Regulation Of Heme Oxygenase-1 Gene Expression Research Articles

Involvement of ELAV RNA-binding proteins in the post-transcriptional regulation of HO-1.

Heme oxygenase-1 (HO-1) is an inducible rate-controlling enzyme of heme catabolism. The cytoprotective function of HO-1 activity has been verified in multiple studies, and together with its by-products is considered a key component of the cellular stress response. The transcriptional induction of HO-1 has been largely studied in response to multiple forms of stressful stimuli but our understanding of HO-1 post-transcriptional control mechanisms in neuronal cells is currently lacking. In the present report we show the involvement of the RNA-binding proteins (RBPs) embryonic lethal abnormal vision (ELAV) in the regulation of HO-1 gene expression. Our study demonstrates a specific binding between HO-1 messenger RNA (mRNA) and ELAV proteins, accompanied by an increased expression of HO-1 at protein level, in a human neuroblastoma cell line treated with hemin. Clarifying the induction of HO-1 expression at post-transcriptional level may open therapeutic perspectives for treatments associated with the modulation of HO-1 expression.

Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

Butylated hydroxyanisole stimulates heme oxygenase-1 gene expression and inhibits neointima formation in rat arteries.

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound that is a potent inducer of phase II genes. Since heme oxygenase-1 (HO-1) is a vasoprotective protein that is upregulated by phase II inducers, the present study examined the effects of BHA on HO-1 gene expression and vascular smooth muscle cell proliferation. The regulation of HO-1 gene expression and vascular cell growth by BHA was studied in cultured rat aortic smooth muscle cells and in balloon injured rat carotid arteries. Treatment of cultured smooth muscle cells with BHA stimulated the expression of HO-1 protein, mRNA and promoter activity in a time- and concentration-dependent manner. BHA-mediated HO-1 expression was dependent on the activation of NF-E2-related factor-2 by p38 mitogen-activated protein kinase. BHA also inhibited cell cycle progression and DNA synthesis in an HO-1-dependent manner. In addition, the local perivascular delivery of BHA immediately after arterial injury of rat carotid arteries induced HO-1 protein expression and markedly attenuated neointima formation. These studies demonstrate that BHA stimulates HO-1 gene expression in vascular smooth muscle cells, and that the induction of HO-1 contributes to the antiproliferative actions of this phenolic antioxidant. BHA represents a potentially novel therapeutic agent in treating or preventing vasculoproliferative disease.

HMG–CoA reductase inhibitors upregulate heme oxygenase-1 expression in murine RAW264.7 macrophages via ERK, p38 MAPK and protein kinase G pathways

Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme catabolism, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. HMG–CoA reductase inhibitors (statins) possess several anti-inflammatory mechanisms and may be beneficial in the treatment of inflammatory diseases. Our previous study has shown that statins can inhibit iNOS gene expression in murine RAW264.7 macrophages. In this study, we showed that lovastatin, fluvastatin, atorvastatin, simvastatin, mevastatin and pravastatin are able to upregulate the mRNA expression of HO-1 gene. This effect of lovastatin was attenuated by farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), a protein kinase G (PKG) inhibitor (KT5823), a soluble guanylyl cyclase inhibitor (ODQ), a p38 MAPK inhibitor (SB203580), and MEK inhibitors (U0126 and PD98059), but not by inhibitors of protein kinase C (PKC), protein kinase A (PKA), c- jun N-terminal kinase (JNK) and Rho kinase. Consistent with this notion, our previous study has reported the ability of statins to activate ERK and p38 MAPK in RAW264.7 macrophages. Here we further found the participation of cyclic guanosine monophosphate (cGMP)/PKG pathway for ERK activation in cells stimulated with statin and the ability of statin to induce AP-1 activity, which is an essential transcription factor in the regulation of HO-1 gene expression. In addition, a Ras inhibitor (manumycin A) treatment also caused a marked induction of HO-1 mRNA followed by a corresponding increase in HO-1 protein; instead, inhibition of Rho activity by toxin B only led to a transient and weak induction of HO-1. The involvement of signal pathways in manumycin A-induced HO-1 gene expression was associated with p38 MAPK, JNK and ERK activation. Taken together, these results demonstrate for the first time that statins might activate PKG to elicit activations of ERK and p38 MAPK pathways and finally induce HO-1 gene expression, which provides a novel anti-inflammatory mechanism in the therapeutic validity.

