Nrf2 pre-recruitment at Enhancer 2 is a hallmark of H2 O2 -induced epigenetic transcriptional memory in the HMOX1 gene in human umbilical artery endothelial cells.

Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity.

Protective Effect of Glycomacropeptide on the Inflammatory Response of U937 Macrophages.

Macrophages play crucial roles in inflammation and oxidative stress associated with noncommunicable diseases, such as cardiovascular diseases, diabetes, and cancer. Glycomacropeptide (GMP) is a bioactive peptide derived from milk κ-casein that contains abundant sialic acid and has shown anti-inflammatory, antioxidative, anti-obesity, and anti-diabetic properties when is orally administered. The aim of this study was to evaluate the effect of GMP on the regulation of the inflammatory response in human macrophages and the participation of sialic acid in this activity. GMP pretreatment decreased by 35%, 35%, and 49% the production of nitrites, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α, respectively, in activated human macrophages U937. The same effect was obtained when cells were pretreated with asialo GMP, and no change on the gene expression of the lectins associated with the recognition of sialic acids, SIGLEC5, 7, and 9, was induced by GMP on macrophages, which suggests that sialic acid might not be involved in this immunoregulatory effect. Interestingly, GMP increased 8.9- and 3.5-fold the gene expression of the canonical anti-inflammatory protein SOCS3 and the antioxidant enzyme HMOX1, respectively, in U937 cells. Thus, GMP exerts anti-inflammatory and antioxidative activities on activated macrophages in a sialic acid-independent manner, which might be related to its in vivo reported bioactivity.

Exploiting Interdata Relationships in Prostate Cancer Proteomes: Clinical Significance of HO-1 Interactors.

Prostate cancer (PCa) cells display abnormal expression of proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown the anti-tumoral role of heme oxygenase 1 (HO-1) in this disease. In this work, we undertook a mass spectrometry-based proteomics study to identify HO-1 molecular interactors that might collaborate with its modulatory function in PCa. Among the HO-1 interactors, we identified proteins with nuclear localization. Correlation analyses, using the PCa GSE70770 dataset, showed a significant and positive correlation between HMOX1 and 6 of those genes. Alternatively, HMOX1 and YWHAZ showed a negative correlation. Univariable analyses evidenced that high expression of HNRNPA2B1, HSPB1, NPM1, DDB1, HMGA1, ZC3HAV1, and HMOX1 was associated with increased relapse-free survival (RFS) in PCa patients. Further, PCa patients with high HSPB1/HMOX1, DDB1/HMOX1, and YWHAZ/HMOX1 showed a worse RFS compared with patients with lower ratios. Moreover, a decrease in RFS for patients with higher scores of this signature was observed using a prognostic risk score model. However, the only factor significantly associated with a higher risk of relapse was high YWHAZ. Multivariable analyses confirmed HSPB1, DDB1, and YWHAZ independence from PCa clinic-pathological parameters. In parallel, co-immunoprecipitation analysis in PCa cells ascertained HO-1/14-3-3ζ/δ (protein encoded by YWHAZ) interaction. Herein, we describe a novel protein interaction between HO-1 and 14-3-3ζ/δ in PCa and highlight these factors as potential therapeutic targets.

Treatment with sodium (S)-2-hydroxyglutarate prevents liver injury in an ischemia-reperfusion model in female Wistar rats.

