Heme Oxygenase-1 Transcription Research Articles

Histone methyltransferase Suv39h1 regulates hepatic stellate cell activation and is targetable in liver fibrosis

ObjectiveLiver fibrosis is a prelude to a host of end-stage liver diseases. Hepatic stellate cells (HSCs), switching from a quiescent state to myofibroblasts, are the major source for excessive production...

EZH2 Promotes Corneal Endothelial Cell Apoptosis by Mediating H3K27me3 and Inhibiting HO-1 Transcription

Purpose This paper aims to explore the molecular mechanism of Enhancer of Zeste Homolog 2 (EZH2)-mediated H3K27me3 in human corneal endothelial cells (HCEC) apoptosis by inhibiting Heme oxygenase-1 (HO-1) transcription to provide a potential target for the treatment of corneal apoptosis. Methods HCECs were cultured in vitro and transfected with si-EZH2, pcDNA3.1-EZH2, pcDNA3.1-HO-1, GSK-J4 (an effective H3K27me3 demethylase inhibitor), and corresponding controls. Western Blot assay was used to detect the levels of EZH2, HO-1, H3K27me3, and apoptosis-related proteins (Bcl-2, Bax, and Cleaved-caspase-3) in HCECs; CCK-8 assay was conducted to detect cell viability and flow cytometry to analyze the apoptosis. HO-1 mRNA levels were detected by RT-qPCR and changes in H3K27me3 levels on the HO-1 promoter were detected by chromatin immunoprecipitation. Results HCECs transfected with si-EZH2 showed significantly lower EZH2 mRNA and protein levels, higher HCEC viability, lower apoptosis rates, higher antiapoptotic protein Bcl-2 expression, lower proapoptotic protein (Bax and Cleaved-caspase-3) levels, and significantly higher HO-1 expression. HCECs transfected with pcDNA3.1-EZH2 showed the opposite results. EZH2 repressed HO-1 transcription by mediating H3K27me3. H3K27me27 was enriched in the HO-1 promoter and overexpression of EZH2 increased H3K27me27 levels. Promotion of H3K27me3 partially reversed the mitigating effect of si-EZH2 on HCEC apoptosis. Overexpression of HO-1 partially reversed the apoptosis-promoting effects of EZH2 and H3K27me3 on HCECs. Conclusions EZH2 promotes HCE cell apoptosis by mediating H3K27me3 to inhibit HO-1 transcription.

EHMT2 affects microglia polarization and aggravates neuronal damage and inflammatory response via regulating HMOX1.

This research was designed to ascertain the function of euchromatic histone lysine methyltransferase 2 (EHMT2) in ischemic stroke-induced neuronal damage and inflammatory response and its regulatory mechanism. Mouse microglia (BV-2 cells) were induced by oxygen glucose deprivation/reoxygenation (OGD/R) to establish a cellular model, and then co-cultured with HT22 hippocampal neurons. After that, HT22 cell viability and apoptosis were evaluated, followed by the measurement of apoptosis-related factors (B-cell lymphoma-2, Bcl-2 associated X, and cleaved-Caspase 3). Meanwhile, the expression of inducible nitric oxide synthase (M1 microglia polarization marker) and arginase 1 (M2 microglia polarization marker) in BV-2 cells was detected, as well as the levels of inflammatory factors (tumor necrosis factor-α, interleukin [IL]-6, IL-10, IL-1β, and IL-4). Additionally, the expression of EHMT2 and heme oxygenase 1 (HMOX1) in BV-2 cells was assessed by quantitative reverse transcription polymerase chain reaction and western blot, and the binding between EHMT2 and HMOX1 was predicted and verified. OGD/R treatment led to decreased cell viability and increased cell apoptosis in HT22 cells, and aggravated inflammatory response in BV-2 cells. In OGD/R-induced BV-2 cells, EHMT2 and HMOX1 were increasingly expressed, and knockdown of EHMT2 or HMOX1 in BV-2 cells could inhibit neuronal damage and inflammatory response. Moreover, EHMT2 promoted HMOX1 transcription level by histone methylation. Collected evidence showed that down-regulation of EHMT2 relieved neuronal damage and inflammatory response by inhibiting HMOX1 expression.

