Hypoxia-inducible factor-1 (HIF-1) is composed of HIF-1alpha and HIF-1beta, and is a master regulator of oxygen homeostasis, playing critical roles in physiological and pathological processes. Normally, the formation and transcriptional activity of HIF-1 depend on the amount of HIF-1alpha, and the expression of HIF-1alpha is tightly controlled by the cellular oxygen tension. Recent progress in the study of its regulation mechanism provided clues as to how HIF-1alpha is regulated by oxygen. It appears that HIF-1alpha is not regulated only by the oxygen tension, but also by various other stimuli, such as transition metals, nitric oxide, reactive oxygen species, growth factors, and mechanical stresses. In this review, we summarize the oxygen-dependent and -independent regulation of HIF-1alpha, and the respective physiological and pathological meanings.

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Ultraviolet (UV) radiation damages human skin and causes skin diseases such as epidermal hyperplasia, sunburn, inflammatory responses and photoaging. Photoaging is associated with upregulated matrix metalloproteinase (MMP) expression and downregulated collagen synthesis. Fucosterol, which is isolated from marine brown algae, has been reported to possess antioxidant and anticancer activities; however, its effects on photoaging are unknown. This study assessed the effects of fucosterol on photoaging and investigated its mechanisms of action in UV-irradiated immortalized human keratinocytes (HaCaT) by enzyme-linked immunosorbent assay, semi-quantitative reverse transcription-polymerase chain reaction, Western blot analysis and 2',7'-dichlorofluorescein diacetate assay. Our results showed that fucosterol attenuated UV-induced MMP and inflammatory cytokine expression by deactivating mitogen-activated protein kinases (MAPKs) induced by reactive oxygen species. Fucosterol also increased type-I procollagen and antioxidant enzyme expression. Taken together, fucosterol regulates the expression of MMPs and type-I procollagen in UV-irradiated HaCaT by modulating MAPK, suggesting it as a potential candidate for prevention and treatment of skin aging.

The cyclin-dependent kinase inhibitor p21(WAF1/Cip1) plays a central role in a spatial and temporal balance of epidermal keratinocyte proliferation and growth arrest. However, what controls p21 expression in keratinocytes remains uncertain. Hypoxia-inducible factor 1alpha (HIF-1alpha) does not only express a variety of genes essential for hypoxic adaptation, but also up-regulates p21 so as to slow down cell cycle under hypoxic conditions. In the present study, we examined the role of HIF-1alpha in p21-mediated growth arrest of keratinocyte. Keratinocyte proliferation was arrested in the G1 phase at a high cell density. p21 was also up-regulated in a cell density-dependent manner and was found to be highly expressed in epidermal keratinocytes of normal human skins. In addition, in the same specimens and cells, we noted robust HIF-1alpha expression. HIF-1alpha siRNAs inhibited p21 expression and released the G1 arrest. In vivo, moreover, the intradermal injection of HIF-1alpha siRNA attenuated p21 expression in rat epidermis and induced skin hyperplasia. Mechanistically, we propose that the production of mitochondrial reactive oxygen species and the activation of the MEK/ERK pathway are involved in the HIF-1alpha stabilization in keratinocytes. These results imply that HIF-1alpha functions as an up-stream player in the p21-mediated growth arrest of keratinocytes.

Atrial natriuretic peptide (ANP) is a cardiac peptide, the transcription of which is up-regulated in the ischaemic ventricle. However, the molecular mechanism of ANP induction is unclear. This study demonstrated that ANP mRNA expression in rat ventricular myocardium is induced in an early phase of ischaemia, preceded by hypoxia-inducible factor-1 (HIF-1) alpha expression. The ANP gene was also induced by hypoxia or HIF-1 inducers such as CoCl2 and desferrioxamine in H9c2 and neonatal cardiomyocytes. The 2307 bp 5'-flanking region of the rat ANP gene was cloned and fused to the luciferase gene. Evidence of the promoter activity was only apparent in the myocytes and was induced by hypoxia and HIF-1 inducers. The overexpression of HIF-1alpha markedly enhanced ANP promoter activity, and a dominant-negative isoform completely suppressed it. We demonstrated that the promoter regions are essential for hypoxic ANP induction. One promoter region, containing the HIF-1-binding sequence, is regulated directly by HIF-1. The other region is also activated by HIF-1 despite having no HIF-1-binding sequence. These results suggest that HIF-1 enhances the transactivation of the ANP gene in hypoxic myocytes, implying that stimulation of the ANP promoter by HIF-1 may in fact be responsible for the induction of the ANP gene in ischaemic ventricular myocardium.

Circulating erythropoietin (EPO) is mainly produced by the kidneys and mediates erythrogenesis in bone marrow and nonhematopoietic cell survival. EPO is also produced in other tissues where it functions as a paracrine. Moreover, the hypoxic induction of EPO is known to be mediated by HIF-1alpha and HIF-2alpha, but it remains obscure as to which of these two mediators mainly contributes to EPO expression. Thus, we designed in vivo experiments to evaluate the contributions made by HIF-1alpha and HIF-2alpha to EPO expression. In mice exposed to mild whole body hypoxia, HIF-1alpha and HIF-2alpha were both induced in all tissues examined. However, EPO mRNA was expressed in kidney and brain, but not in liver and lung. Likewise, chromatin immunoprecipitation (CHIP) analyses demonstrated that HIF-1alpha or HIF-2alpha binding to the EPO gene increased under hypoxic conditions only in kidney and brain. A comparison of CHIP data and EPO mRNA levels suggested that, during mild hypoxia, renal EPO transcription is induced equally by HIF-1alpha and HIF-2alpha, but that brain EPO is mainly induced during hypoxia by HIF-2alpha. Thus, HIF-1alpha and HIF-2alpha appear to contribute to EPO expression tissue specifically.

Amphotericin B (AmB) is widely used for treating severe systemic fungal infections. However, long-term AmB treatment is invariably associated with adverse effects such as anemia. The erythropoietin (EPO) suppression by AmB has been proposed to contribute to the development of anemia. However, the mechanism whereby EPO is suppressed remains obscure. In this study, we investigated the possibility that AmB inhibits the transcription of the EPO gene by inactivating HIF-1, which is a known key transcription factor and regulator of EPO expression. EPO mRNA levels were markedly attenuated by AmB treatment both in rat kidneys and in Hep3B cells. AmB inactivated the transcriptional activity of HIF-1␣, but did not affect the expression or localization of HIF-1 subunits. Moreover, AmB was found to specifically repress the C-terminal transactivation domain (CAD) of HIF-1␣, and this repression by AmB required Asn803, a target site of the factor-inhibiting HIF-1 (FIH); moreover, this repressive effect was reversed by FIH inhibitors. Furthermore, AmB stimulated CAD-FIH interaction and inhibited the p300 recruitment by CAD. We propose that this mechanism underlies the unexplained anemia associated with AmB therapy.

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