Bach1 Expression Research Articles

Aging-related decline in the induction of Nrf2-regulated antioxidant genes in human bronchial epithelial cells

Evidence from animal studies suggests that stress-induced increases in Nrf2-regulated antioxidant gene expression, a critical mechanism of cellular protection, declines with aging. This study examined whether this also occurs in humans. We measured the basal and inducible levels of Nrf2-regulated antioxidant genes in human bronchial epithelial (HBE) cells from subjects of young adult (21–29 years) and older (60–69 years) non-smokers, and explored factors affecting expresion. The basal expression of three representative Nrf2-regulated genes, the catalytic and modulator subunits of glutamate cysteine ligase (GCLC and GCLM, respectively), and NAD(P)H quinone oxidoreductase 1 (NQO1), was higher in cells from the older donors compared with cells from the young adult donors. Upon exposure to the Nrf2 activator, sulforaphane (SF), the expression of these antioxidant genes was increased in cells from both the young adults and the older donors; however, the induction by SF in older donor cells was significantly less than that seen in young adult cells. In addition, the activation of an EpRE-driven reporter by SF was lower in cells from older donors compared to cells from young adults. The basal expression of Nrf2 protein was also lower in cells from older donors than cells from young adults. Furthermore, we found that the basal expression of both Bach1 and c-Myc, two Nrf2 suppressors, was higher in cells from older adults than from young adult donors. In summary, our data suggest that, as in other species, basal expression of Nrf2-regulated genes increases with aging, while inducibility declines with aging. The increased expression of Nrf2 suppressors such as Bach1 and c-Myc may contribute to the impaired inducibility of the Nrf2-regulated antioxidant genes with aging in human bronchial epithelial cells.

Dynamic changes in Bach1 expression in the kidney of rhabdomyolysis-associated acute kidney injury.

Free heme, a pro-oxidant released from myoglobin, is thought to contribute to the pathogenesis of rhabdomyolysis-associated acute kidney injury (RM-AKI), because renal overexpression of heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, confers protection against RM-AKI. BTB and CNC homology 1 (Bach1) is a heme-responsive transcription factor that represses HO-1. Here, we examined the changes with time in the gene expression of Bach1, HO-1, and δ-aminolevulinate synthase (ALAS1, a heme biosynthetic enzyme) in the rat kidney using an RM-AKI model induced by the injection of 50% glycerol (10 mL/kg body weight) into bilateral limbs. We also examined the protein expression of Bach1 in the nucleus and cytosol, and HO-1 in the rat kidney. Glycerol treatment induced significant elevation of serum creatinine kinase and aspartate aminotransferase levels followed by the marked elevation of serum blood urea nitrogen and creatinine levels, which caused serious damage to renal tubules. Following glycerol treatment, HO-1 mRNA and protein levels were significantly up-regulated, while ALAS1 mRNA expression was down-regulated, suggesting an increase in the free renal heme concentration. The Bach1 mRNA level was drastically increased 3 h after glycerol treatment, and the increased level was maintained for 12 h. Nuclear Bach1 protein levels were significantly decreased 3 h after treatment. Conversely, cytosolic Bach1 protein levels abruptly increased after 6 h. In conclusion, we demonstrate the dynamic changes in Bach1 expression in a rat model of RM-AKI. Our findings suggest that the increase in Bach1 mRNA and cytosolic Bach1 protein expression may reflect de novo Bach1 protein synthesis to compensate for the depletion of nuclear Bach1 protein caused by the induction of HO-1 by free heme.

Silencing of bach1 gene by small interfering RNA-mediation regulates invasive and expression level of miR-203, miR-145, matrix metalloproteinase-9, and CXCR4 receptor in MDA-MB-468 breast cancer cells.

Recently experimental validation of the networks revealed bach1, a basic leucine zipper transcription factor, as the common regulator of several functional invasive genes. The expression of bach1 and its target genes was linked to the higher risk of breast cancer recurrence in patients. The aim of this study was to investigate the effect of specific bach1 small interfering RNAs, on the invasive and expression level of miR-203, miR-145, matrix metalloproteinase-9, and CXCR4 receptor which play a role in cancer metastasis, in MDA-MB-468 cell lines. Small interfering RNA transfection was performed with transfection regent. The survival effects of small interfering RNA were determined using trypan blue assay cells. The expression level of messenger RNA and matrix metalloproteinase-9 to assess cell invasion and the expression level of miR-203, miR-145, and CXCR4 receptor were measured by quantitative real-time polymerase chain reaction analysis on the MDA-MB-468 cell lines. Transfection with small interfering RNA significantly suppressed the expression of bach1 gene in dose-dependent manner after 48 h ( p < 0.0001). A significant reduction in cell invasion and CXCR4 receptor, matrix metalloproteinase-9 expression were observed ( p < 0.0001). It was also a dramatic increase in the expression level of miR-203 and miR-145 ( p < 0.0001). Our results suggest that the bach1-specific small interfering RNA effectively decrease CXCR4 receptor, matrix metalloproteinase-9 expression and breast adenocarcinoma cells invasive, also increased the expression of tumor-suppressive microRNA-203 and miR-145. Thus, these microRNAs may play a role in invasive/metastasis of carcinogenic breast cancer cells. Therefore, bach1 knockdown can be considered as a potent adjuvant in breast cancer therapy.