Anti-inflammatory activity of 2-methoxy-4-vinylphenol involves inhibition of lipopolysaccharide-induced inducible nitric oxidase synthase by heme oxygenase-1

Background 2-Methoxy-4-vinylphenol (2M4VP) is a natural anti-inflammatory compound derived from red wine, but its underlying mechanism remains unclear. Heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, inhibits NO gene expression, while nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor involved in HO-1 production, binds to the antioxidant response element (ARE) in the nucleus and promotes HO-1 transcription. Based on the hypothesis that the inhibitory effect of 2M4VP on NO production is mediated by HO-1, we examined the possible mechanism of the anti-inflammatory activity of 2M4VP in this study. Materials and methods The anti-inflammatory activity of 2M4VP was analyzed by Griess method, ELISA, qPCR, and Western blotting using LPS-treated macrophage lineage RAW264.7 cells. The impact of 2M4VP on the Nrf2/ARE pathway was also analyzed using immunocytochemistry and an ARE luciferase reporter using HEK293 cells. Results The results showed that 2M4VP reduced the production of LPS-induced NO and inducible nitric oxidase synthase (iNOS). In addition, 2M4VP increased the expression of HO-1, while pretreatment with the Nrf2 inhibitor ML385 downregulated HO-1 expression. 2M4VP induced Kelch-like ECH-associated protein 1 (Keap1) degradation. Furthermore, it promoted Nrf2 nuclear translocation and increased luciferase activity by binding to the ARE. Conclusions 2M4VP induces Keap1 degradation and promotes Nrf2 nuclear translocation. Activation of Nrf2/ARE pathway enhances HO-1 expression and leads to iNOS inhibition for anti-inflammatory function.

Photooxidation of A2E by Blue Light Regulates Heme Oxygenase 1 Expression via NF-κB and Lysine Methyltransferase 2A in ARPE-19 Cells.

Background: N-retinylidene-N-retinylethanolamine (A2E) is a component of drusen that accumulates in retinal cells and induces oxidative stress through photooxidation, such as blue light (BL). We found that the heme oxygenase 1 (HMOX1) gene responds sensitively to photooxidation by the BL of A2E in retinal pigment epithelial (RPE) cells, and we sought to identify the transcription factors and coactivators involved in the upregulation of HMOX1 by A2E and BL. Methods: A2E-laden human RPE cells (ARPE-19) were exposed to BL (430 nm). RNA sequencing was performed to identify genes responsive to BL exposure. Chromatin immunoprecipitation and RT-qPCR were performed to determine the regulation of HMOX1 transcription. Clinical transcriptome data were used to evaluate HMOX1 expression in patients with age-related macular degeneration (AMD). Results: In ARPE-19 cells, the expression of HMOX1, one of the NF-κB target genes, was significantly increased by A2E and BL. The binding of RELA and RNA polymerase II to the promoter region of HMOX1 was significantly increased by A2E and BL. Lysine methyltransferase 2A (MLL1) plays an important role in H3K4me3 methylation, NF-κB recruitment, chromatin remodeling at the HMOX1 promoter, and, subsequently, HMOX1 expression. The retinal tissues of patients with late-stage AMD showed significantly increased expression of HMOX1 compared to normal retinal tissues. In addition, the expression levels of MLL1 and HMOX1 in retinal tissues were correlated. Conclusions: Taken together, our results suggest that BL induces HMOX1 expression by activating NF-κB and MLL1 in RPE cells.

FOXO3a Alleviates the Inflammation and Oxidative Stress via Regulating TGF-β and HO-1 in Ankylosing Spondylitis.

