucosal injury induced by lipopolysaccharide in rats. Intragastric administration of HO-1-secreting Lactococcus lactis strain led to bioactive delivery of HO-1 at intestinal mucosa and significantly decreased mucosal damage, myeloperoxidase activity, bacterial translocation, and tumor necrosis factor-α levels when compared with rats treated with the wild-type strain.

Among products of HO-1, CO may be an important mediator of the host defense response to sepsis.⁽⁹⁵⁾ Chung et al.⁽⁹⁵⁾ have demonstrated that targeting HO-1 to smooth muscle cells and myofibroblasts of blood vessels and bowel ameliorates sepsis-induced death associated with Enterococcus faecalis in association with enhancement of bacterial clearance by increasing phagocytosis and the endogenous antimicrobial response, and that injection of a CO-releasing molecule into wild type mice increases phagocytosis and rescues HO-1-deficient mice from sepsis-induced lethality. More interstingly, it has been reported that CO-releasing molecule ameliorates postoperative ileus and muscularis inflammation, and that these protective effects are, at least in part, mediated through induction of HO-1, in a p38-dependent manner, as well as reduction of ERK1/2 activation. These findings shown here may be of significant importance in clinical small bowel transplantation, post-operative condition for small intestine, or sepsis-related intestinal failure.

Wang et al.⁽⁹⁶⁾ used a rat model of inflammatory bowel disease induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) to investigate whether the expression of HO-1 is an endogenous mechanism responsible for host defense against inflammatory injury in colonic tissue. They demonstrated that HO activity and HO-1 gene expression increased markedly after TNBS induction, and that administration of tin mesoporphyrin (SnMP), an HO inhibitor, potentiated the colonic damage as well as decreased HO-1 activity. These results indicate that HO-1 plays a protective role in the colonic damage induced by TNBS enema. Using a dextran sulfate sodium (DSS)-induced colitis model of mice, we have demonstrated that HO-1 mRNA is markedly induced in inflamed colonic tissue, whereas HO-2 mRNA is constitutively expressed.⁽⁹⁷⁾ Co-administration with ZnPP, an HO inhibitor, also enhanced intestinal inflammation and increased the disease activity index as determined by a calculated score based on changes in body weight, stool consistency, and intestinal bleeding.

Recent investigation has shown that upregulation of HO-1 by several HO-1 inducers significantly reduces the intestinal injuries induced by DSS^(98–100) or TNBS^(101–107) (Table 1). In these studies, HO-1 inducers increase HO-1 expression in intestinal mucosa, and ameliorate mucosal injury as well as inflammatory cell accumulation by decreasing infiltrating neutrophils and lymphocytes via the inhibition of NF-κB-dependent proinflammatory cytokines. To further assess the anti-inflammatory mechanisms, Zhong et al.⁽¹⁰⁰⁾ have examined whether hemin enhanced the proliferation of Treg cells and suppressed the production of interleukin (IL)-17 in a DSS-colitis model. Flow cytometry analysis has revealed that hemin markedly expands the CD4⁺ CD25⁺ Foxp3⁺ Treg population and attenuates IL-17 and TH17-related cytokines. It has been also demonstrated that HO-1 exerts immunoregulatory effects by modulating Treg cell function,⁽¹⁰⁸⁾ and that HO-1 activity in antigen-presenting cells is important for Treg-mediated suppression, providing an explanation for the apparent defect in immune regulation in HO-1-deficient mice.⁽¹⁰⁹⁾

The biological significance of HO-1 up-regulation in gastro-intestinal inflammation remains to be fully elucidated. However, there is no doubt that CO derived from HO-1 exerts significant effects on many pathways of cellular metabolism. In inflamed intestinal cells CO may inhibit the inflammatory response, by consequently influencing the synthesis of cytokines, expression of adhesion molecules, and cell proliferation. Although the mechanisms underlying HO-1 activity on gene expression are not well known, the results obtained in recent years have demonstrated its importance in modulation of the inflammatory reaction. Recent experimental studies clearly demonstrated that HO-1 expression is a self-defense mechanism against inflammation. These data suggest that HO-1 is a possible therapeutic target in several kinds of gastrointestinal diseases.