Nrf2- and Bach1 May Play a Role in the Modulation of Ultraviolet A-Induced Oxidative Stress by Acetyl-11-Keto-β-Boswellic Acid in Skin Keratinocytes

Background: Exposure of human skin to solar ultraviolet A (UVA) irradiation causes severe oxidative stress with damage to various cellular components and concomitant inflammation and carcinogenesis. Objective: The aim of this study is to investigate the protective effect of acetyl-11-keto-β-boswellic acid (AKBA) against UVA radiation on human skin keratinocytes. Methods: HaCaT cells were pretreated with AKBA followed by UVA irradiation. Radiation effects on cell morphology, cell viability, intracellular reactive oxygen species (ROS) levels, and antioxidant enzymes were examined. Results: AKBA reduces UVA irradiation-induced cell viability loss, accompanied by a decreased production of UVA-induced ROS, decreased malondialdehyde, and increased superoxide dismutase expression. In addition, AKBA increased basal and UVA-induced levels of Nrf2 (NF-E2-related factor 2), the redox-sensitive factor, and its target genes NQO1 and heme oxygenase-1 (HO-1), whereas expression of the transcriptional repressor Bach1 (BTB and CNC homology 1) was reduced. Furthermore, the cytoprotective effects of AKBA against UVA-derived oxidative damage were accompanied by modulating expression of inflammatory mediators (i.e., cyclooxygenase-2 and nuclear factor-κB) and NOX1. Conclusions: AKBA protects skin cells from UVA-induced damage by modulating inflammatory mediators and/or ROS production. Therefore, AKBA has potential in the development of skin care products.

Neuroprotective effects of erythropoietin against sevoflurane-induced neuronal apoptosis in primary rat cortical neurons involving the EPOR-Erk1/2-Nrf2/Bach1 signal pathway.

Erythropoietin (EPO)-induced activation of the EPO receptor (EPOR) against sevoflurane-induced neuronal apoptosis is an effective intervention, but the underlying mechanism is poorly understood. Previous studies have shown that alteration of the nuclear factor erythroid 2-related factor (Nrf2)/BTB-to-CNC homology 1 (Bach1) ratio by extracellular signal-related kinases (Erk) 1/2 ameliorates the oxidative stress which occurs in human macrophages. In this study, we determined whether or not EPO attenuates sevoflurane-induced neuronal apoptosis via the EPOR-Erk1/2-Nrf2/Bach1 signal pathway. Primary rat cortical neurons were exposed to 4% sevoflurane for 6h. Neuronal viability, injury, apoptosis, and oxidative stress were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), propidium iodide uptake (PI), malondialdehyde (MDA), and superoxide dismutase (SOD) assays. A real-time PCR assay was used to measure EPOR expression. Western blotting was used to assess Nrf2, Bach1, and heme oxygenase-1 (HO-1) expression. Sevoflurane exposure increased cell apoptosis, injury, and MDA (P<0.05), but decreased cell viability and the Nrf2:Bach1 ratio (P<0.05), and down-regulated superoxide dismutase (SOD; P<0.05), while EPO partially rescued the neurotoxicity induced by sevoflurane (P<0.05). Inhibition of EPOR via EPO-specific monoclonal antibody or Erk1/2 phosphorylation via PD98059 reversed the protective effect of EPO (P<0.05). The neuroprotective effects of EPO against sevoflurane-induced neuronal apoptosis in primary rat cortical neurons involves the EPOR-Erk1/2-Nrf2/Bach1 signal pathway.

The effects of emodin on cell viability, respiratory burst and gene expression of Nrf2-Keap1 signaling molecules in the peripheral blood leukocytes of blunt snout bream (Megalobrama amblycephala)

We determined the effects of emodin on the cell viability, respiratory burst activity, mRNA levels of antioxidative enzymes (Cu-Zn SOD, CAT and NOX2), and gene expressions of the Nrf2-Keap1 signaling molecules in the peripheral blood leukocytes of blunt snout bream. Triplicate groups of cultured cells were treated with different concentrations of emodin (0.04–25 μg/ml) for 24 h. Results showed that the emodin caused a dramatic loss in cell viability, and occurred in a dose-dependent manner. Emodin exposure (1–25 μg/ml) were significantly induced the ROS generation compared to the control. The respiratory burst and NADPH oxidase activities were significantly induced at a concentration of 0.20 μg/ml, and inhibited at 25 μg/ml. Besides, mRNA levels of antioxidant enzyme genes were dramatically regulated by emodin exposure for 24 h. During low concentrations of exposure, mRNA levels of Cu-Zn SOD in the cells treated with 0.04, 0.20 μg/ml, CAT, NOX2 and Nrf2 in the cells treated with 1 μg/ml were sharply increased, respectively. Whereas, high concentrations were dramatically down-regulated the gene expressions of CAT in the cells treated with 5, 25 μg/ml and NOX2 in the cells treated with 25 μg/ml. Furthermore, sharp increase in Keap1and Bach1 expression levels were observed a dose-dependent manner. In conclusion, this study demonstrated that emodin could induce antioxidant defenses which were involved in cytotoxic activities, respiratory burst and the transcriptional regulation levels of antioxidant enzymes and Nrf2-Keap1 signaling molecules.

