Nitric oxide affects the production of reactive oxygen species in hepatoma cells: implications for the process of oxygen sensing

Treatment of human hepatoma cells (HepG2) with NO-donors for 24 h inhibited hypoxia-induced erythropoietin (EPO) gene activation. NO was found to increase the production of reactive oxygen species (ROS), the putative signaling molecules between a cellular O2-sensor and hypoxia inducible factor 1 (HIF-1). HIF-1 is the prime regulator of O2-dependent genes such as EPO. NO-treatment for more than 20 h reduced HIF-1-driven reporter gene activity. In contrast, immediately after the addition of NO, ROS levels in HepG2 cells decreased below control values for as long as 4 h. Corresponding to these lowered ROS-levels, HIF-1 reporter gene activity and EPO gene expression transiently increased but were reduced when ROS levels rose thereafter. Our findings of a bimodal effect of NO on ROS production shed new light on the involvement of ROS in the mechanism of O2-sensing and may explain earlier conflicting data about the effect of NO on O2-dependent gene expression.     

动物抗低氧胁迫相关基因的表达调控机制

体内氧浓度的稳定是机体维持自身功能的一个必要条件。在低氧条件下,机体内部在低氧信号的刺激下形成一个强大的防御体系以保护自己的组织。在采取防御的过程中,低氧诱导因子-1 (hypoxia inducible factor-1,HIF-1)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、促红细胞生成素(erythropoietin, EPO)、核因子-κB (nuclear factor-κB, NF-κB)等基因表达上调。HIF-1是一个与低氧胁迫相关的转录因子,它的激活与体内氧浓度相关。VEGF是HIF-1下游的一个靶基因,它是至今发现的一个在促血管新生方面起着最关键作用的因子。NF-κB能够抑制由低氧引起的细胞凋亡。以上这些基因在动物抗低氧胁迫方面起着重要作用,综述了低氧条件下HIF-1、VEGF、EPO、NF-κB的功能、表达特性以及调控机制。     

Zinc induces the accumulation of hypoxia-inducible factor (HIF)-1alpha, but inhibits the nuclear translocation of HIF-1beta, causing HIF-1 inactivation

The replacement of heme iron by cobalt or nickel in a putative oxygen sensor is supposed to reduce oxygen binding to the heme protein, resulting in HIF-1 activation and erythropoietin (EPO) induction. According to this hypothesis, zinc might be another example of a transition metal which is capable of stimulating EPO production. By substituting for heme iron, zinc protoporphyrin IX is produced, which has a known low oxygen affinity. However, it has been reported that zinc fails to induce EPO in normoxia, and that it suppresses EPO production in hypoxic cells. This unexpected effect of zinc on EPO production is not understood. In this study, we found that zinc induced the accumulation and nuclear translocation of hypoxia-inducible factor (HIF)-1alpha but inhibited the nuclear translocation of HIF-1beta, which inactivated HIF-1 and suppressed EPO mRNA induction in hypoxic cells.     

Hypoxia signalling in the control of erythropoietin gene expression in rat hepatocytes

This study aimed to investigate the sequence of events involved in the stimulation of erythropoietin (EPO) gene expression by hypoxia in hepatocytes. To this end, primary cultures of rat hepatocytes were kept at either high (40% O₂) or low (3% O₂) oxygen tensions for 2.5 h. Hypoxia increased EPO mRNA about fifteen-fold, whilst the divalent cation cobalt (50–100 μM) or the iron chelator desferrioxamine (10–200 μM) did not increase EPO mRNA levels. Addition of hydrogen peroxide (100–500 μM) to the culture medium did also not change EPO mRNA levels at high or low oxygen tension. Addition of catalase (50–200 μg/ml) to the culture medium resulted in a lower level of hypoxia-induced EPO mRNA. Inhibition of protein synthesis by cycloheximide (100 μM) completely abolished the increase of EPO mRNA in response to hypoxia. Hypoxia but not cobalt increased the appearance of the hypoxia-inducible factor 1 (HIF-1), and this increase was blunted by cycloheximide. Taken together, these findings suggest that a classic heme protein and a related oxygen-dependent production of oxygen radicals is less likely to be involved in the regulation of the EPO gene by oxygen in hepatocytes. On the other hand, intact protein synthesis is an absolute requirement for the hypoxia-induced appearance of HIF-1 and for hypoxia-stimulated expression of the EPO gene in hepatocytes. © 1996 Wiley-Liss, Inc.     

