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Curcumin (diferuloylmethane), a component of turmeric, has been shown to have therapeutic properties. Induction of phase 2 detoxifying enzymes is a potential mechanism through which some of the actions of curcumin could proceed. Heme oxygenase-1 (HO-1), an antioxidant phase 2 enzyme, has been reported to have cytoprotective effects in pancreatic [beta]-cells. Curcumin on further purification yields demethoxy curcumin (DMC) and bisdemethoxy curcumin (BDMC). The objective of the present study was to determine the mechanism by which these purified curcuminoids induce HO-I in MIN6 cells, a mouse [beta]-cell line. Demethoxy curcuminoids induced HO-1 promoter linked to the luciferase reporter gene more effectively than curcumin. The induction was dependent on the presence of antioxidant response element (ARE) sites containing enhancer regions (E1 and E2) in HO-1 promoter and nuclear translocation of nuclear factor-E2-related factor (Nrf2), the transcription factor that binds to ARE. Curcuminoids stimulated multiple signaling pathways that are known to induce HO-1. Inhibition of specific signaling pathways with pharmacological inhibitors and cotransfection experiments suggested the involvement of phosphotidylinositol 3-kinase and Akt. Real-time quantitative RTPCR analysis showed significant elevation in the mRNA levels of HO-1 and two other phase 2 enzymes, the regulatory subunit of glutamyl cysteine ligase, which is needed for the synthesis of glutathione, and NAD(P)H:quinone oxidoreductase, which detoxifies quinones. DMC and BDMC induced the expression of HO-1 and translocated Nrf2 to nucleus in [beta]-cells of mouse islets. Our observations suggest that demethoxy curcuminoids could be used to induce a cellular defense mechanism in [beta]-cells under conditions of stress as seen in diabetes. phase 2 enzymes; diabetes; pancreatic [beta]-cells; oxidative stress

Our published studies show that the distribution of the ANG II type 1 (A[T.sub.1]) receptor (A[T.sub.1]R), expressed as a enhanced yellow fluorescent fusion (YFP) protein (A[T.sub.1]R/EYFP), is altered upon cellular treatment with ANG II or coexpression with intracellular ANG II. A[T.sub.1]R accumulates in nuclei of cells only in the presence of ANG II. Several transmembrane receptors are known to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. The present study was designed to determine whether the A[T.sub.1]R is cleaved before nuclear transport. A plasmid encoding a rat A[T.sub.1]R labeled at the amino terminus with enhanced cyan fluorescent protein (CFP) and at the carboxy terminus with EYFP was employed. Image analyses of this protein in COS-7 cells, CCF-STTG1 glial cells, and A10 vascular smooth muscle cells show the two fluorescent moieties to be largely spatially colocalized in untreated cells. ANG II treatment, however, leads to a separation of the fluorescent moieties with yellow fluorescence accumulating in more than 30% of cellular nuclei. Immunoblot analyses of extracts and conditioned media from transfected cells indicate that the CFP domain fused to the extracellular amino-terminal A[T.sub.1]R domain is cleaved from the membrane and that the YFP domain, together with the intracellular cytoplasmic carboxy terminus of the A[T.sub.1]R, is also cleaved from the membrane-bound receptor. The carboxy terminus of the A[T.sub.1]R is essential for cleavage; cleavage does not occur in protein deleted with respect to this region. Overexpressed native A[T.sub.1]R (nonfusion) is also cleaved; the intracellular 6-kDa cytoplasmic domain product accumulates to a significantly higher level with ANG II treatment. nuclear angiotensin II type 1 receptor; intracrine; intracellular