This study aimed to investigate whether Forkhead box O3a (FOXO3a) modulates inflammation and oxidative stress in ankylosing spondylitis (AS). We applied bioinformatics analysis, quantitative real-time polymerase chain reaction, immunoblotting, enzyme linked immunosorbent assay, chromatin immunoprecipitation, and dual-luciferase reporter assay. Gene overexpression and knockdown of FOXO3a were conducted via lentivirus and small interfering RNA, respectively. Downregulated FOXO3a expression was first confirmed in AS patients. Interleukin-8 (IL-8) and IL-17A were highly expressed and negatively related with FOXO3a in AS. Total antioxidant capacity (T-AOC) were markedly decreased and positively associated with FOXO3a in AS. Overexpression of FOXO3a inhibited the secretion of inflammatory cytokines and promoted the production of antioxidant enzymes in Jurkat cells. Transforming growth factor-β (TGF-β) and heme oxygenase 1 (HO-1), which had binding sites to FOXO3a based on bioinformatics analysis, were abnormally expressed and positively related with FOXO3a. Accordingly, FOXO3a obviously elevated the protein and transcription levels of TGF-β and HO-1 in Jurkat cells. The above results were verified by silencing FOXO3a. Moreover, FOXO3a directly interacted with and promoted the transcription of TGF-β and HO-1. In summary, the modulation of cellular inflammation and oxidative stress via FOXO3a-mediated TGF-β and HO-1 activation is partly involved in the pathogenesis of AS.

Early Effects of Extracellular Vesicles Secreted by Adipose Tissue Mesenchymal Cells in Renal Ischemia Followed by Reperfusion: Mechanisms Rely on a Decrease in Mitochondrial Anion Superoxide Production.

Acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is characterized by intense anion superoxide (O2•−) production and oxidative damage. We investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administered during reperfusion can suppress the exacerbated mitochondrial O2•− formation after I/R. We used Wistar rats subjected to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received EVs (I/R + EVs group) or saline (I/R group) in the kidney subcapsular space. The third group consisted of false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and used immediately. Amplex Red™ was used to measure mitochondrial O2•− formation and MitoTracker™ Orange to evaluate inner mitochondrial membrane potential (Δψ). In vitro studies were carried out on human renal proximal tubular cells (HK-2) co-cultured or not with EVs under hypoxic conditions. Administration of EVs restored O2•− formation to SHAM levels in all mitochondrial functional conditions. The gene expression of catalase and superoxide dismutase-1 remained unmodified; transcription of heme oxygenase-1 (HO-1) was upregulated. The co-cultures of HK-2 cells with EVs revealed an intense decrease in apoptosis. We conclude that the mechanisms by which EVs favor long-term recovery of renal structures and functions after I/R rely on a decrease of mitochondrial O2•− formation with the aid of the upregulated antioxidant HO-1/Nuclear factor erythroid 2-related factor 2 system, thus opening new vistas for the treatment of AKI.

Protective Effects of Anthocyanins Extracted from Vaccinium Uliginosum on 661W Cells Against Microwave-Induced Retinal Damage.

To study the protective effect of anthocyanins extracted from Vaccinium Uliginosum (VU) on retinal 661W cells against microwave radiation induced retinal injury. 661W cells were divided into 6 groups, including control, model [661W cells radiated by microwave (30 mW/cm2, 1 h)] and VU groups [661W cells pretreated with anthocyanins extracted from VU (25, 50, 100 and 200 µg/mL, respectively) for 48 h, and radiated by microwave 30 mW/cm2, 1 h]. After treatment with different interventions, the cell apoptosis index (AI) was determined using Heochst staining; contents of malonaldehyde (MDA), glutataione (GSH), and activity of superoxide dismutase (SOD) were measured. mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1(HO-1) were detected by real time quantitative polymerase chain reaction, and the expression of HO-1 protein was examined by Western blot analysis. Nucleus and cytoplasm were separated and Nrf2 protein expression was further verified by Western blot analysis. There was significant difference in AI among the groups (F=322.83, P<;0.05). Compared with the control group, AI was significantly higher in the model group and was lower in 4 VU-pretreated groups (P<;0.05). Linear regression analysis showed the decline of AI was in a dose-dependent manner with VU treatment (r=0.8419, P<;0.05). The MDA and GSH contents of 661W cells in VU-treated groups were significantly lower than the model group (P<;0.05). Compared with the model group, the SOD activity in the VU-treated groups (50, 100 and 200 µg/mL) was significantly higher (all P<;0.05). The Nrf2 and HO-1 mRNA expressions were slightly increased after irradiation, and obviously increased in 100 µg/mL VU-treated group. After irradiation, the relative expressions of HO-1 and Nrf2 proteins in nucleus were slightly increased (P<;0.05), and the changes in cytoplasm were not obvious, whereas it was significantly increased in both nucleus and cytoplasm in the VU treatment groups. Anthocyanins extracted from VU could reduce apoptosis, stabilize cell membrane, and alleviate oxidant injury of mouse retinal photoreceptor 661W cells. The mechanism might be through activating Nrf2/HO-1 signal pathway and inducing HO-1 transcription and translation.

