Induced Heme Oxygenase-1 mRNA Research Articles

Cytoprotective effects of heme oxygenase-1 induction by 3- O-caffeoyl-1-methylquinic acid

The novel antioxidant 3- O-caffeoyl-one-methylquinic acid (MCGA3) is a methyl chlorogenic acid derivative isolated from bamboo leaves. MCGA3 scavenges reactive oxygen species (ROS) and inhibits lipid peroxidation and xanthine oxidase in vitro. In this study, we evaluated the cytoprotective effect of MCGA3, which occurs via heme oxygenase-1 (HO-1) induction in bovine vascular endothelial cells exposed to tert-butylhydroperoxide (tBHP). Cells treated with 1 mM tBHP (6–18 h) generated substantial ROS and concomitantly lost most intracellular lactate dehydrogenase (LDH), which then caused necrotic cell death. Of the several MCGA antioxidants and structurally related phenolic acids examined in this study, MCGA3 (0.01–0.15 mM) was found to completely block this necrosis and generation of ROS by tBHP. Surprisingly, MCGA3 by itself was found to be a potent inducer of HO-1. We observed the time- and dose-dependent induction of HO-1 mRNA and protein, which was closely associated with decreased intracellular ROS and necrosis against tBHP. Deesterified or Al-chelated MCGA3 or co-treatment with MCGA3 and actinomycin D abolished HO-1 induction and the antinecrotic effect of MCGA3. Zinc protoporphyrin IX and cycloheximide attenuated the cytoprotection afforded by MCGA3, but did not reduce HO-1 mRNA. Interestingly, N-acetylcysteine (1 mM) enhanced the HO-1 induction of MCGA3, but N-acetylcysteine itself did not induce HO-1. These results suggested that not only ortho-dihydroxyl groups but also aromatic ester and methoxyl ester moieties are necessary for full HO-1 induction and cytoprotection against toxic tBHP-derived ROS. Ferritin mRNA was also upregulated during all HO-1 induction by MCGA3, which might decrease iron and lower ROS levels. Consequently, the combined action of HO-1 and ferritin may protect cells from toxic tBHP-mediated necrosis.

Gastroprotection by vitamin C—a heme oxygenase-1-dependent mechanism?

Free oxygen radicals contribute to gastric mucosal damage induced by acetylic–salicylic acid (ASA). Vitamin C has been shown to reduce gastric toxicity of ASA in humans. We intended to assess the role of heme oxygenase-1 (HO-1) in this process by application of these substances to AGS and KATO III cells. HO-1 expression was monitored by real-time RT-PCR, Western blot, and HO activity measurement. HO-1 mRNA was significantly elevated by either ASA or vitamin C in gastric epithelial cells, combination of both substances further increased expression. HO-1 protein and enzyme activity rose in cells exposed to vitamin C alone or combined with ASA, but not after stimulation with ASA alone. In contrast to endothelia, in which ASA simultaneously induces HO-1 mRNA and protein expression, gastric epithelial cells require vitamin C to translate HO-1 mRNA into active protein, which then may exert gastroprotection by its antioxidant and vasodilative properties.

Increased Constitutive c-Jun N-terminal Kinase Signaling in Mice Lacking Glutathione S-Transferase Pi

Glutathione S-transferase Pi (GSTP) detoxifies electrophiles by catalyzing their conjugation with reduced glutathione. A second function of this protein in cell defense has recently been proposed that is related to its ability to interact with c-Jun N-terminal kinase (JNK). The present study aimed to determine whether this interaction results in increased constitutive JNK activity in the absence of GSTP in GstP1/P2(-/-) mice and whether such a phenomenon leads to the up-regulation of genes that are relevant to cell defense. We found a significant increase in constitutive JNK activity in the liver and lung of GstP1/P2-/- compared with GstP1/P2(+/+) mice. The greatest increase in constitutive JNK activity was observed in null liver and was accompanied by a significant increase in activator protein-1 DNA binding activity (8-fold) and in the mRNA levels for the antioxidant protein heme oxygenase-1 compared with wild type. Furthermore UDP-glucuronosyltransferase 1A6 mRNA levels were significantly higher in the livers of GstP1/P2(-/-) compared with GstP1/P2(+/+) mice, which correlated to a 2-fold increase in constitutive activity both in vitro and in vivo. There was no difference in the gene expression of other UDP-glucuronosyltransferase isoforms, manganese superoxide dismutase, microsomal epoxide hydrolase, or GSTA1 between GstP1/P2(-/-) and GstP1/P2(+/+) mice. Additionally there was no phenotypic difference in the induction of heme oxygenase-1 mRNA after acetaminophen administration. This study not only demonstrates the role of GSTP as a direct inhibitor of JNK in vivo but also its role in regulating the constitutive expression of specific downstream molecular targets of the JNK signaling pathway.