Proteinuria contributes to chronic kidney disease by stimulating renal tubular epithelial cells to produce cytokines such as monocyte chemoattractant protein-1 (MCP-1). The present study determined whether cellular overexpression of heine oxygenase-1 (HO-1) can influence albumin-stimulated MCP-1 production. In response to bovine serum albumin, NRK-52E cells constitutively overexpressing HO-1 (HO-1 OE cells) exhibit less induction of MCP-1 mRNA and less production of MCP-1 protein compared with similarly treated, control NRK-52E cells (CON cells). In wild-type NRK-52E cells, and under these conditions, we demonstrate that the induction of MCP-1 is critically dependent on intact NF-[kappa]B binding sites in the MCP-1 promoter. In response to albumin, CON cells exhibit activation of NF-[kappa]B, and this is reduced in HO-1 OE cells. Albumin also activates ERK1/2 and increases ERK activity, both of which are exaggerated in HO-1 OE cells. Studies with an inhibitor of MAPK/ERK kinase (U0126) demonstrate that the inhibitory effects of U0126 on MCP-1 production are attenuated in HO-1 OE cells. We conclude that HO-1 overexpression in the proximal tubule reduces MCP-1 production in response to albumin, and this occurs, at least in part, by inhibiting an ERK-dependent, NF-[kappa]B-dependent pathway at a site that is distal to the activation of ERK. These findings suggest that the induction of HO-1 in the proximal tubule, as occurs in chronic kidney disease, may be a countervailing response that reduces albumin-stimulated production of cytokines such as MCP-1. proteinuria; extracellular signal-regulated kinase 1/2; tubulointerstitial disease; cytoprotection

I. Perspective II. Heme Oxygenase Isozymes A. Enzymatic activity and its measurement B. Biochemical properties C. Genetics D. Tissue distribution E. Subcellular localization F. Phylogeny III. Regulation of Heme Oxygenases [...]

Heme oxygenase-1 (HO-1), a cytoprotective gene, is commonly induced in renal tubules in the diseased kidney. Because proteinuria is a hallmark for kidney disease, we examined the relationship between proteinuria and tubular induction of HO-1, specifically questioning whether increased trafficking of protein across the renal tubular epithelium, as a consequence of proteinuria, induces tubular expression of HO-1. We examined a model of glomerular proteinuria induced by daily injections of BSA, which is associated with increased tubular uptake of filtered protein, and a model of tubular proteinuria induced by maleate, the latter exhibiting decreased tubular uptake and trafficking of protein. The BSA model of glomerular proteinuria failed to exhibit induction of HO-1; HO-1 was not induced in proximal tubular epithelial cells exposed to BSA. In contrast, in maleate nephropathy wherein tubular uptake of protein is decreased because of generalized proximal tubular injury induced by maleate, HO-1 was strongly induced in proximal tubules; inhibition of HO activity in maleate nephropathy worsened proteinuria, renal histological injury, and apoptosis. In renal proximal tubular epithelial cells, maleate induced HO-1 and caused apoptosis, the latter increased when HO activity was inhibited. From these studies, we conclude that expression of HO-1 in the diseased kidney cannot be ascribed to the tubular uptake and metabolism of protein such as albumin, and that the expression of HO-1 in a model of tubular proteinuria reflects a functionally significant stress response to toxin-induced proximal tubular injury. albumin; maleate; cytoprotection; apoptosis

Heme oxygenase-1 (HO-1) and p21 influence cell fate, and genetic HO-1 overexpression upregulates p21 and confers resistance to apoptosis. The present study examined the effects of heme, a metabolite incriminated in renal injury, on sensitivity to apoptosis and cell growth in conjunction with cellular expression of HO-1 and p21. Immortalized rat proximal tubular epithelial cells (IRPTCs) were exposed to hemin (10 [micro]M) in serum-deplete media (0.1% FBS) and in standard cell culture media (5.0% FBS). In the presence of 0.1% FBS media, hemin induced p21 through an HO-dependent, p53-independent mechanism; certain products of HO activity (iron and carbon monoxide), but not others (ferritin, apoferritin, bilirubin), recapitulated these inductive effects on p21 expression. Along with this inductive effect on HO-1 and p21, heroin worsened apoptosis, the latter exacerbated by the inhibition of HO activity and loss of p21 expression. In IRPTCs maintained in 5% FBS, hemin induced HO-dependent p21 expression, provoked cell cycle arrest, and inhibited cell growth without inducing apoptosis; this inhibitory effect of heroin on cell growth was blocked by the concomitant inhibition of HO activity and loss of p21 expression. We conclude that hemin is a potent HO-dependent inducer of p21 and that hemin increases the sensitivity to apoptosis in serum-deplete conditions and decreases cell growth in serum-replete conditions; inhibiting HO activity and concomitantly ablating p21 expression exacerbate apoptosis and reverse the growth-inhibitory actions of hemin. We suggest that these effects of heme may influence the nature of, and recovery from, ischemic and nephrotoxic insults to the kidney. apoptosis; cell growth; iron; carbon monoxide; renal injury