Expression pattern and regulation of heme oxygenase-1/heat shock protein 32 in human liver cells.

Heme oxygenase-1 (HO-1) is a stress response protein that is highly inducible under various conditions, such as oxidative or heat stress. The present study investigated expression pattern and regulation of HO-1 in human liver. Expression pattern of HO-1 immunoreactive protein was studied in liver biopsies by immunohistochemistry, revealing constitutive expression in Kupffer cells but not in hepatocytes. HO-1 was, however, inducible in hepatocytes and vascular tissue under pathological conditions, e.g. associated with fatty degeneration or liver malignancies. Regulation of HO-1 gene expression was further studied by Northern blot analysis in HepG2 cells and freshly isolated peripheral blood mononuclear cells as model systems of parenchymal and nonparenchymal liver cell populations, respectively. HO-1 mRNA was inducible in HepG2 cells and mononuclear cells by various agents inducing oxidative stress. However, HO-1 gene expression was not inducible by heat shock. Pyrrolidine dithiocarbamate, an inhibitor of nuclear factor kappaB-dependent gene expression, dose dependently decreased HO-1 mRNA transcripts in human mononuclear cells subjected to oxidative stress while slightly increasing HO-1 gene expression in HepG2 cells. In contrast, HO-1 induction upon oxidative stress was attenuated in HepG2 cells by cycloheximide and dexamethasone. Although activator protein-1 has been reported as the predominant redox-sensitive transcription factor inducing HO-1 expression in murine macrophages, nuclear factor kappaB seems to play a significant role in human mononuclear cells. Our data are consistent with a role for activator protein-1 in HO-1 induction in human HepG2 hepatoma cells. These data suggest a differential regulation of HO-1 gene expression in parenchymal and non-parenchymal human liver cells and may provide a topographic basis for the understanding of the role of the heme oxygenase/carbon monoxide pathway in human liver disease.

Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation.

Gram-negative sepsis is the most common cause of the adult respiratory distress syndrome (ARDS). Lipopolysaccharide (LPS) when administered in vivo produces pathophysiologic changes similar to those seen in ARDS. The pathogenesis of these changes is mediated in part by oxidative stress. We demonstrate that LPS induces high mRNA levels of the stress-inducible gene heme oxygenase-1 (HO-1) in the rat lung. Increased HO-1 mRNA levels correlate with increased HO-1 protein and enzyme activity. Immunohistochemical analyses of lung tissues from rats treated with LPS reveal abundant HO-1 expression in inflammatory and bronchoalveolar epithelial cells. We further examined the molecular regulation of HO-1 gene expression after exposure of RAW 264.7 macrophage cells to LPS in vitro. These cells respond to LPS with increased HO-1 mRNA expression and HO-1 gene transcription. Transcriptional activation of the mouse HO-1 gene by LPS is mediated by a 5' distal enhancer fragment located approximately 4 kbp upstream from the transcription site. Electrophoretic mobility shift assays show increased activator protein-1 (AP-1) binding activity in RAW 264.7 cells after LPS treatment. Mutation of the AP-1 binding site in this enhancer fragment completely abolishes HO-1 gene activation while mutation of CCAAT/enhancer-binding protein (C/EBP) binding site exerts negligible effect, suggesting that the AP-1 family of transcription factors plays a critical role in regulating HO-1 gene activation following LPS treatment. Furthermore, upstream phosphorylation events modulate this AP-1-dependent expression of the HO-1 gene after LPS treatment.