Transcriptional Regulation of Heme Oxygenase-1 Gene Expression by MAP Kinases of the JNK and p38 Pathways in Primary Cultures of Rat Hepatocytes

Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with -754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38alpha, beta, and delta decreased and p38gamma increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (-668/-654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (-47/-42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediates HO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box.

Nerve Growth Factor Protects against 6-Hydroxydopamine-induced Oxidative Stress by Increasing Expression of Heme Oxygenase-1 in a Phosphatidylinositol 3-Kinase-dependent Manner

The survival signal elicited by the phosphatidylinositol 3-kinase (PI3K)/Akt1 pathway has been correlated with inactivation of pro-apoptotic proteins and attenuation of the general stress-induced increase in reactive oxygen species (ROS). However, the mechanisms by which this pathway regulates intracellular ROS levels remain largely unexplored. In this study, we demonstrate that nerve growth factor (NGF) prevents the accumulation of ROS in dopaminergic PC12 cells challenged with the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism that involves PI3K/Akt-dependent induction of the stress response protein heme oxygenase-1 (HO-1). The effect of NGF was mimicked by induction of HO-1 expression with CoCl(2); by treatment with bilirubin, an end product of heme catabolism; and by infection with a retroviral expression vector for human HO-1. The relevance of HO-1 in NGF-induced ROS reduction was further demonstrated by the evidence that cells treated with the HO-1 inhibitor tin-protoporphyrin or infected with a retroviral expression vector for antisense HO-1 exhibited enhanced ROS release in response to 6-OHDA, despite the presence of the neurotrophin. Inhibition of PI3K prevented NGF induction of HO-1 mRNA and protein and partially reversed its protective effect against 6-OHDA-induced ROS release. By contrast, cells transfected with a membrane-targeted active version of Akt1 exhibited increased HO-1 expression, even in the absence of NGF, and displayed a greatly attenuated production of ROS and apoptosis in response to 6-OHDA. These observations indicate that the PI3K/Akt pathway controls the intracellular levels of ROS by regulating the expression of the antioxidant enzyme HO-1.

Tissue-dependent induction of heme oxygenase-1 and metallothionein-1/2 by methyl methanesulfonate.

Methyl methanesulfonate (MMS), a methylating agent, is known to be a genotoxicant in testis. The purpose of this study was to investigate roles of oxidative stress-responsive proteins, heme oxygenase-1 (HO-1) and metallothionein-1/2 (MT-1/2), in genotoxicity of MMS. Cadmium, a potent genotoxicity inducer, induced HO-1 and MT-1/2 in rat livers and kidneys. Then we comparatively investigated MMS-induced HO-1 and MT-1/2 in rat livers, kidneys and testes. We found that a single administration of MMS (40 mg/kg) resulted in the induction of MT-1/2 mRNA in the liver, but not HO-1 mRNA, reaching maximum level at 6 hr and returning to the control levels by 24 hr. Interestingly, MMS induced both HO-1 and MT-1/2 mRNAs in the kidney. In contrast, MMS induced HO-1 mRNA, but not MT-1/2 mRNA in the testis. Since HO-1 and MT-1/2 have been recognized to respond to various oxidative stimuli, we further examined the inducing effect of MMS on these two proteins. MMS at dosages of 20 to 40 mg/kg for 2 consecutive weeks induced HO-1 mRNA (123 to 187% of the control) and protein (274 to 404% of the control) in rat testes. However, MT-1/2 mRNA was not induced by MMS administration, although a high level of expression was observed in comparison with the liver and kidney. These findings suggest that MMS induces HO-1 and/or MT-1/2 mRNA and its protein tissue-dependently, and the heme catabolites by HO-1 in the testis may contribute in some manner to its genotoxicity.