Transcription Factor Bach1 and Bach2 Operate Erythro-Myeloid Competitive Differentiation By Responding to Environmental Changes

Hematopoietic system is maintained by the differentiation and proliferation of hematopoietic stem/progenitor cells (HSPCs) and their commitment to the mature blood cells should be tightly controlled by gene regulatory networks (GRNs) governed by transcription factors (TFs). To keep the homeostasis, GRNs should respond to the environmental changes, such as infection. However, the precise mechanism of such a system remains to be elucidated.TFs Bach1 and Bach2 belong to the basic region-leucine zipper family and recognize Maf-recognition elements containing AP-1 site (Oyake et al., 1996). We have previously shown that Bach1-/-Bach2-/-(DKO; double knockout) mice show erythropoiesis disorders with increased myelopoiesis from common myeloid progenitors (CMPs), which is an erythro-myeloid bifurcation point (ASH2015; Abstract ID# 81562) (Akashi et al., 2000). Since this phenotype is similar to that of LPS treated mice (O'Connell et al., 2008), we hypothesized that Bach factors work as sensors for infection.First, to evaluate the cell-intrinsic function of Bach factors, WT or DKO bone marrow cells were depleted of mature differentiated cells and transplanted to lethally irradiated WT mice. After 8 weeks, DKO donor cells showed greater myelopoiesis and lesser lymphogenesis compared to WT, suggesting Bach factors are necessary to suppress myelopoiesis to the appropriate level in regenerating hematopoiesis.To reveals the function of Bach factors in HSPCs from other aspect, LSKs (Lin-Sca1+c-kit+) were infected with retroviruses expressing Bach1-IRES-eGFP or Bach2-IRES-eGFP and transplanted to lethally irradiated WT mice. Cells derived from transgene induced LSKs were monitored by GFP fluorescence. After 2 weeks, Bach1 overexpressing LSKs did not show any difference in erythropoiesis and myelopoiesis. This might be explained by the high Bach1 expression levels in HSPCs according to the previous report (Lara-Astiaso et al., 2014). On the other hand, Bach2 overexpressing LSKs showed increased erythropoiesis and decreased myelopoiesis, suggesting that Bach2 regulates the erythro-myeloid lineage specification as expected by the observations of DKO mice.To assess the function of Bach factors under infection, we used M1 murine myeloid leukemia cells that differentiate to macrophage-like cells by LPS stimulation. LPS stimulation reduced expressions of Bach1, Bach2 and erythroid gene Gata1, and induced those of myeloid genes such as Cebpb and Csf1rin a dose-dependent manner. To determine if down-regulation of Bach factors is necessary for myeloid differentiation, Bach1 or Bach2 were transgenically overexpressed in M1 cells. Both of the M1 cells overexpressing Bach1 or Bach2 showed lower expression levels of myeloid marker CD11b compared to control under LPS stimulation. Thus, reductions of the expression of Bach factors in response to LPS were necessary for appropriate myeloid differentiation.To identify the direct target genes of Bach factors, Bach1 or Bach2 ChIP-seq data of M1 cells (Ebina-Shibuya et al., 2016) were merged with results of expression profile of WT and DKO CMPs. Several myeloid or inflammatory genes such as Cebpb, Fcgr1 and Tlr4 were identified as putative repressed target genes and several erythroid or lymphoid genes such as Klf1, Rag1 and Rag2 were identified as putative activated target genes. In addition, when Bach1 or Bach2 ChIP-seq data were merged by that of C/EBPb, which also possesses AP-1 site as its target motif, obtained from ENCODE database (ENCSR000AIB), we found that there were several co-localized regions of Bach and C/EBPb near the myeloid genes such as Cebpa, Il6 and Fcgr1. These observations suggest that Bach factors repress myeloid genes by competitively working with C/EBPb at same genomic regions. This is particularly interesting in the light of the latest findings showing the Bach2 function on AP-1 site in lymphoid cells (Sidwell et al., 2016).These results reveal a novel mechanism by which how the differentiation of erythro-myeloid bifurcation is controlled by responding to environmental changes. Bach factors regulate erythro-myeloid competitive differentiation by promoting and repressing erythroid and myeloid differentiation, respectively. We suggest that infection promote myelopoiesis at the expense of erythropoiesis by reducing the expression of Bach factors. Therefore, Bach factors may function as sensors for environmental changes. DisclosuresNo relevant conflicts of interest to declare.

MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung.