Protective Effect of Chrysanthemum morifolium cv. Fubaiju Hot-Water Extracts Against ARPE-19 Cell Oxidative Damage by Activating PI3K/Akt-Mediated Nrf2/HO-1 Signaling Pathway

Chrysanthemum morifolium cv. Fubaiju is a kind of widely consumed herb tea with multiple health benefits. The present study was aimed to evaluate the protective capacity of C. morifolium cv. Fubaiju hot-water extracts (CMs) against ARPE-19 cell oxidative damage. The results showed that pretreatment with 100 μg/mL CM could significantly reduce cell oxidative damage and apoptosis. Proapoptotic protein expression such as Bax, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP) was significantly decreased after CM addition, while the expression level of antioxidant enzymes including catalase, glutamate-cysteine ligase catalytic subunit (GCLc), superoxide dismutase 2 (SOD2), and NAD(P)H:quinone oxidoreductase 1 (NQO-1) was significantly promoted. Meanwhile, CM treatment upregulated Akt phosphorylation, nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, and the expression level of antioxidant gene heme oxygenase-1 (HO-1) in a dose-dependent manner under oxidative stress. Knockdown of Nrf2 by targeted small interfering RNA (siRNA) alleviated CM-mediated HO-1 transcription and almost abolished CM-mediated protection against hydrogen peroxide (H2O2)-induced cell damage. Correspondingly, the protective effect of CM was dramatically blocked after interference with phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor LY294002, indicating that the protective effect of CM on cell oxidative damage was attributed to PI3K/Akt-mediated Nrf2/HO-1 signaling pathway.

Long-Term Consumption of Anthocyanin-Rich Fruit Juice: Impact on Gut Microbiota and Antioxidant Markers in Lymphocytes of Healthy Males.

Polyphenols are considered protective against diseases associated with oxidative stress. Short-term intake of an anthocyanin-rich fruit juice resulted in significantly reduced deoxyribonucleic acid (DNA) strand-breaks in peripheral blood lymphocytes (PBLs) and affected antioxidant markers in healthy volunteers. Consequently, effects of long-term consumption of fruit juice are of particular interest. In focus was the impact on nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), the Nrf2-regulated genes NAD(P)H quinone oxidoreductase 1 (NQO-1) and heme oxygenase 1 (HO-1) as well as effects on the gut microbiota. In a nine-week placebo-controlled intervention trial with 57 healthy male volunteers, consumption of anthocyanin-rich juice significantly increased NQO-1 and HO-1 transcript levels in PBLs compared to a placebo beverage as measured by real-time polymerase chain reaction (PCR). Three Nrf2-promotor single nucleotide polymorphisms (SNPs), analyzed by pyrosequencing, indicated an association between individual Nrf2 transcript levels and genotype. Moreover, the Nrf2 genotype appeared to correlate with the presence of specific microbial organisms identified by 16S-PCR and classified as Spirochaetaceae. Furthermore, the microbial community was significantly affected by the duration of juice consumption and intake of juice itself. Taken together, long-term consumption of anthocyanin-rich fruit juice affected Nrf2-dependent transcription in PBLs, indicating systemic effects. Individual Nrf2 genotypes may influence the antioxidant response, thus requiring consideration in future intervention studies focusing on the Nrf2 pathway. Anthocyanin-rich fruit juice had an extensive impact on the gut microbiota.