TGF-beta1 stimulates HO-1 via the p38 mitogen-activated protein kinase in A549 pulmonary epithelial cells.

In lung injury and progressive lung diseases, the multifunctional cytokine transforming growth factor-beta1 (TGF-beta1) modulates inflammatory responses and wound repair. Heme oxygenase-1 (HO-1) is a stress-inducible protein that has been demonstrated to confer cytoprotection against oxidative injury and provide a vital function in maintaining tissue homeostasis. Here we report that TGF-beta1 is a potent inducer of HO-1 and examined the signaling pathway by which TGF-beta1 regulates HO-1 expression in human lung epithelial cells (A549). TGF-beta1 (1-5 ng/ml) treatment resulted in a marked time-dependent induction of HO-1 mRNA in A549 cells, followed by corresponding increases in HO-1 protein and HO enzymatic activity. Actinomycin D and cycloheximide inhibited TGF-beta1-responsive HO-1 mRNA expression, indicating a requirement for transcription and de novo protein synthesis. Furthermore, TGF-beta1 rapidly activated the p38 mitogen-activated protein kinase (p38 MAPK) pathway in A549 cells. A chemical inhibitor of p38 MAPK (SB-203580) abolished TGF-beta1-inducible HO-1 mRNA expression. Both SB-203580 and expression of a dominant-negative mutant of p38 MAPK inhibited TGF-beta1-induced ho-1 gene activation, as assayed by luciferase activity of an ho-1 enhancer/luciferase fusion construct (pMHO1luc-33+SX2). These studies demonstrate the critical intermediacy of the p38 MAPK pathway in the regulation of HO-1 expression by TGF-beta1.

Induction of Heme Oxygenase 1 by Nitrosative Stress: A ROLE FOR NITROXYL ANION

Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO(+)), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997) J. Biol. Chem. 272, 18411-18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613-13620). Here, we report that Angeli's salt (AS) (0.25-2 mm), a compound that releases nitroxyl anion (NO(-)) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO(-)-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 degrees C (t(12) = 2.3 min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 microm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO(-), not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO(-) oxidation. N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO(-). We conclude that NO(-), in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress.

Tin chloride pretreatment prevents renal injury in rats with ischemic acute renal failure.

To investigate whether tin chloride pretreatment ameliorates renal injury in rats with ischemic acute renal failure (IARF) by virtue of its kidney-specific heme oxygenase-1 induction. Randomized, masked, controlled animal study. University-based animal research facility. Sprague-Dawley male rats, weighing 200-230 g (n = 359). Rats were injected with tin chloride subcutaneously, because subcutaneous administration of tin chloride is known to specifically and potently induce renal heme oxygenase activity in the rat. Anesthetized rats were subjected to bilateral flank incisions, and the right kidney was removed. Renal ischemia for 40 mins was performed by left renal microvascular clamping, followed by reflow of the blood. Tin chloride treatment specifically induced heme oxygenase-1 mRNA and protein in the proximal tubular epithelial cells of the kidney without apparent cell injury in the rat. Tin chloride treatment before renal ischemia augmented the induction of heme oxygenase-1 in IARF rats at both transcriptional and protein concentrations in the renal epithelial cells compared with IARF animals. Tin chloride pretreatment, which decreased microsomal heme concentration, ameliorated the ischemic renal injury as judged by the significant decrease in serum creatinine and blood urea nitrogen concentrations and the lesser tubular epithelial cell injuries. In contrast, inhibition of heme oxygenase activity by treatment with tin mesoporphyrin, which increased microsomal heme concentration, abolished the beneficial effect of tin chloride pretreatment. These findings indicate that tin chloride pretreatment significantly ameliorates renal injury in rats with IARF by virtue of its specific heme oxygenase-1 induction in renal epithelial cells. These findings also suggest that heme oxygenase-1 induction plays an important role in protecting renal cells from oxidative damage caused by heme.