In the lung, heme oxygenase-1 (HO-1) is developmentally regulated, with its highest expression in the first days of life. In addition, neonatal mice have limited HO-1 induction in hyperoxia compared with adults. However, few reports have addressed the functional effect of microRNAs (miRNAs) in the regulation of HO-1 in vivo. The aims of the present study were to characterize changes in lung miRNA expression during postnatal development and in response to hyperoxic exposure, and to identify miRNAs that target lung HO-1 gene expression. Neonatal (<12 h old) and adult (2 mo old) mice were exposed to room air or hyperoxia (95% oxygen) for 72 h. TaqMan low-density array rodent miRNA assays were used to calculate miRNA expression changes between control and hyperoxia groups in neonatal and adult lungs. In neonates, we identified miR-196a, which binds to the 3'-untranslated region of the transcriptional repressor BTB and CNC homology 1 (Bach1) and regulates its expression, and subsequently leads to higher levels of lung HO-1 mRNA compared with levels in adults. Despite the increase at baseline, miR-196a was degraded in hyperoxia resulting in limited HO-1 induction in neonatal mice lungs. Furthermore, the developmental differences in lung HO-1 gene expression can be explained in part by the variation in miRNA-196a and its effect on Bach1. This report is the first to show developmental differences in lung miR-196a and its effect on Bach1 and HO-1 expression at baseline and in hyperoxia.

Bach1 Induces Endothelial Cell Apoptosis and Cell-Cycle Arrest through ROS Generation.

The transcription factor BTB and CNC homology 1 (Bach1) regulates genes involved in the oxidative stress response and cell-cycle progression. We have recently shown that Bach1 impairs cell proliferation and promotes apoptosis in cultured endothelial cells (ECs), but the underlying mechanisms are largely uncharacterized. Here we demonstrate that Bach1 upregulation impaired the blood flow recovery from hindlimb ischemia and this effect was accompanied both by increases in reactive oxygen species (ROS) and cleaved caspase 3 levels and by declines in the expression of cyclin D1 in the injured tissues. We found that Bach1 overexpression induced mitochondrial ROS production and caspase 3-dependent apoptosis and its depletion attenuated H2O2-induced apoptosis in cultured human microvascular endothelial cells (HMVECs). Bach1-induced apoptosis was largely abolished when the cells were cultured with N-acetyl-l-cysteine (NAC), a ROS scavenger. Exogenous expression of Bach1 inhibited the cell proliferation and the expression of cyclin D1, induced an S-phase arrest, and increased the expression of cyclin E2, which were partially blocked by NAC. Taken together, our results suggest that Bach1 suppresses cell proliferation and induces cell-cycle arrest and apoptosis by increasing mitochondrial ROS production, suggesting that Bach1 may be a promising treatment target for the treatment of vascular diseases.

Transcription Factor Bach1 and Bach2 Control Common Myeloid Progenitor Cell Differentiation Under Infectious Stimuli

Background: Erythrocyte and granulocyte/macrophage develop from common myeloid progenitor (CMP) (Akashi et al., 2000). Differentiation of hematopoietic progenitor cells is precisely controlled by multiple transcription factors, among which GATA1, C/EBPα, C/EBPβ and Spi-C play pivotal roles in erythrocyte and granulocyte/macrophage differentiation (Mancini et al., 2012; Pongubala et al., 2008; Hirai et al., 2006; Haldar et al., 2014). However, the mechanism by which the differentiation of CMP controlled under infectious condition has been unclear. Bach1 and Bach2 belong to the basic region-leucine zipper family and recognize Maf-recognition elements (Oyake et al., 1996). They promote B cell development by repressing the myeloid genes such as Cebpb and Spic in common lymphoid progenitor cells (Itoh-Nakadai et al., 2014). In addition, Bach1 regulates several target genes related to iron/heme homeostasis such as globin genes and hemeoxygenase-1, and Bach2 may similarly regulate these genes (Igarashi, 2014). Therefore, it is expected that both Bach1 and Bach2 play redundant roles in erythropoiesis. To figure out their roles in erythroid and myeloid cell differentiation, we performed hematological and transcriptomics analyses using Bach1-/- Bach2-/- (double-deficient; DD) mice.Methods: The generation of DD mice on the C57BL/6J background and Bach2 reporter mice with red fluorescent protein coding cDNA inserted in the Bach2 locus were described previously (Itoh-Nakadai et al., 2014). Mice between 8-12 weeks old were analyzed in the present study. Bone marrow (BM) cells were stained with specific combinations of antibodies to identify erythroid/myeloid progenitor and mature cells (Sheila et al., 2008; Cornelis et al., 2007; Socolovsky et al., 2001). Flow cytometry analysis and cell sorting were performed by using FACSAriaⅡ(BD) and FlowJo software (TreeStar). For infectious simulation of CMP, sorted CMPs were incubated with 1μg/ml LPS (Sigma) for 48h and RNA was purified with RNeasy micro kit (Qiagen). Quantitative PCR by using SuperscriptⅢ reverse transcriptase (Invitrogen) and Light Cycler system (Roche) was performed according to manufacturer's instructions. Microarray analysis by using Sure-Print G3 mouse GE microarray slide (Agilent) was performed as previously described (Itoh-Nakadai et al., 2014) and the results were analyzed by using GeneSpring software (Agilent). We used Gene Set Enrichment Analysis (GSEA) to interpret gene expression data (Subramanian et al., 2005; Mootha et al., 2003). LPS stimulation (50 μg/body) of mice was performed as previously described (Ryan et al., 2008). Data were analyzed by the two-sided Student's t-test and p - values of <0.05 were considered statistically significant.Results: DD mice show mild normocytic anemia compered to wild-type (WT), Bach1-/-, and Bach2-/- mice (hemoglobin; 14.4±0.2, 14.0±0.3, 13.5±0.3 and 11.9±0.7 g/dl, for WT, Bach1-/-, Bach2-/- and DD, respectively, p<0.05 for comparison between DD and other genotypes, n=7). Immature and mature erythroblast populations were significantly decreased in BM of DD (immature; 25.8±1.78, 15.6±1.4, mature; 27.6±3.3, 17.4±2.3×106/body for WT and DD, respectively, p<0.05, n=6). Megakaryocyte-erythroid progenitor (MEP)/granulocyte-monocyte progenitor (GMP) ratio was significantly decreased in BM of DD (MEP/GMP: 0.13±0.01, 0.07±0.01 for WT and DD, respectively, p<0.05, n=5). Bach2 expression was detected in CMP, MEP and even GMP by using Bach2-RFP mice. LPS stimulation of WT CMP significantly decreased mRNA levels of Bach1, Bach2 and Gata1. On the other hand, Cebpb and Spic mRNA levels were significantly increased. LPS stimulation of WT mice induced significant increase of granulocyte and decrease of erythrocyte and B lymphocyte in BM, which was consistent with previous reports. It was also shown that LPS stimulation significantly decreased MEP/ GMP ratio. According to the clustering analysis of the microarray data of CMP sorted from WT and DD mice, they showed clearly different expression profiles. GSEA showed that CMP of DD skewed to myeloid cell lineage and lost the erythroid gene expression compared to WT.Conclusions: Bach1 and Bach2 control the differentiation of CMP to erythroid cell or myeloid cell by repressing myeloid genes such as Cebpb and Spic. Infectious stimuli may promote myeloid cell differentiation by reducing the expression of Bach1 and Bach2 in CMP. DisclosuresFujiwara:Chugai Pharmaceutical CO., LTD: Research Funding. Harigae:Chugai Pharmaceutical CO., LTD: Research Funding.