The mechanism of heme oxygenase-1 gene (ho-1) activation by heme in immortalized rat proximal tubular epithelial cells was examined. Analysis of the ho-1 promoter identified the heme-responsive sequences as the stress-response element (StRE), multiple copies of which are present in two enhancer regions, E1 and E2. Electrophoretic mobility shift assays identified Nrf2, MafG, ATF3, and Jun and Fos family members as StRE-binding proteins; binding of Nrf2, MafG, and ATF3 was increased in response to heme. Dominant-negative mutants of Nrf2 and Maf, but not of c-Fos and c-Jun, inhibited basal and heme-induced expression of an E1-controlled luciferase gene. Heme did not affect the transcription activity of Nrf2, dimerization between Nrf2 and MafG, or the level of MafG, but did stimulate expression of Nrf2. Heme did not influence the level of Nrf2 mRNA but increased the half-life of Nrf2 protein from ~10 min to nearly 110 min. These results indicate that heme promotes stabilization of Nrf2, leading to accumulation of Nrf2*MafG dimers that bind to StREs to activate the ho-1 gene. Nrf2 transcription factor; stress-response element; protein stabilization

This study examined the effect of hemin on the expression of heme oxygenase-1 (HO-1) and monocyte chemoattractant protein-1 (MCP-1) in immortalized rat proximal tubular epithelial cells (IRPTCs). Hemin elicited a dose- and time-dependent induction of HO-1 and MCP-1 mRNA. HO activity contributed to MCP-1 mRNA expression at early time points (4-6 h) because inhibition of HO activity by zinc protoporphyrin (ZnPP) prevented hemin-induced expression of MCP-1 mRNA. Catalytically active intracellular iron was markedly increased in hemin-treated IRPTCs and contributed to the induction of HO-1 and MCP-1 mRNA because an iron chelator blocked hemin-induced upregulation of both genes, whereas a cell-permeant form of iron directly induced these genes. N-acetylcysteine completely blocked hemin-induced expression of HO-1 and MCP-1 mRNA, thereby providing added evidence for redox regulation of expression of these genes. The redox-sensitive transcription factor NF-[kappa]B was recruited in hemin-induced upregulation of MCP-1 because two different compounds that abrogate the activation of NF-[kappa]B (TPCK and BAY 11-7082) completely blocked hemin-induced upregulation of MCP-1 mRNA. In contrast to this HO-mediated induction of MCP-1 through redox-sensitive, iron-dependent, and NF-[kappa]B-involved pathways observed after 4-6 h, hemin also elicited a delayed induction of MCP-1 at 18 h through HO-independent pathways. We conclude that hemin is a potent inducer of MCP-1 in IRPTCs: HO-dependent, hemedegrading pathways lead to an early, robust, and selfremitting induction of MCP-1, whereas HO-independent mechanisms lead to a delayed expression of MCP-1. heme oxygenase; monocyte chemoattractant protein-1; iron; oxidant stress