1,2,3,4,6-Penta- O-galloyl-beta- d-glucose protects rat neuronal cells (Neuro 2A) from hydrogen peroxide-mediated cell death via the induction of heme oxygenase-1

The root of Paeonia suffruticosa ANDREWS is an important Chinese crude drug used in many traditional prescriptions. 1,2,3,4,6-Penta- O-galloyl-β- d-glucose (PGG), a major component of this crude drug, has been shown to possess potent anti-oxidant, anti-mutagenic and anti-proliferative effects. In the present study, we examined the effect of PGG on the expression of neuronal heme oxygenase-1 (HO-1), an inducible stress protein that degrades heme to the neuroactive molecule, carbon monoxide and the anti-oxidant, biliverdin. Exposure of Neuro 2A cells to PGG (10–50 μM) resulted in a concentration- and time-dependent induction of HO-1 mRNA, and protein expressions and heme oxygenase activity. Interestingly, pretreatment of the neuronal cells with PGG resulted in enhanced cellular resistance to hydrogen peroxide. This cytoprotective effect was reversed by zinc protoporphyrin IX, an inhibitor of heme oxygenase. This study showed that PGG could protect neuronal cells from oxidative stress via the induction of HO-1 gene expression.

Effects of modulators of protein phosphorylation on heme metabolism in human hepatic cells: induction of delta-aminolevulinic synthase mRNA and protein by okadaic acid.

Effects of modulators of protein phosphorylation on delta-aminolevulinic acid (ALA) synthase and heme oxygenase-1 mRNA were analyzed in the human hepatic cell lines Huh-7 and HepG2 using a quantitative RNase protection assay. Okadaic acid was found to induce ALA synthase mRNA in a concentration-dependent fashion in both Huh-7 and HepG2 cells. The EC(50) for induction of ALA synthase mRNA in Huh-7 cells was 13.5 nM, with maximum increases occurring at okadaic acid concentrations of 25-50 nM. The EC(50) for induction of ALA synthase mRNA in HepG2 cells was 35.5 nM, with maximum increases occurring at okadaic acid concentrations of 50 nM. Concentration-dependent induction of ALA synthase mRNA paralleled the increase in ALA synthase protein. Maximum induction of ALA synthase was observed between 5 and 10 h post-treatment in both cell lines. Induction of ALA synthase mRNA in Huh-7 cells, but not HepG2 cells, was associated with an increase in ALA synthase mRNA stability. Okadaic acid also induced heme oxygenase-1 mRNA in both cell lines, but the magnitude of induction was only twofold, and was rapid and transient. Okadaic acid and phorbol 12-myristate 13-acetate significantly decreased heme-mediated induction of heme oxygenase-1 mRNA in both Huh-7 and HepG2 cells. Wortmannin diminished the heme-mediated induction of heme oxygenase-1 mRNA in HepG2 cells, but not Huh-7 cells. These results report a novel property of okadaic acid to affect heme metabolism in human cell lines.

Involvement of tumor necrosis factor α, rather than interleukin-1α/β or nitric oxides in the heme oxygenase-1 gene expression by lipopolysaccharide in the mouse liver

Heme oxygenase-1 (HO-1) is induced under various oxidative stress conditions, such as lipopolysaccharide (LPS) insult. Induction of HO-1 by LPS is reported to be mediated through interleukin-1β (IL-1β), rather than other inflammatory cytokines in the mouse liver. However, we found that IL-1α/β knockout (KO) mice responded well to LPS insult, as did wild-type mice with respect to HO-1 mRNA induction (about 30-fold increase). In contrast, tumor necrosis factor α KO (TNFαKO) mice responded very weakly to LPS in the HO-1 mRNA expression, but not metallothionein mRNA. Recent studies reveal that nitric oxide from Kupffer cells is involved in HO-1 induction in the liver produced by LPS. Therefore, nitrite and nitrate concentrations in the liver were also measured and these parameters did not increase in either IL-1KO or TNFαKO. In addition, the phosphorylation of c-JUN N-terminal kinase (JNK) and p38, but not extracellular signal-regulated kinase, was very low in TNFαKO mice due to LPS administration. All of these findings indicate that TNFα is a major candidate to trigger HO-1 induction in response to LPS stimulation, and that its message is likely transduced through JNK and p38 pathways.