Heme oxygenase promotes B-Raf-dependent melanosphere formation.

Biosynthesis and degradation of heme, an iron-bound protoporphyrin molecule utilized by a wide variety of metabolic processes, are tightly regulated. Two closely related enzymes, heme oxygenase 1 (HMOX1) and heme oxygenase 2 (HMOX2), degrade free heme to produce carbon monoxide, Fe2+ , and biliverdin. HMOX1 expression is controlled via the transcriptional activator, NFE2L2, and the transcriptional repressor, Bach1. Transcription of HMOX1 and other NFE2L2-dependent genes is increased in response to electrophilic and reactive oxygen species. Many tumor-derived cell lines have elevated levels of NFE2L2. Elevated expression of NFE2L2-dependent genes contributes to tumor growth and acquired resistance to therapies. Here, we report a novel role for heme oxygenase activity in melanosphere formation by human melanoma-derived cell lines. Transcriptional induction of HMOX1 through derepression of Bach1 or transcriptional activation of HMOX2 by oncogenic B-RafV600E results in increased melanosphere formation. Genetic ablation of HMOX1 diminishes melanosphere formation. Further, inhibition of heme oxygenase activity with tin protoporphyrin markedly reduces melanosphere formation driven by either Bach1 derepression or B-RafV600E expression. Global transcriptome analyses implicate genes involved in focal adhesion and extracellular matrix interactions in melanosphere formation.

Sulforaphane ameliorates serum starvation-induced muscle atrophy via activation of the Nrf2 pathway in cultured C2C12 cells.

Oxidative stress, an imbalance of redox homeostasis, contributes to the pathogenesis and progress of muscle atrophy. However, it is debated whether oxidative stress is a cause or consequence of muscle atrophy. In this study, we investigated the relationship between menadione-induced oxidative stress and serum starvation-induced muscle atrophy in C2C12 myotubes. We found that atrophic phenotypes including myotube diameter decrease, protein ubiquitination, and the expression of atrogenes were detected under oxidative stress as well as during serum starvation. Oxidative stress during serum starvation was assessed to confirm the correlation. Both intracellular reactive oxygen species (ROS) and protein oxidation were increased in atrophic myotubes. These results indicate that menadione-induced oxidative stress triggers muscle atrophy and vice versa. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular response to oxidative stress and it is considered to have a cytoprotective role in the mitigation of muscle atrophy. Transcription of heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase-1, target genes of Nrf2, was decreased during serum starvation, which is related to decreased nuclear translocation of Nrf2. Pre-treatment of sulforaphane (SFN), a known Nrf2 inducer, before serum starvation showed a protective effect via Nrf2/HO-1 upregulation. SFN can liberate Nrf2 from Keap1, enabling the nuclear translocation of Nrf2. Consequently, the expression of HO-1 increased and intracellular ROS was significantly reduced by SFN pre-treatment. These results demonstrate that oxidative stress mediates the pathophysiology of muscle atrophy, which can be improved via upregulation of the Nrf2-mediated antioxidant response.

Albumin Protects Lung Cells against Acrolein Cytotoxicity and Acrolein-Adducted Albumin Increases Heme Oxygenase 1 Transcripts.