BACKGROUND: Ischemia-reperfusion (IR) injury is one of the leading causes of early graft dysfunction in liver transplantation. Techniques such as ischemic preconditioning protect the graft through the activation of the hypoxia-inducible factors (HIF), which are downregulated by the EGLN family of prolyl-4-hydroxylases, a potential biological target for the development of strategies based on pharmacological preconditioning. For that reason, this study aims to evaluate the effect of the EGLN inhibitor sodium (S)-2-hydroxyglutarate [(S)-2HG] on liver IR injury in Wistar rats. METHODS: Twenty-eight female Wistar rats were divided into the following groups: sham (SH, n = 7), non-toxicity (HGTox, n = 7, 25 mg/kg of (S)-2HG, twice per day for two days), IR (n = 7, total liver ischemia: 20 minutes, reperfusion: 60 minutes), and (S)-2HG+IR (HGIR, n = 7, 25 mg/kg of (S)-2HG, twice per day for two days, total liver ischemia as the IR group). Serum ALT, AST, LDH, ALP, glucose, and total bilirubin were assessed. The concentrations of IL-1ß, IL-6, TNF, malondialdehyde, superoxide dismutase, and glutathione peroxidase were measured in liver tissue, as well as the expression of Hmox1, Vegfa, and Pdk1, determined by RT-qPCR. Sections of liver tissue were evaluated histologically, assessing the severity of necrosis, sinusoidal congestion, and cytoplasmatic vacuolization. RESULTS: The administration of (S)-2HG did not cause any alteration in the assessed biochemical markers compared to SH. Preconditioning with (S)-2HG significantly ameliorated IR injury in the HGIR group, decreasing the serum activities of ALT, AST, and LDH, and the tissue concentrations of IL-1ß and IL-6 compared to the IR group. IR injury decreased serum glucose compared to SH. There were no differences in the other biomarkers assessed. The treatment with (S)-2HG tended to decrease the severity of hepatocyte necrosis and sinusoidal congestion compared to the IR group. The administration of (S)-2HG did not affect the expression of Hmox1 but decreased the expression of both Vegfa and Pdk1 compared to the SH group, suggesting that the HIF-1 pathway is not involved in its mechanism of hepatoprotection. In conclusion, (S)-2HG showed a hepatoprotective effect, decreasing the levels of liver injury and inflammation biomarkers, without evidence of the involvement of the HIF-1 pathway. No hepatotoxic effect was observed at the tested dose.

HO-1 Modulates Aerobic Glycolysis through LDH in Prostate Cancer Cells.

Prostate cancer (PCa) is the second most diagnosed malignancy and the fifth leading cause of cancer associated death in men worldwide. Dysregulation of cellular energetics has become a hallmark of cancer, evidenced by numerous connections between signaling pathways that include oncoproteins and key metabolic enzymes. We previously showed that heme oxygenase 1 (HO-1), a cellular homeostatic regulator counteracting oxidative and inflammatory damage, exhibits anti-tumoral activity in PCa cells, inhibiting cell proliferation, migration, tumor growth and angiogenesis. The aim of this study was to assess the role of HO-1 on the metabolic signature of PCa. After HO-1 pharmacological induction with hemin, PC3 and C4-2B cells exhibited a significantly impaired cellular metabolic rate, reflected by glucose uptake, ATP production, lactate dehydrogenase (LDH) activity and extracellular lactate levels. Further, we undertook a bioinformatics approach to assess the clinical significance of LDHA, LDHB and HMOX1 in PCa, identifying that high LDHA or low LDHB expression was associated with reduced relapse free survival (RFS). Interestingly, the shortest RFS was observed for PCa patients with low HMOX1 and high LDHA, while an improved prognosis was observed for those with high HMOX1 and LDHB. Thus, HO-1 induction causes a shift in the cellular metabolic profile of PCa, leading to a less aggressive phenotype of the disease.

Heme Oxygenase-1 Is a Pivotal Modulator of Bone Turnover and Remodeling: Molecular Implications for Prostate Cancer Bone Metastasis.

Aims: Bone is the most frequent site of prostate cancer (PCa) metastasis. Tumor cells interact with the bone microenvironment interrupting tissue balance. Heme oxygenase-1 (HO-1; encoded by Hmox1) appears as a potential target in PCa maintaining the cellular homeostasis. Our hypothesis is that HO-1 is implicated in bone physiology and modulates the communication with PCa cells. Here we aimed at (i) assessing the physiological impact of Hmox1 gene knockout (KO) on bone metabolism in vivo and (ii) determining the alterations of the transcriptional landscape associated with tumorigenesis and bone remodeling in cells growing in coculture (PCa cells with primary mouse osteoblasts [PMOs] from BALB/c Hmox1+/+, Hmox1+/-, and Hmox1-/- mice). Results: Histomorphometric analysis of Hmox1-/- mice bones exhibited significantly decreased bone density with reduced remodeling parameters. A positive correlation between Hmox1 expression and Runx2, Col1a1, Csf1, and Opg genes was observed in PMOs. Flow cytometry studies revealed two populations of PMOs with different reactive oxygen species (ROS) levels. The high ROS population was increased in PMOs Hmox1+/- compared with Hmox1+/+, but was significantly reduced in PMOs Hmox1-/-, suggesting restrained ROS tolerance in KO cells. Gene expression was altered in PMOs upon coculture with PCa cells, showing a pro-osteoclastic profile. Moreover, HO-1 induction in PCa cells growing in coculture with PMOs resulted in a significant modulation of key bone markers such as PTHrP and OPG. Innovation and Conclusion: We here demonstrate the direct implications of HO-1 expression in bone remodeling and how it participates in the alterations in the communication between bone and prostate tumor cells.