Glucose Deprivation Induces Heme Oxygenase-1 Gene Expression by a Pathway Independent of the Unfolded Protein Response

Nutrients such as glucose regulate the expression of genes that are involved in plasma membrane transport, metabolic functions, and protein trafficking in the endoplasmic reticulum. Depletion of nutrients results in cellular stress, which evokes adaptive and protective responses, one of which is the induction of heme oxygenase-1 (HO-1), a 32-kDa endoplasmic reticulum enzyme that catalyzes the rate-limiting step in heme degradation. Incubation of HepG2 human hepatoma cells in glucose-free medium resulted in an increased HO-1 mRNA content, reaching a maximum of approximately 25-fold over control cells after 12 h. The glucose-dependent induction of HO-1 mRNA was concentration-dependent (k(12) approximately 0.5 mm) and was attenuated by fructose, galactose, mannose, and 2-deoxyglucose, but not by the non-metabolizable glucose analog, 3-O-methylglucose. Tunicamycin, thapsigargin, or azetidine 2-carboxylate, each of which activates the unfolded protein response pathway, did not induce HO-1 mRNA expression, whereas glucose-regulated protein 78 mRNA was increased. These results demonstrate that glucose availability regulates transcription of the HO-1 gene via a pathway that is different from the unfolded protein response. The induction of HO-1 may serve as a protective response in hypoglycemic circumstances and underscores the importance of understanding nutrient control of the HO-1 gene.

Mechanism of heme oxygenase-1 gene induction by curcumin in human renal proximal tubule cells

First published August 9, 2001; 10.1152/ajprenal.00140.2001.—Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, releasing iron, carbon monoxide, and biliverdin. Induction of HO-1 occurs as an adaptive and protective response to several inflammatory stimuli. The transcription factor activator protein-1 (AP-1) has been implicated in the activation of the HO-1 gene. To elucidate the molecular mechanism of HO-1 induction, we examined the effects of diferuloylmethane (curcumin), an inhibitor of the transcription factor AP-1. Surprisingly, curcumin by itself was a very potent inducer of HO-1. Curcumin has anti-inflammatory, antioxidant, and renoprotective effects. To evaluate the mechanism of curcumin-mediated induction of HO-1, confluent human renal proximal tubule cells were exposed to curcumin (1–8 μM). We observed a time- and dose-dependent induction of HO-1 mRNA that was associated with increased HO-1 protein. Coincubation of curcumin with actinomycin D completely blocked the upregulation of HO-1 mRNA. Blockade of nuclear factor-κB (NF-κB) with an IκBα phosphorylation inhibitor attenuated curcumin-mediated induction of HO-1 mRNA and protein. These data demonstrate that curcumin induces HO-1 mRNA and protein in renal proximal tubule cells. HO-1 induction by curcumin is mediated, at least in part, via transcriptional mechanisms and involves the NF-κB pathway.

Mechanism of heme oxygenase-1 gene induction by curcumin in human renal proximal tubule cells.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, releasing iron, carbon monoxide, and biliverdin. Induction of HO-1 occurs as an adaptive and protective response to several inflammatory stimuli. The transcription factor activator protein-1 (AP-1) has been implicated in the activation of the HO-1 gene. To elucidate the molecular mechanism of HO-1 induction, we examined the effects of diferuloylmethane (curcumin), an inhibitor of the transcription factor AP-1. Surprisingly, curcumin by itself was a very potent inducer of HO-1. Curcumin has anti-inflammatory, antioxidant, and renoprotective effects. To evaluate the mechanism of curcumin-mediated induction of HO-1, confluent human renal proximal tubule cells were exposed to curcumin (1-8 microM). We observed a time- and dose-dependent induction of HO-1 mRNA that was associated with increased HO-1 protein. Coincubation of curcumin with actinomycin D completely blocked the upregulation of HO-1 mRNA. Blockade of nuclear factor-kappaB (NF-kappaB) with an IkappaBalpha phosphorylation inhibitor attenuated curcumin-mediated induction of HO-1 mRNA and protein. These data demonstrate that curcumin induces HO-1 mRNA and protein in renal proximal tubule cells. HO-1 induction by curcumin is mediated, at least in part, via transcriptional mechanisms and involves the NF-kappaB pathway.

Increase in cyclooxygenase-2 and nitric oxide-synthase-2 mRNAs in pouchitis without modification of inducible isoenzyme heme-oxygenase-1.