Albumin is an abundant protein in the lung lining fluid that forms an interface between lung epithelial cells and the external environment. In the lung, albumin can be targeted for adduction by inhaled acrolein. Acrolein, an α,β-unsaturated aldehyde, reacts with biomolecules via Michael addition at the β-carbon or Schiff base formation at the carbonyl carbon. To gain insight into acrolein's mode of action, we investigated in vitro albumin-acrolein reactivity and the consequence of albumin adduction by acrolein on cytotoxicity and transcript changes in NCI-H441 and human airway epithelial cells (HAEC). Albumin protected NCI-H441 cells from acrolein toxicity. In addition, albumin inhibited acrolein-induced increase of transcripts associated with cellular stress response, activating transcription factor 3 (ATF3), and antioxidant response, heme oxygenase 1 (HMOX1) in HAEC cells. Acrolein-adducted albumin itself increased HMOX1 transcripts but not ATF3 transcripts. The HMOX1 transcript increase was inhibited by hydralazine, a carbonyl scavenger, suggesting that the carbonyl group of acrolein-adducted albumin mediated HMOX1 transcript increase. In acutely exposed C57BL/6J mice, bronchoalveolar lavage protein carbonylation increased. Acrolein-adducted albumin Cys34 was identified by nLC-MS/MS. These findings indicate that adduction of albumin by acrolein confers a cytoprotective function by scavenging free acrolein, decreasing a cellular stress response, and inducing an antioxidant gene response. Further, these results suggest that β-carbon reactivity may be required for acrolein's cytotoxicity and ATF3 transcript increase, and the carbonyl group of acrolein-adducted albumin can induce HMOX1 transcript increase.

574-P: Antioxidant Defence and Long Noncoding RNA in Diabetic Retinopathy

Retinal oxidative stress is increased and the antioxidant defence system is compromised in diabetic retinopathy. Expression of many key antioxidant genes is regulated by a master regulator, nuclear factor erythroid 2-related factor (Nrf2). The transcriptional activity of Nrf2 is mediated by an intracellular inhibitor Kelch-like ECH-associated protein 1 (Keap1), which regulates its nuclear movement. In diabetes, Nrf2 transcriptional activity is decreased and Keap1 levels are increased. A long noncoding RNA (IncRNA), Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), plays a role in regulating Keap1 expression, and retinal MALAT1 levels are upregulated in diabetes. Our aim is to investigate the role of MALAT1 in maintaining the antioxidant defence system in diabetic retinopathy. Human retinal endothelial cells (untransfected or transfected with MALAT1-siRNA) were incubated in 20mM glucose for 96 hours. MALAT1 expression was analysed by q-RTPCR and RNA sequencing, and role of MALAT1 in Keap1 transcription was quantified using MALAT1 RNA pull down assay. Simultaneous occupancy of MALAT1 and transcription factor Sp1 on Keap1 promoter was quantified by Chromatin Isolation by RNA Purification (ChIRP)-reChIP technique, nuclear translocation of Nrf2 by immunofluorescence imaging, and Nrf2-responsive genes superoxide dismutase 2 (Sod2) and heme oxygenase 1 (HO1) by q-RTPCR. Compared to cells in 5mM glucose, high glucose increased co-occupancy of MALAT1 and Sp1 at Keap1 promoter. MALAT1-siRNA ameliorated glucose-induced increase in co-occupancy of MALAT1-Sp1 at its promoter and decreased Keap1 expression. MALAT1-siRNA also restored nuclear localization of Nrf2 and the transcription of Sod2 and HO1. Thus, IncRNA MALAT1, via Keap1, modulates the transcriptional activity of Nrf2 and the antioxidant defence system in diabetic retinopathy. Regulation of MALAT1 can protect the retina from oxidative damage, and prevent/slow down the development of diabetic retinopathy. Disclosure R. Kowluru: None. R. Radhakrishnan: None. Funding EY014370, EY017313, EY022230

Non-invasive Assessment of Fecal Stress Biomarkers in Hunting Dogs During Exercise and at Rest.