Lung ischemia-reperfusion (I-R) is an important model of oxidant-mediated acute lung and vascular injury. Heme oxygenase-1 (HO-1) is a cytoprotective gene that is markedly induced by lung I-R injury. HO-1 mRNA is increased in mouse lung after 30 min of lung hilar clamping (ischemia) followed by 2-6 h of unclamping (reperfusion) compared with control mice. In a variety of vascular cell types, HO-1 mRNA is induced after 24 h of anoxia followed by 30 min-1 h of reoxygenation (A-R). Transfection studies reveal that the promoter and 5'-distal enhancer E1 are necessary and sufficient for increased HO-1 gene transcription after A-R. Immunoblotting studies show all three subfamilies of MAPKs (ERK, JNK, and p38) are activated by 15 min of reperfusion. We also demonstrate that HO-1 gene transcription after A-R involves ERK, JNK, and p38 MAPK pathways. Together, our data show that I-R not only induces HO-1 gene expression in mouse lungs and vascular cells but that gene transcription occurs via the promoter and E1 enhancer and involves upstream MAPK pathways. oxidant injury; gene regulation; heme oxygenase; mitogen-activated protein kinases

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress. The anti-inflammatory properties of heme oxygenase-1 may serve as a basis for this cytoprotection. We demonstrate here that carbon monoxide, a by-product of heme catabolism by heme oxygenase, mediates potent anti-inflammatory effects. Both in vivo and in vitro, carbon monoxide at low concentrations differentially and selectively inhibited the expression of lipopolysaccharide-induced pro-inflammatory cytokines tumor necrosis factor-[alpha], interleukin-1[beta], and macrophage inflammatory protein-1[beta] and increased the lipopolysaccharide-induced expression of the anti-inflammatory cytokine interleukin-10. Carbon monoxide mediated these anti-inflammatory effects not through a guanylyl cyclase-cGMP or nitric oxide pathway, but instead through a pathway involving the mitogen-activated protein kinases. These data indicate the possibility that carbon monoxide may have an important protective function in inflammatory disease states and thus has potential therapeutic uses., Author(s): Leo E. Otterbein [1, 3]; Fritz H. Bach [7]; Jawed Alam [6]; Miguel Soares [7]; Hong Tao Lu [4]; Mark Wysk [5]; Roger J. Davis [5]; Richard A. Flavell [...]

Author(s): Prachetash Ghosh[sup.1] , Avijit Sarkar[sup.1] , Mou Ganguly[sup.1] , Raghwan[sup.1] , Jawed Alam[sup.1] , Ronita De[sup.1] and Asish K. Mukhopadhyay[sup.1] Background Helicobacter pylori is a Gram-negative, genetically diverse spiral [...]

The role of nitric oxide in the regulation of heme oxygenase (HO)-1 gene expression in vascular smooth muscle cells (aSMC) was investigated. Primary cultures of rat aSMC were used in an experiment to test whether NO donors stimulate HO-1 messenger RNA (mRNA) and protein expression in aortic aSMC via a guanosine 3',5'-cyclic monophosphate (cGMP)-independent pathway. Results showed that overexpression of HO-1 in pulmonary epithelial cells retarded cell growth and enhanced resistance to hyperoxia. It was concluded that nitric oxide strongly stimulates HO-1 gene expression.