Downregulation of Nuclear Factor Erythroid 2-Related Factor and Associated Antioxidant Genes Contributes to Redox-Sensitive Vascular Dysfunction in Hypertension.

Oxidative stress is implicated in vascular dysfunction in hypertension. Although mechanisms regulating vascular pro-oxidants are emerging, there is a paucity of information on antioxidant systems, particularly nuclear factor erythroid 2-related factor (Nrf2), a master regulator of antioxidants enzymes. We evaluated the vascular regulatory role of Nrf2 in hypertension and examined molecular mechanisms, whereby Nrf2 influences redox signaling in small arteries and vascular smooth muscle cells from Wistar Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Cells were stimulated with angiotensin II in the absence/presence of Nrf2 activators (bardoxolone/L-sulforaphane). Increased vascular reactive oxygen species production (chemiluminescence and amplex red) was associated with reduced Nrf2 activity in arteries (18%) and vascular smooth muscle cells (48%) in SHRSP (P<0.05 versus WKY). Expression of antioxidant enzymes, including superoxide dismutase-1 (64%), catalase (60%), peroxiredoxin 1 (75%), and glutathione peroxidase (54%), was reduced in SHRSP. L-sulforaphane reversed these effects. Angiotensin II increased nuclear accumulation of Nrf2 in vascular smooth muscle cells from WKY (197% versus vehicle), with blunted effects in SHRSP (44% versus vehicle). These responses were associated with increased antioxidant expression (superoxide dismutase-1, 32%; catalase, 42%; thioredoxin, 71%; peroxiredoxin, 1%-90%; quinone oxidoreductase, 84%; P<0.05 versus vehicle) and increased activity of superoxide dismutase-1, catalase, and thioredoxin in WKY but not in SHRSP, which exhibited increased Bach1 expression. Nrf2 activators blocked angiotensin II-induced reactive oxygen species generation. Vascular function demonstrated increased contractility (Emax WKY 113.4±5.6 versus SHRSP 159.0±8.3) and decreased endothelial-dependent relaxation (Emax WKY 88.6±3.1 versus SHRSP 74.6±3.2, P<0.05) in SHRSP, effects corrected by L-sulforaphane. Our findings suggest that Nrf2 downregulation contributes to redox-sensitive vascular dysfunction in hypertension.

Bach1 Represses Wnt/β-Catenin Signaling and Angiogenesis.

Wnt/β-catenin signaling has an important role in the angiogenic activity of endothelial cells (ECs). Bach1 is a transcription factor and is expressed in ECs, but whether Bach1 regulates angiogenesis is unknown. This study evaluated the role of Bach1 in angiogenesis and Wnt/β-catenin signaling. Hind-limb ischemia was surgically induced in Bach1(-/-) mice and their wild-type littermates and in C57BL/6J mice treated with adenoviruses coding for Bach1 or GFP. Lack of Bach1 expression was associated with significant increases in perfusion and vascular density and in the expression of proangiogenic cytokines in the ischemic hindlimb of mice, with enhancement of the angiogenic activity of ECs (eg, tube formation, migration, and proliferation). Bach1 overexpression impaired angiogenesis in mice with hind-limb ischemia and inhibited Wnt3a-stimulated angiogenic response and the expression of Wnt/β-catenin target genes, such as interleukin-8 and vascular endothelial growth factor, in human umbilical vein ECs. Interleukin-8 and vascular endothelial growth factor were responsible for the antiangiogenic response of Bach1. Immunoprecipitation and GST pull-down assessments indicated that Bach1 binds directly to TCF4 and reduces the interaction of β-catenin with TCF4. Bach1 overexpression reduces the interaction between p300/CBP and β-catenin, as well as β-catenin acetylation, and chromatin immunoprecipitation experiments confirmed that Bach1 occupies the TCF4-binding site of the interleukin-8 promoter and recruits histone deacetylase 1 to the interleukin-8 promoter in human umbilical vein ECs. Bach1 suppresses angiogenesis after ischemic injury and impairs Wnt/β-catenin signaling by disrupting the interaction between β-catenin and TCF4 and by recruiting histone deacetylase 1 to the promoter of TCF4-targeted genes.