Intense exercise causes to organisms to have oxidative stress and inflammation at the gastrointestinal (GI) level. The reduction in intestinal blood flow and the exercise-linked thermal damage to the intestinal mucosa can cause intestinal barrier disruption, followed by an inflammatory response. Furthermore, the adaptation to exercise may affect the gut microbiota and the metabolome of the biofluids. The aim of the present research was to evaluate the presence of a GI derangement in hunting dogs through a non-invasive sampling as a consequence of a period of intense exercise in comparison with samples collected at rest. The study included nine dogs that underwent the same training regime for hunting wild boar. In order to counterbalance physiological variations, multiple-day replicates were collected and pooled at each experimental point for each dog. The samples were collected immediately at rest before the training (T0), after 60 days of training (T1), after 60 days of hunting wild boar (T2), and finally, at 60 days of rest after hunting (T3). A number of potential stress markers were evaluated: fecal cortisol metabolites (FCMs) as a major indicator of altered physiological states, immunoglobulin A (IgA) as an indicator of intestinal immune protection, and total antioxidant activity [total antioxidant capacity (TAC)]. Since stool samples contain exfoliated cells, we investigated also the presence of some transcripts involved in GI permeability [occludin (OCLN), protease-activated receptor-2 (PAR-2)] and in the inflammatory mechanism [interleukin (IL)-8, IL-6, IL-1b, tumor necrosis factor alpha (TNFα), calprotectin (CALP), heme oxygenase-1 (HO-1)]. Finally, the metabolome and the microbiota profiles were analyzed. No variation in FCM and IgA content and no differences in OCLN and CALP gene expression between rest and training were observed. On the contrary, an increase in PAR-2 and HO-1 transcripts, a reduction in total antioxidant activity, and a different profile of microbiota and metabolomics data were observed. Collectively, the data in the present study indicated that physical exercise in our model could be considered a mild stressor stimulus.

Heme Oxygenases: Cellular Multifunctional and Protective Molecules against UV-Induced Oxidative Stress.

Ultraviolet (UV) irradiation can be considered as a double-edged sword: not only is it a crucial environmental factor that can cause skin-related disorders but it can also be used for phototherapy of skin diseases. Inducible heme oxygenase-1 (HO-1) in response to a variety of stimuli, including UV exposure, is vital to maintain cell homeostasis. Heme oxygenase-2 (HO-2), another member of the heme oxygenase family, is constitutively expressed. In this review, we discuss how heme oxygenase (HO), a vital rate-limiting enzyme, participates in heme catabolism and cytoprotection. Phylogenetic analysis showed that there may exist a functional differentiation between HO-1 and HO-2 during evolution. Furthermore, depending on functions in immunomodulation and antioxidation, HO-1 participates in disease progression, especially in pathogenesis of skin diseases, such as vitiligo and psoriasis. To further investigate the particular role of HO-1 in diseases, we summarized the profile of the HO enzyme system and its related signaling pathways, such as Nrf2 and endoplasmic reticulum crucial signaling, both known to regulate HO-1 expression. Furthermore, we report on a C-terminal truncation of HO-1, which is generally considered as a signal molecule. Also, a newly identified alternative splice isoform of HO-1 not only provides us a novel perspective on comprehensive HO-1 alternative splicing but also offers us a basis to clarify the relationship between HO-1 transcripts and oxidative diseases. To conclude, the HO system is not only involved in heme catabolism but also involved in biological processes related to the pathogenesis of certain diseases, even though the mechanism of disease progression still remains sketchy. Further understanding the role of the HO system and its relationship to UV is helpful for revealing the HO-related signaling networks and the pathogenesis of many diseases.

Hepatocellular HO-1 mediated iNOS-induced hepatoprotection against liver ischemia reperfusion injury

Hepatocyte-derived inducible nitric oxide synthase (iNOS) was proved to impart protection against liver ischemia reperfusion (I/R) injury in our prior analysis. However, the mechanism for this hepatoprotection remains incompletely understood. Bone marrow chimeric mice were generated using expression of iNOS in a hepatocyte-selective manner against an iNOS-knockout background. The function of heme oxygenase 1 (HO-1) in iNOS-stimulated hepatoprotection and the molecular mechanisms were explored in vitro and in vivo, respectively. Hepatocyte-derived iNOS conferred protection from I/R injury and anoxia/reoxygenation stimulation. Mechanistically, iNOS activated nuclear factor erythroid 2-related factor 2 (Nrf-2) and subsequently, stimulated the transcription of HO-1. Results from our study led to the conclusion that HO-1 is another potent mediator of iNOS-mediated protection after liver I/R.