The pathophysiology of pouchitis occurring after ileal pouch-anal anastomosis remains controversial. Prostaglandins and nitric oxide synthesized in excess by cyclooxygenase-2 and nitric oxide synthase-2 are thought to be involved in the inflammatory process. Because heme oxygenase-1, by its antioxidant properties, could modulate inflammatory reaction, we analyzed mRNAs of the three enzymes (cyclooxygenase-2, nitric oxide synthase-2, and heme oxygenase-1) in patients with ileal pouch-anal anastomosis. Endoscopic biopsies were obtained in eight patients with normal ileal pouch-anal anastomosis, in eight patients with pouchitis, and in normal ileum of six healthy subjects. A relative quantitative RT-PCR was performed to determine the levels of cyclooxygenase-2, nitric oxide synthase-2, and heme oxygenase-1 mRNAs. Cyclooxygenase-2 and nitric oxide synthase-2 mRNAs were increased both in normal ileal pouch-anal anastomosis and in pouchitis, compared with healthy subjects. Pouchitis disease activity index was correlated with mRNA levels of cyclooxygenase-2 (r = 0.71; p < 0.01) and nitric oxide synthase-2 (r = 0.51; p < 0.05). Heme oxygenase-1 mRNA levels were not significantly different in patients versus healthy subjects. The increase in cyclooxygenase-2 and nitric oxide synthase-2 mRNA levels both in pouchitis and normal ileal pouch-anal anastomosis demonstrates that a latent inflammatory process occurs in the ileal pouch mucosa. This inflammatory process was not found to be associated with an induction of heme oxygenase mRNA, a possible regulator of the inflammatory response.

Increase in cyclooxygenase-2 and nitric oxide–synthase-2 mRNAs in pouchitis without modification of inducible isoenzyme heme-oxygenase-1

OBJECTIVES: The pathophysiology of pouchitis occurring after ileal pouch–anal anastomosis remains controversial. Prostaglandins and nitric oxide synthesized in excess by cyclooxygenase-2 and nitric oxide synthase-2 are thought to be involved in the inflammatory process. Because heme oxygenase-1, by its antioxidant properties, could modulate inflammatory reaction, we analyzed mRNAs of the three enzymes (cyclooxygenase-2, nitric oxide synthase-2, and heme oxygenase-1) in patients with ileal pouch–anal anastomosis. METHODS: Endoscopic biopsies were obtained in eight patients with normal ileal pouch–anal anastomosis, in eight patients with pouchitis, and in normal ileum of six healthy subjects. A relative quantitative RT-PCR was performed to determine the levels of cyclooxygenase-2, nitric oxide synthase-2, and heme oxygenase-1 mRNAs. RESULTS: Cyclooxygenase-2 and nitric oxide synthase-2 mRNAs were increased both in normal ileal pouch–anal anastomosis and in pouchitis, compared with healthy subjects. Pouchitis disease activity index was correlated with mRNA levels of cyclooxygenase-2 ( r = 0.71; p < 0.01) and nitric oxide synthase-2 ( r = 0.51; p < 0.05). Heme oxygenase-1 mRNA levels were not significantly different in patients versus healthy subjects. CONCLUSIONS: The increase in cyclooxygenase-2 and nitric oxide synthase-2 mRNA levels both in pouchitis and normal ileal pouch–anal anastomosis demonstrates that a latent inflammatory process occurs in the ileal pouch mucosa. This inflammatory process was not found to be associated with an induction of heme oxygenase mRNA, a possible regulator of the inflammatory response.

Density-dependent expression of FGF-2 in response to oxidative stress in RPE cells in vitro

Purpose. The purpose of this study is to demonstrate the effect of culture density on the steady state mRNA levels of fibroblast growth factor-2 (FGF-2) when retinal pigment epithelial (RPE) cells are subjected to oxidative stress in vitro. Methods. Subconfluent and confluent cultures of the established RPE cell line ARPE-19, were treated with increasing concentrations of tert-butyl hydroperoxide (tBH) or hydrogen peroxide (H 2 O 2) . Cell viability was measured using the WST-1 assay, and intracellular reactive oxygen intermediate (ROI) production was quantified by dichlorofluoroscein (DCF) fluorescence. Steady state changes in heme oxygenase-1 (HO-1) and FGF-2 mRNAs were measured by Northern blot analysis. Results. Confluent cultures of ARPE-19 cells were less susceptible than subconfluent cultures to the toxic effects of the chemical oxidants. The intracellular reactive oxygen intermediate production was higher in subconfluent than confluent cultres with increasing tBH concentration. At nontoxic concentrations of tBH and H 2 O 2, a dose dependent increase in FGF-2 expression was seen as a function of culture density. FGF-2 mRNA expression was induced after tBH treatment in subconfluent, but not confluent cells. On the other hand, FGF-2 mRNA induction was observed after H 2 O 2 treatment in confluent, but not subconfluent cultures. In contrast, no density dependent induction of HO-1 mRNA was seen after treatment with either tBH or H 2 O 2. Conclusions. These results suggest that care should be taken to control for cell density in similar types of in vitro experiments.