Background The duodenal ulcer promoting gene (dupA) and dupA cluster in Helicobacter pylori have been described as a risk factor for duodenal ulcer development in some populations. Polymorphic gene dupA can be divided into two groups, intact dupA1 (long or short type based on the presence or absence of 615-bp extra sequences at the 5' region) having complete reading frame and other truncated dupA2 having frame-shift mutation. This study was aimed to elucidate the role of dupA of H. pylori and their clusters in the disease manifestation of Indian population. Methods A total of 170 H. pylori strains were screened for the presence of dupA, dupA alleles and dupA cluster by PCR and sequencing. Pro-inflammatory cytokine (IL-8) with different dupA variant H. pylori stimulated gastric epithelial cells (AGS cells) was measured by ELISA. Results A total of 50 strains (29.4%) were positive for dupA among the tested 170 strains. The prevalence of dupA1 in duodenal ulcer (DU) and non-ulcer dyspepsia (NUD) populations was found to be 25.5% (25/98) and 11.1% (8/72), respectively and 16.4% (28/170) of the tested strains had dupA1, cagA and vacAs1m1 positive. The distribution of long and short type dupA1 has not been significantly associated with the disease outcome. The dupA cluster analysis showed that 10.2% (10/98) and 8.3% (6/72) strains were positive among DU and NUD, respectively. IL-8 production was significantly higher in dupA1.sup.+, cagA.sup.+, vacA.sup.+ (902.5 [+ or -] 79.01 pg/mL) than dupA2.sup.+, cagA.sup.+, vacA.sup.+ (536.0 [+ or -] 100.4 pg/mL, P = 0.008) and dupA.sup.-, cagA.sup.+, vacA.sup.+ (549.7 [+ or -] 104.1 pg/mL, P = 0.009). Phylogenetic analysis of dupA indicated that the Indian H. pylori strains clustered with East Asian strains but distinct from Western strains. This is the first known genetic element of Indian H. pylori that is genetically closer to the East Asian strains but differed from the Western strains. Conclusions The intact dupA1 was significantly associated with DU than NUD (P = 0.029) but the dupA1 cluster has no role in the disease manifestation at India (P = 0.79). Thus, dupA1 can be considered as a biomarker for DU patients in India. Keywords: Helicobacter pylori, Duodenal ulcer, dupA, dupA cluster, Non-ulcer dyspepsia, Disease association, Author(s): Jawed Alam[sup.1] , Prachetash Ghosh[sup.1] , Mou Ganguly[sup.1] , Avijit Sarkar[sup.1] , Ronita De[sup.1] and Asish K Mukhopadhyay[sup.1] Introduction Infection caused by H. pylori is a growing concern as [...]

Abstract Background Infection with Helicobacter pylori strains that express CagA is associated with gastritis, peptic ulcer disease, and gastric adenocarcinoma. The biological function of CagA depends on tyrosine phosphorylation by a cellular kinase. The phosphate acceptor tyrosine moiety is present within the EPIYA motif at the C-terminal region of the protein. This region is highly polymorphic due to variations in the number of EPIYA motifs and the polymorphism found in spacer regions among EPIYA motifs. The aim of this study was to analyze the polymorphism at the C-terminal end of CagA and to evaluate its association with the clinical status of the host in West Bengal, India. Results Seventy-seven H. pylori strains isolated from patients with various clinical statuses were used to characterize the C-ternimal polymorphic region of CagA. Our analysis showed that there is no correlation between the previously described CagA types and various disease outcomes in Indian context. Further analyses of different CagA structures revealed that the repeat units in the spacer sequences within the EPIYA motifs are actually more discrete than the previously proposed models of CagA variants. Conclusion Our analyses suggest that EPIYA motifs as well as the spacer sequence units are present as distinct insertions and deletions, which possibly have arisen from extensive recombination events. Moreover, we have identified several new CagA types, which could not be typed by the existing systems and therefore, we have proposed a new typing system. We hypothesize that a cagA gene encoding higher number EPIYA motifs may perhaps have arisen from cagA genes that encode lesser EPIYA motifs by acquisition of DNA segments through recombination events.

We previously demonstrated that protein expression of both [[Alpha].sub.x]- and [[Alpha].sub.2]-catalytic subunits of the [Na.sup.+] -[K.sup.+] -ATPase is elevated after a 2- to 4-day chronic cyclic stretch regimen in cultured aortic smooth muscle cells (ASMC). In the present study, we investigated whether cyclic stretch affects mRNA expression of the a-isoforms of the [Na.sup.+] -[K.sup.+] -ATPase. Using a stretch apparatus, rat ASMC were cyclically stretched 10 or 20% of their length for 1, 3, or 6 h. [Alpha]-Isoform mRNA levels were measured using Northern analysis. A 3-h 10% stretch had no significant affect on mRNA expression for either isoform, but a 20% stretch increased mRNA of both isoforms approximately twofold. Whereas a 6-h 20% stretch increased [[Alpha].sub.1] mRNA by 3.3-fold, [[Alpha].sub.2] was not affected any further. Actinomycin D blocked the stretch-induced stimulation of mRNA expression of both [Alpha]-subunits. In conclusion, cyclic stretch stimulates the mRNA expression of both [[Alpha].sub.1]- and [[Alpha].sub.2]-subunits of [Na.sup.+] -[K.sup.+] -ATPase. The sensitivity of the two genes to the degree and duration of stretch is different. The stretch-induced increase of mRNA may be a result of increased transcription. sodium pump; transcription