Hypoxia-induced erythropoietin expression in human neuroblastoma requires a methylation free HIF-1 binding site

The glycoprotein hormone Erythropoietin (EPO) stimulates red cell production and maturation. EPO is produced by the kidneys and the fetal liver in response to hypoxia (HOX). Recently, EPO expression has also been observed in the central nervous system where it may be neuroprotective. It remained unclear, however, whether EPO is expressed in the peripheral nervous system and, if so, whether a neuronal phenotype is required for its regulation. Herein, we report that EPO expression was induced by HOX and a HOX mimetic in two cell lines derived from neuroblastoma (NB), a tumor of the peripheral nervous system. Both cell lines with inducible EPO expression, SH-SY5Y and Kelly cells, expressed typical neuronal markers like neuropeptide Y (NPY), growth-associated protein-43 (GAP-43), and neuron-specific enolase (ENO). NB cells with a more epithelial phenotype like SH-SHEP and LAN-5 did not show HOX inducible EPO gene regulation. Still, oxygen sensing and up-regulation of hypoxia-inducible factor-1 (HIF-1) were intact in all cell lines. We found that CpG methylation of the HIF binding site (HBS) in the EPO gene 3' enhancer was only present in the SH-SHEP and LAN-5 cells but not in SH-SY5Y and Kelly cells with regulated EPO expression. The addition of recombinant EPO to all NB cells, both under normoxic and hypoxic conditions, had no effect on cell proliferation. We conclude that the ability to respond to HOX with an increase in EPO expression in human NB may depend on CpG methylation and the differentiation status of these embryonic tumor cells but does not affect the proliferative characteristics of the cells.     

Localization of erythropoietin in and around growing cartilage

Erythropoietin (EPO) is known to be a hematopoietic growth factor and a regulator of red blood cell production. Recently, EPO has also been reported to function as a tissue-protective cytokine and as an angiogenesis promoting factor. EPO is mainly regulated by hypoxia through the action of hypoxia inducible factors (HIF-1α and HIF-2α). The localization of the EPO protein and the HIF-2α protein were immunohistochemically analyzed in developing porcine embryos. Both proteins were localized in developing cartilage tissue. HIF-2α and EPO protein were expressed in the peripheral chondrocytes of cartilage anlagen, in the perichondrium and in the cell condensations that will eventually differentiate into cartilage tissue. The results of this study reveal that EPO might play a role as a survival factor or as a mitogen in developing cartilage tissue. Moreover, the presence of both proteins at the same locations supports the hypothesis that EPO expression is regulated by HIF-2α.     

Development of Lymphoproliferative Diseases by Hypoxia Inducible Factor-1alpha Is Associated with Prolonged Lymphocyte Survival

Hypoxia-inducible factor-1alpha (HIF-1 alpha) plays an essential role in the regulation of various genes associated with low oxygen consumption. Elevated expression of HIF-1alpha has been reported to be associated with tumor progression, invasion and metastasis in many cancers. To investigate the role of HIF-1alpha in tumor development and metastasis, we established transgenic mice constitutively expressing HIF1A gene under regulation of the cytomegalovirus gene promoter. Although HIF-1alpha protein levels varied among organs, expression of HIF1A mRNA in most organs gradually increased in an age-dependent manner. The transgenic mice showed no gross morphological abnormality up to 8 weeks after birth, although they subsequently developed tumors in the lymphoid, lung, and breast; the most prominent tumor was lymphoma appearing in the intestinal mucosa and intra-mesenchymal tissues. The prevalence of tumors reached 80% in 13 months after birth. The constitution of lymphocyte populations in the transgenic mice did not differ from that in wild-type mice. However, lymphocytes of the transgenic mice revealed prolonged survival under long-term culture conditions and revealed increased resistance to cytotoxic etoposide. These results suggest that HIF-1alpha itself is not oncogenic but it may play an important role in lymphomagenesis mediated through the prolonged survival of lymphocytes in this transgenic mouse model.