Exogenous Apo-Transferrin Increases Monoferric Transferrin, Decreasing Cytosolic Iron Uptake and Heme and Globin Synthesis in β-Thalassemic Mice

β-thalassemia is an inherited blood disorder caused by reduced or absence of β-globin expression which results in imbalanced globin synthesis, ineffective erythropoiesis, and anemia. How the imbalance between α- and β-globin results in ineffective erythropoiesis, if apoptosis or dysfunctional differentiation of erythroid precursors results in ineffective erythropoiesis, and whether disrupted iron regulation and / or iron overload in β-thalassemia is directly involved in the pathophysiology of ineffective erythropoiesis is incompletely understood. Iron is critical for hemoglobin synthesis and erythropoiesis is dependent on transferrin (Tf) bound iron. Tf functions as the main iron transporter in circulation, where it exists in three forms: as iron-free apo-transferrin (apoTf), monoferric Tf, or diferric Tf (holoTf). Typically, iron is bound to 30% of all Tf binding sites in circulation at which point monoferric Tf is found in the highest concentration relative to holoTf. . We have previously shown that exogenous apoTf ameliorates anemia in a mouse model of β-thalassemia intermedia (th1/th1), resulting in reduced splenomegaly, reticulocytosis, and α-globin precipitation on circulatory red blood cells (RBC). We also observe a decrease in mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), serum iron, Tf saturation, together suggesting that relative iron deficiency improves iron metabolism and ineffective erythropoiesis in apoTf-treated th1/th1 mice. We hypothesize that exogenous apoTf decreases cytosolic iron and heme as a consequence of increased monoferric Tf which results in less iron entering cells via Tf:TfR1 binding. Our current data reveals that in vitro incubation of purified apoTf and holoTf at 37C results in the formation of monoferric Tf, and injection of wild-type (WT) mice with a single intraperitoneal dose of apoTf (10mg) decreases holoTf (P=0.01) and increases monoferric Tf (P=0.02) in the serum 6 hours after injection. Using both calcium mobilization and anti-Tf antibodies in flow cytometry, we demonstrate that apoTf results in no TfR1 binding relative to holoTf in CHO cells (P<0.0001), and an equal concentration apoTf:holoTf mixture results in intermediate binding between apoTf and holoTf (P=0.004). We also demonstrate a dose dependent decrease in cytosolic iron (P<0.001) and heme (P<0.01) in MEL cells treated with escalating concentrations of apoTf, an effect abrogated by the additional of DFO, an iron chelator; urea gel of MEL cell supernatants reveals that monoferric Tf increases with increasing doses of apoTf and decreased in concurrently DFO treated cells (Figure 1). These findings together suggest that changes in iron uptake result from increased monoferric Tf (rather than competition of apoTf for TfR1 binding sites). In addition, in vivo experiments demonstrate a decrease in heme concentration (56 vs. 67 uM, P<0.0001) in circulating RBCs as well as α-globin (1.3 vs. 3.3-fold, P=0.009) and β-globin (Figure 2) mRNA expression in sorted bone marrow orthochromatophilic erythroblasts from apoTf- vs. PBS-treated th1/th1 mice. As heme is known to regulate globin expression through transcriptional and translational routes, we evaluate bach1, heme oxygenase 1 (HO-1), and heme-regulated eIF2α kinase (HRI). We demonstrate no difference in bach1 and HO-1 mRNA expression but a significant and unexpected decrease in HRI expression (1.0 vs. 2.8-fold, P=0.01) in sorted bone marrow orthochromatophilic erythroblasts from apoTf- vs. PBS-treated th1/th1 mice (similar findings in all stages of terminal erythroid differentiation), suggesting that HRI is regulated by mechanisms independent of cellular iron and heme. Furthermore, western blots of HRI and its target eIF2α (total and phosphorylated) are also normalized in sorted bone marrow erythroid precursors from apoTf- vs. PBS-treated th1/th1 mice (Figure 3). Taken together, our data suggests that exogenous apoTf in th1/th1 mice results in decreased cytosolic iron and heme as well as α- and β-globin synthesis as a consequence of increased monoferric Tf in circulation and provides a novel system to explore the regulation of globin in β-thalassemia and how iron deficiency benefits this disease. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Homocysteine downregulates gene expression of heme oxygenase-1 in hepatocytes.