AP-1 and STAT mediate hyperoxia-induced gene transcription of heme oxygenase-l. Am J Physiol Lung Cell Mol Physiol 279: L175-L182, 2000.--We have previously shown marked induction of the stress-inducible gene heme oxygenase-1 (HO-1) in vivo and in vitro after hyperoxia. In RAW 264.7 cells, HO-1 induction is transcriptionally regulated and dependent on cooperation between the HO-1 gene promoter and the 5' distal enhancer element SX2. In our present study, further deletional and mutational analyses demonstrate that signal transducer and activator of transcription (STAT) DNA binding sites located in the promoter of HO-1 and activator protein (AP)-1 DNA binding sites in the distal enhancer element SX2 are necessary for optimal HO-1 gene activation after hyperoxia. Interestingly, a second 5' distal enhancer element, AB1, located 10 kb upstream from the HO-1 promoter, alone is activated after hyperoxia but cannot confer maximal hyperoxia-induced HO-1 gene transcription. Mutational analysis of the AB1 enhancer shows that AP-1 is essential for ABl-mediated HO-1 gene transcription after hyperoxia. Electromobility shift assays show increased STAT1, STAT3, STAT5, and AP-1 DNA binding activity in RAW 264.7 cells after hyperoxia. Taken together, our data suggest that the 5' distal enhancer elements of the HO-1 gene in concert with the promoter regulate HO-1 gene induction and highlight the complexity of HO-1 gene transcription in response to hyperoxia. signal transducer and activator of transcription; activator protein-1; gene regulation; oxidative stress; transcription factor; oxygen

Petrache, Irina, Leo E. Otterbein, Jawed Alam, Gordon W. Wiegand, and Augustine M. K. Choi. Heme oxygenase-1 inhibits TNF-a-induced apoptosis in cultured fibroblasts. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L312-L319, 2000.--Heine oxygenase (HO)-1 catalyzes the oxidative cleavage of heme to yield equimolar amounts of biliverdin, iron, and carbon monoxide. HO-1 is a stress response protein, the induction of which is associated with protection against oxidative stress. The mechanism(s) of protection is not completely elucidated, although it is suggested that one or more of the catalytic by-products provide antioxidant functions either directly or indirectly. The involvement of reactive oxygen species in apoptosis raised the question of a possible role for HO-1 in programmed cell death. Using the tetracycline-regulated expression system, we show here that conditional overexpression of HO-1 prevents tumor necrosis factor-[Alpha]-induced apoptosis in murine L929 fibroblasts. Inhibition of apoptosis was not observed in the presence of tin protoporphyrin, a specific inhibitor of HO activity, and in cells overexpressing antisense HO-1. Interestingly, exogenous administration of a low concentration of carbon monoxide also prevented tumor necrosis factor-[Alpha]-induced apoptosis in L929 fibroblasts. Inhibition of tumor necrosis factor-[Alpha]-induced apoptosis by HO-1 overexpression was reversed by 1H-(1,2,4)oxadiazolo(4,3-[Alpha])quinoxalin-1-one, an inhibitor of guanylate cyclase, which is a target enzyme for carbon monoxide. Taken together, our data suggest that the antiapoptotic effect of HO-1 may be mediated via carbon monoxide. tumor necrosis factor-[Alpha]; programmed cell death; carbon monoxide; oxidants; reactive oxygen species