Role of thyroid transcription factor-1 in transcriptional regulation of heme oxygenase-1

Here, we report thyroid transcription factor 1 (TTF-1) as an important transcription factor for the expression of heme oxygenase-1 (HO-1). HO-1 is a well-known cytoprotective enzyme against inflammation. We observed that HO-1 co-expressed with TTF-1 in mouse hypothalamic cells. Results from luciferase and chromatin immunoprecipitation assays revealed that TTF-1 directly activated HO-1 transcription by binding to binding domains in the 5′-flanking region of the HO-1 gene. A proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α), induced nuclear translocation of TTF-1 and increased binding affinity of TTF-1 to its binding sites on the HO-1 gene. HO-1 mRNA increased with TTF-1 overexpression but decreased with RNA interference of TTF-1 expression in rat astroglial C6 cells. Together with results showing involvement of TTF-1 in the TNF-α-induced increase in interleukin 1 beta and monocyte chemotactic protein 1 production, this study suggests that TTF-1 plays an important role in the mouse hypothalamus TNF-α-induced inflammatory response for regulating HO-1 gene expression.

Anti-neuroinflammatory effect of curcumin on Pam3CSK4-stimulated microglial cells.

Curcumin is the main curcuminoid present in Curcumalonga and it has been previously reported to exhibit a wide range of pharmacological activities. In the present study, the inhibitory effects of curcumin on the inflammatory mediators released by Pam3CSK4-stimulated BV-2 microglial cells were investigated. The production of pro-inflammatory mediators and cytokines, including tumor necrosis factor-α(TNF-α) and prostaglandinE2(PGE2), were measured by enzyme‑linked immunosorbent assay(ELISA). The expression of inflammatory genes, including inducible nitric oxide synthase and cyclooxygenase-2, were further investigated using reverse transcription-quantitative polymerase chain reaction. The effects of curcumin on heme oxygenase-1(HO-1), nuclear factor (erythroid-derived2)-like2(Nrf2), mitogen-activated protein kinase(MAPK) and nuclear factor-κB(NF-κB) signaling pathways were analyzed by western blotting. The results revealed that curcumin dose-dependently inhibited Pam3CSK4-induced nitric oxide, PGE2, and TNF-α secretion. Curcumin suppressed the secretion of inflammatory mediators through an increase in the expression of HO-1. Curcumin induced HO-1 transcription and translation through the Nrf2/antioxidant response element signaling pathway. Inhibitory experiments revealed that HO-1 was required for the anti-inflammatory effects of curcumin. Further mechanistic studies demonstrated that curcumin inhibited neuroinflammation by suppressing NF-κB and MAPK signaling pathways in Pam3CSK4-activated microglial cells. The results of the present study suggest that curcumin may be a novel treatment for neuroinflammation-mediated neurodegenerative disorders.

MiR-1254 suppresses HO-1 expression through seed region-dependent silencing and non-seed interaction with TFAP2A transcript to attenuate NSCLC growth.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, which direct post-transcriptional gene silencing (PTGS) and function in a vast range of biological events including cancer development. Most miRNAs pair to the target sites through seed region near the 5’ end, leading to mRNA cleavage and/or translation repression. Here, we demonstrated a miRNA-induced dual regulation of heme oxygenase-1 (HO-1) via seed region and non-seed region, consequently inhibited tumor growth of NSCLC. We identified miR-1254 as a negative regulator inhibiting HO-1 translation by directly targeting HO-1 3’UTR via its seed region, and suppressing HO-1 transcription via non-seed region-dependent inhibition of transcriptional factor AP-2 alpha (TFAP2A), a transcriptional activator of HO-1. MiR-1254 induced cell apoptosis and cell cycle arrest in human non-small cell lung carcinoma (NSCLC) cells by inhibiting the expression of HO-1, consequently suppressed NSCLC cell growth. Consistently with the in vitro studies, mouse xenograft studies validated that miR-1254 suppressed NSCLC tumor growth in vivo. Moreover, we found that HO-1 expression was inversely correlated with miR-1254 level in human NSCLC tumor samples and cell lines. Overall, these findings identify the dual inhibition of HO-1 by miR-1254 as a novel functional mechanism of miRNA, which results in a more effective inhibition of oncogenic mRNA, and leads to a tumor suppressive effect.