Hypoxic Gene Signature of Primary and Metastatic Melanoma Cell Lines: Focusing on HIF-1ß and NDRG-1

Background: Hypoxia is an important microenvironmental factor significantly affecting tumor proliferation and progression. The importance of hypoxia is, however, not well known in oncogenesis of malignant melanoma. Aims: To evaluate the difference of hypoxic gene expression signatures in primary melanoma cell lines and metastatic melanoma cell lines and to find the expression changes of hypoxia-related genes in primary melanoma cell lines at experimental hypoxic conditions. Study Design: Cell study. Methods: The mRNA expression levels of hypoxia-related genes in primary melanoma cell lines and metastatic melanoma cell lines and at experimental hypoxic conditions in primary melanoma cell lines were evaluated by using real-time polymerase chain reaction. Depending on the experimental data, we focused on two genes/proteins, the hypoxia-inducible factor-1 beta and the N-myc downstream regulated gene-1. The expression levels of the two proteins were investigated by immunohistochemistry methods in 16 primary and metastatic melanomas, 10 intradermal nevi, and a commercial tissue array comprised of 208 cores including 192 primary and metastatic malignant melanomas. Results: The real-time polymerase chain reaction study showed that hypoxic gene expression signature was different between metastatic melanoma cell lines and primary melanoma cell lines. Hypoxic experimental conditions significantly affected the hypoxic gene expression signature. In immunohistochemical study, N-myc downstream regulated gene-1 expression was found to be lower in primary cutaneous melanoma compared to in intradermal nevi (p=0.001). In contrast, the cytoplasmic expression of hypoxia-inducible factor-1 beta was higher in primary cutaneous melanoma than in intradermal nevi (p=0.001). We also detected medium/strong significant correlations between the two proteins studied in the study groups. Conclusion: Hypoxic response consists of closely related proteins in more complex pathways. These findings will shed light on hypoxic processes in melanoma and unlock a Pandora's box for development of new therapeutic strategies.

COPII proteins exhibit distinct subdomains within each ER exit site for executing their functions.

Secretory proteins are exported from special domains of the endoplasmic reticulum (ER) termed ER exit sites, via COPII-coated carriers. We recently showed that TANGO1 and Sec16 cooperatively organize mammalian ER exit sites for efficient secretion. However, the detailed spatial organization of mammalian ER exit sites is yet to be revealed. Here, we used super-resolution confocal live imaging microscopy (SCLIM) to investigate the localization of endogenous proteins, and we identified domains abundant in transmembrane complexes (TANGO1/cTAGE5/Sec12) juxtaposed to Sec16. Interestingly, this domain can be distinguished from the inner and the outer coats of COPII proteins within each mammalian ER exit site. Cargoes are partially concentrated in the domain for secretion. Our results suggest that mammalian ER exit sites compartmentalize proteins according to their function in COPII vesicle formation.

Telomere elongation protects heart and lung tissue cells from fatal damage in rats exposed to severe hypoxia.

BACKGROUND: The effects of acute hypoxia at high altitude on the telomere length of the cells in the heart and lung tissues remain unclear. This study aimed to investigate the change in telomere length of rat heart and lung tissue cells in response to acute exposure to severe hypoxia and its role in hypoxia-induced damage to heart and lung tissues. METHODS: Forty male Wistar rats (6-week old) were randomized into control group (n = 10) and hypoxia group (n = 30). Rats in control group were kept at an altitude of 1500 m, while rats in hypoxia group were exposed to simulated hypoxia with an altitude of 5000 m in a low-pressure oxygen chamber for 1, 3, and 7 days (n = 10). The left ventricular and right middle lobe tissues of each rat were collected for measurement of telomere length and reactive oxygen species (ROS) content, and the mRNA and protein levels of telomerase reverse transcriptase (TERT), hypoxia-inducible factor1α (HIF-1α), and hypoxia-inducible factor1α (HIF-2α). RESULTS: Increased exposure to hypoxia damaged rat heart and lung tissue cells and increased ROS production and telomere length. The mRNA and protein levels of TERT and HIF-1α were significantly higher in rats exposed to hypoxia and increased with prolonged exposure; mRNA and protein levels of HIF-2α increased only in rats exposed to hypoxia for 7 days. TERT was positively correlated with telomere length and the levels of HIF-1α but not HIF-2α. CONCLUSIONS: Acute exposure to severe hypoxia causes damage to heart and lung tissues due to the production of ROS but promotes telomere length and adaptive response by upregulating TERT and HIF-1α, which protect heart and lung tissue cells from fatal damage.