BackgroundHyperhomocysteinemia (HHcy) is an independent risk factor for liver diseases, such as fatty liver and hepatic fibrosis. However, the mechanisms underlying this pro-oxidative effect of homocysteine (Hcy) in hepatocytes remain largely unknown. Thus, we investigated the effect of Hcy on the gene expression of heme oxygenase-1 (HO-1), the primary rate-limiting enzyme in heme catabolism and a key anti-oxidant detoxification enzyme in maintaining cellular redox homeostasis.MethodsIn vivo, twenty male C57BL/6 mice at 8 weeks of age were randomly divided into two groups. One group was fed a chow diet (chow group; n = 10), the other group of mice was fed a methionine-supplemented diet (Met group, 1 mg kg−1 day−1 L-methionine in drinking water; n = 10) for 4 weeks. In vitro, HepG2 cells were stimulated with different doses of homocysteine (Hcy).ResultsFour weeks’ methionine supplementation caused a significant increase of plasma Hcy concentration and a decrease of HO-1 expression in the liver of C57BL/6 mice than mice received chow diet. Besides, SOD enzyme activities were impaired and the level of oxidative stress markers, such as malondialdehyde (MDA) were elevated in the liver from mice supplemented with methionine compared with control mice. In cultured hepatocytes, Hcy treatment reduced both the mRNA and protein levels of HO-1 dose-dependently. However, Hcy had no effect on the gene expression of Nrf2, the major transcriptional regulator of HO-1. Instead, Hcy induced the expression of Bach1, a transcriptional repressor of HO-1. In addition, Hcy stimulated the nuclear localization of Bach1 but prevented that of Nrf2. Furthermore, we found that knockdown of Bach1 attenuated the suppression of the HO-1 expression by Hcy.ConclusionsCollectively, our results demonstrated that Bach1 plays an important role in Hcy-triggered ROS generations through inhibiting HO-1 expression, likely, resulting from the disturbed interplay between Bach1 and Nrf2.Electronic supplementary materialThe online version of this article (doi:10.1186/1743-7075-11-55) contains supplementary material, which is available to authorized users.

Cell-type-specific downregulation of heme oxygenase-1 by lipopolysaccharide via Bach1 in primary human mononuclear cells

Heme oxygenase (HO)-1 is the inducible isoform of the heme-degrading enzyme HO, which is upregulated by multiple stress stimuli. HO-1 has major immunomodulatory and anti-inflammatory effects via its cell-type-specific functions in mononuclear cells. Contradictory findings have been reported on HO-1 regulation by the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) in these cells. Therefore, we reinvestigated the effects of LPS on HO-1 gene expression in human and murine mononuclear cells in vitro and in vivo. Remarkably, LPS downregulated HO-1 in primary human peripheral blood mononuclear cells (PBMCs), CD14+ monocytes, macrophages, dendritic cells, and granulocytes, but upregulated this enzyme in primary murine macrophages and human monocytic leukemia cell lines. Furthermore, experiments with human CD14+ monocytes revealed that activation of other TLRs including TLR1, -2, -5, -6, -8, and -9 decreased HO-1 mRNA expression. LPS-dependent downregulation of HO-1 was specific, because expression of cyclooxygenase-2, NADP(H)-quinone oxidoreductase-1, and peroxiredoxin-1 was increased under the same experimental conditions. Notably, LPS upregulated expression of Bach1, a critical transcriptional repressor of HO-1. Moreover, knockdown of this nuclear factor enhanced basal and LPS-dependent HO-1 expression in mononuclear cells. Finally, downregulation of HO-1 in response to LPS was confirmed in PBMCs from human individuals subjected to experimental endotoxemia. In conclusion, LPS downregulates HO-1 expression in primary human mononuclear cells via a Bach1-mediated pathway. As LPS-dependent HO-1 regulation is cell-type- and species-specific, experimental findings in cell lines and animal models need careful interpretation.

Analysis of mtDNA, miR-155 and BACH1 expression in hearts from donors with and without Down syndrome

Cancer patients with Down syndrome (DS) are at increased risk for anthracycline-related cardiotoxicity. Mitochondrial DNA (mtDNA) alterations in hearts with-DS may contribute to anthracycline-related cardiotoxicity. Cardiac mtDNA and the mtDNA4977 deletion were quantitated in samples with- (n = 11) and without-DS (n = 31). Samples with-DS showed 30% lower mtDNA (DSMT-ND1/18Sratio: 1.48 ± 0.72 versus non-DSMT-ND1/18Sratio: 2.10 ± 1.59; p = 0.647) and 30% higher frequency of the mtDNA4977 deletion (DS% frequency mtDNA4977 deletion: 0.0086 ± 0.0166 versus non-DS% frequency mtDNA4977 deletion: 0.0066 ± 0.0124, p = 0.514) than samples without-DS. The BACH1 and microRNA-155 (miR-155) genes are located in chromosome 21, and their products have demonstrated roles during oxidative stress. BACH1 and miR-155 expression did not differ in hearts with- and without-DS. An association between BACH1 and miR-155 expression was detected in hearts without-DS, suggesting alterations between BACH1-miR-155 interactions in the DS settings.

Matrix conditions and KLF2-dependent induction of heme oxygenase-1 modulate inhibition of HCV replication by fluvastatin.