High expression of HMOX1 in blue-shelled chickens is associated with a TG haplotype

HMOX1 is an important gene in biosynthesis of the eggshell pigment of blue eggs. Previous studies found that HMOX1 is highly expressed in the shell gland of hens laying blue eggs (BlueH) compared with hens laying brown eggs (BrownH); however, the reasons for the differential expression are unclear. In this study five single nucleotide polymorphism (SNP) in HMOX1 were genotyped in 111 BlueH and 115 BrownH. The association of haplotypes of these SNP with the blue egg phenotype was tested. Haplotype-specific expression of HMOX1 was detected in the shell gland. The interaction of sequence variants and transcription factors was analyzed using electrophoretic mobility shift assay (EMSA). A TG haplotype covering upstream 1.4 kb region of HMOX1 was significantly associated with blue eggs (p 0.05). Furthermore, the birds (n=12) with the haplotype expressed 3.8 fold more transcripts than those (n=12) without the haplotype (p 0.05). After re-sequencing a 2.2 kb region harboring the TG haplotype, a total of 26 SNP were found, of which a SNP was predicted to create a binding site of Nrf2, a transcription factor initiating HMOX1 expression. However, subsequent EMSA failed to confirm the Nrf2-DNA interaction. Taken together, the data suggested that the TG haplotype is not directly involved in regulation of HMOX1 expression; a regulatory mutation located near the haplotype and linked with the haplotype may exist and be responsible for the differential expression of HMOX1.

High expression of HMOX1 in blue-shelled chickens is associated with a TG haplotype

HMOX1 is an important gene in biosynthesis of the eggshell pigment of blue eggs. Previous studies found that HMOX1 is highly expressed in the shell gland of hens laying blue eggs (BlueH) compared with hens laying brown eggs (BrownH); however, the reasons for the differential expression are unclear. In this study five single nucleotide polymorphism (SNP) in HMOX1 were genotyped in 111 BlueH and 115 BrownH. The association of haplotypes of these SNP with the blue egg phenotype was tested. Haplotype-specific expression of HMOX1 was detected in the shell gland. The interaction of sequence variants and transcription factors was analyzed using electrophoretic mobility shift assay (EMSA). A TG haplotype covering upstream 1.4 kb region of HMOX1 was significantly associated with blue eggs (p 0.05). Furthermore, the birds (n=12) with the haplotype expressed 3.8 fold more transcripts than those (n=12) without the haplotype (p 0.05). After re-sequencing a 2.2 kb region harboring the TG haplotype, a total of 26 SNP were found, of which a SNP was predicted to create a binding site of Nrf2, a transcription factor initiating HMOX1 expression. However, subsequent EMSA failed to confirm the Nrf2-DNA interaction. Taken together, the data suggested that the TG haplotype is not directly involved in regulation of HMOX1 expression; a regulatory mutation located near the haplotype and linked with the haplotype may exist and be responsible for the differential expression of HMOX1.(AU)

Comparison of HMOX1 expression and enzyme activity in blue-shelled chickens and brown-shelled chickens.

Blue egg coloring is attributed to biliverdin derived from the oxidative degradation of heme through catalysis by heme oxygenase (HO). The pigment is secreted into the eggshell by the shell gland. There is uncertainty as to whether the pigment is synthesized in the shell gland or in other tissues. To investigate the site of pigment biosynthesis, the expression of heme oxygenase (decycling) 1 (HMOX1), a gene encoding HO, and HO activity in liver and spleen were compared between blue-shelled chickens (n = 12) and brown-shelled chickens (n = 12). There were no significant differences in HMOX1 expression and HO activity in these tissues between the two groups. Since the liver and spleen, two important sites outside the shell gland where heme is degraded into biliverdin, CO and Fe(2+), did not differ in HO expression and activity we conclude that the pigment is most likely synthesized in the shell gland.