Smad7-dependent Regulation of Heme Oxygenase-1 by Transforming Growth Factor-β in Human Renal Epithelial Cells

Heme oxygenase-1 (HO-1), a 32-kDa microsomal enzyme, is induced as a beneficial and adaptive response in cells/tissues exposed to oxidative stress. Transforming growth factor-beta1 (TGF-beta1) is a regulatory cytokine that has been implicated in a variety of renal diseases where it promotes extracellular matrix deposition and proinflammatory events. We hypothesize that the release of TGF-beta1 via autocrine and/or paracrine pathways may induce HO-1 and serve as a protective response in renal injury. To understand the molecular mechanism of HO-1 induction by TGF-beta1, we exposed confluent human renal proximal tubule cells to TGF-beta1 and observed a significant induction of HO-1 mRNA at 4 h with a maximal induction at 8 h. This induction was accompanied by increased expression of HO-1 protein. TGF-beta1 treatment in conjunction with actinomycin D or cycloheximide demonstrated that induction of HO-1 mRNA requires de novo transcription and, in part, protein synthesis. Exposure to TGF-beta1 resulted in marked induction of Smad7 mRNA with no effect on Smad6 expression. Overexpression of Smad7, but not Smad6, inhibited TGF-beta1-mediated induction of endogenous HO-1 gene expression. We speculate that the induction of HO-1 in the kidney is an adaptive response to the inflammatory effects of TGF-beta1 and manipulations of the Smad pathway to alter HO-1 expression may serve as a potential therapeutic target.

15-Deoxy-Δ12,14-prostaglandin J2facilitates thyroglobulin production by cultured human thyrocytes

A cyclopentenone-type prostaglandin, 15-deoxy-Delta(12, 14)-prostaglandin J(2) (15-d-PGJ(2)), has been shown to induce the cellular stress response and to be a ligand for the peroxisome proliferator-activated receptor (PPAR)-gamma. We studied its effect on the basal and thyrotropin (TSH)-induced production of thyroglobulin (TG) by human thyrocytes cultured in the presence of 10% FBS. In 15-d-PGJ(2)-treated cells in which the agent itself did not stimulate cAMP production, both the basal production of TG and the response to TSH were facilitated, including the production of TG and cAMP, whereas such production was decreased in untreated cells according to duration of culture. PGD(2) and PGJ(2), which are precursors to 15-d-PGJ(2), exhibited an effect similar to 15-d-PGJ(2). However, the antidiabetic thiazolidinediones known to be specific ligands for PPAR-gamma, and WY-14643, a specific PPAR-alpha ligand, lacked this effect. 15-d-PGJ(2) and its precursors, but not the thiazolidinediones, induced gene expression for heme oxygenase-1 (HO-1), a stress-related protein, and strongly inhibited interleukin-1 (IL-1)-induced nitric oxide (NO) production. Cyclopentenone-type PGs have been recently shown to inhibit nuclear factor-kappaB (NF-kappaB) activation via a direct and PPAR-independent inhibition of inhibitor-kappaB kinase, suggesting that, in human thyrocytes, such PGs may inhibit IL-1-induced NO production, possibly via an inhibition of NF-kappaB activation. On the other hand, sodium arsenite, a known activator of the stress response pathway, induced HO-1 mRNA expression but lacked a promoting effect on TG production. Thus 15-d-PGJ(2) and its precursors appear to facilitate TG production via a PPAR-independent mechanism and through a different pathway from the cellular stress response that is available to cyclopentenone-type PGs. Our findings reveal a novel role of these PGs associated with thyrocyte differentiation.