Background & AimsHMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication.MethodsHO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue.ResultsAll statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease.ConclusionsStatin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease.

Hepatic HMOX1 expression positively correlates with Bach-1 and miR-122 in patients with HCV mono and HIV/HCV coinfection.

AimTo analyze the expression of HMOX1 and miR-122 in liver biopsy samples obtained from HCV mono-and HIV/HCV co-infected patients in relation to selected clinical parameters, histological examination and IL-28B polymorphism as well as to determine whether HMOX1 expression is dependent on Bach-1.Materials and MethodsThe study group consisted of 90 patients with CHC: 69 with HCV mono and 21 with HIV/HCV co-infection. RT-PCR was used in the analysis of HMOX1, Bach-1 and miR-122 expression in liver biopsy samples and in the assessment of IL-28B single-nucleotide polymorphism C/T (rs12979860) in the blood. Moreover in liver biopsy samples an analysis of HO-1 and Bach-1 protein level by Western Blot was performed.ResultsHCV mono-infected patients, with lower grading score (G<2) and higher HCV viral load (>600000 IU/mL) demonstrated higher expression of HMOX1. In patients with HIV/HCV co-infection, the expression of HMOX1 was lower in patients with lower lymphocyte CD4 count and higher HIV viral load. IL28B polymorphism did not affect the expression of either HMOX1 or miR-122. Higher HMOX1 expression correlated with higher expression of Bach-1 (Spearman’s ρ = 0.586, p = 0.000001) and miR-122 (Spearman’s ρ = 0.270, p = 0.014059).ConclusionsHMOX1 and miR-122 play an important role in the pathogenesis of CHC in HCV mono-and HIV/HCV co-infected patients. Reduced expression of HMOX1 in patients with HIV/HCV co-infection may indicate a worse prognosis in this group. Our results do not support the importance of Bach-1 in repression of HMOX1 in patients with chronic hepatitis C.

Hemopexin-dependent heme uptake via endocytosis regulates the Bach1 transcription repressor and heme oxygenase gene activation

BackgroundIntracellular heme plays versatile roles in a variety of physiological processes including mitochondrial respiration. Heme also induces the expression of genes such as heme oxygenase-1 (HO-1) by inactivating the transcription repressor Bach1 through direct binding. However, the source of heme for the regulation of the Bach1–HO-1 axis has been unclear. Considering that extracellular heme exists as a complex with hemopexin (Hx) in serum under the physiological conditions, heme–Hx complex may deliver heme for the gene regulation. MethodsUsing a mammalian expression system, high secretory recombinant Hx (rHx) was developed. We examined the effects of rHx-bound heme on HO-1 expression and Bach1 in Hepa-1c1c7 liver cells and THP-1 macrophage cells. We investigated the uptake pathway of rHx-bound heme by treating cells with chlorpromazine (CPZ). ResultsrHx-bound heme induced the expression of HO-1 and decreased the level of Bach1 protein. CPZ inhibited the induction of the HO-1 expression by rHx-bound heme. ConclusionrHx-bound heme was internalized into the cells via endocytosis, resulting in HO-1 expression and inactivation of Bach1. General significanceThe Bach1-dependent repression of the HO-1 expression is under the control of the Hx-dependent uptake of extracellular heme. Heme may regulate Bach1 as an extracellular signaling molecule.

Bach1 : A possible player in HbE/beta thalassaemia?

IntroductionThe basic leucine zipper transcription factor 1 (Bach1) is known to be among the crucial molecules that has the ability to regulate gene expression when faced with oxidative stress. HbE/ beta thalassaemia patients have increased oxidative stress due to the presence of reactive oxygen species formed as a result of precipitation of excess alpha globins. MethodsA total of 62 suspected HbE/β-thalassaemia individuals from Thalassaemia Clinic of Hospital Ampang were collected. Only 47 samples could be used in our study after performing an exclusion exercise. Full blood count was performed. HbE/beta-thalassaemia screening was done via high performance liquid chromatography (HPLC) while plasma ferritin study was performed to exclude iron deficiency anaemia (IDA). As for genomic studies, PCR were carried out to identify alpha and beta mutations, if any. Gene expression analysis using TaqMan real-time PCR was used to study Bach1, HO-1 and various globin gene expressions. Results & DiscussionBach1 expression was significantly correlated to reticulocytes count, age, HO-1, alpha-, gamma- and beta globin gene expression. The positive correlation of Bach1 expression with age could be due to the increasing oxidative stress caused by natural cellular ageing process. Excess precipitated alpha chains and unstable beta globin chains cause Bach1 expression to increase whereas Bach1 is correlated indirectly with gamma globin chains to ameliorate the clinical severity of HbE/ beta thalassaemia. The compensatory mechanism of Bach1 trying to counter-react with the increased reticulocyte counts and the indirect link of Bach1 with HO-1 as a cytoprotective vehicle further substantiate the role of Bach1 in modifying the phenotypic severity in HbE/ beta thalassaemia. ConclusionBach1 could well be a potential modifier by modifying the clinical severity among HbE/beta thalassaemia patients as it has the ability to regulate the globins genes and the level of oxidative stress produced.