Differential distribution of hypoxia-inducible factor 1-beta (ARNT or ARNT2) in mouse substantia nigra and ventral tegmental area.

Hypoxia has been proposed as a mechanism underlying gene-environment interactions in the neurodevelopmental model of schizophrenia, and hypoxia-inducible factor 1 (HIF-1) could mediate the interactions. In the current study, we analyzed the HIF-1 beta subunit, as formed by aryl hydrocarbon receptor nuclear translocator (ARNT) or ARNT2, in the mouse substantia nigra (SN) and the ventral tegmental area (VTA). We performed immunohistochemical studies of ARNT and ARNT2 in the adult mouse brain, and colocalization analyses, with specific emphasis on dopaminergic cells, i.e. tyrosine hydroxylase (TH) immunoreactive cells. Bioinformatic analyses identified shared protein partners for ARNT and ARNT2. ARNT immunoreactivity showed widespread neuronal labeling without overt regional specificity. We observed co-localization of ARNT and TH in the SN compacta and VTA. Nuclei strongly labeled for ARNT2 were observed in the SN reticulata, while only weak immunoreactivity for ARNT2 was found in TH-immunoreactive neurons in SN compacta and VTA. Stereological analysis showed that ARNT was preferentially expressed in dopaminergic neurons in SN compacta and VTA. Nuclei strongly labeled for ARNT2 were present in neocortex and CA1 of hippocampus. Differential expression of ARNT and ARNT2 in dopaminergic neurons may relate to the vulnerability of distinct dopaminergic projections to hypoxia and to functional vulnerability in schizophrenia and other neuropsychiatric disorders.

Loss of Arnt (Hif1ß) in mouse epidermis triggers dermal angiogenesis, blood vessel dilation and clotting defects.

Targeted ablation of Aryl hydrocarbon receptor nuclear translocator (Arnt) in the mouse epidermis results in severe abnormalities in dermal vasculature reminiscent of petechia induced in human skin by anticoagulants or certain genetic disorders. Lack of Arnt leads to downregulation of Egln3/Phd3 hydroxylase and concomitant hypoxia-independent stabilization of hypoxia-induced factor 1α (Hif1α) along with compensatory induction of Arnt2. Ectopic induction of Arnt2 results in its heterodimerization with stabilized Hif1α and is associated with activation of genes coding for secreted proteins implicated in control of angiogenesis, coagulation, vasodilation and blood vessel permeability such as S100a8/S100a9, S100a10, Serpine1, Defb3, Socs3, Cxcl1 and Thbd. Since ARNT and ARNT2 heterodimers with HIF1α are known to have different (yet overlapping) downstream targets our findings suggest that loss of Arnt in the epidermis activates an aberrant paracrine regulatory pathway responsible for dermal vascular phenotype in K14-Arnt KO mice. This assumption is supported by a significant decline of von Willebrand factor in dermal vasculature of these mice where Arnt level remains normal. Given the essential role of ARNT in the adaptive response to environmental stress and striking similarity between skin vascular phenotype in K14-Arnt KO mice and specific vascular features of tumour stroma and psoriatic skin, we believe that further characterization of Arnt-dependent epidermal-dermal signalling may provide insight into the role of macro- and micro-environmental factors in control of skin vasculature and in pathogenesis of environmentally modulated skin disorders.

Expression of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocators in human adenoid tissue.

OBJECTIVE: The aim of this study was to determine whether aryl hydrocarbon receptor (AhR) and AhR nuclear translocators (ARNTs) are expressed in human adenoid tissue. METHODS: Paraffin-embedded human adenoid tissue specimens were obtained from eight children with adenoid hypertrophy. Sections were stained immunohistochemically with specific polyclonal antibodies to evaluate the expression pattern of AhR, ARNT1 and ARNT2 were evaluated. RESULTS: AhR-immunoreactivity was ubiquitously seen in human adenoid tissue such as the epithelium, subepithelial layer, germinal center, mantle zone and interfollicular area. ARNT1 was also widely expressed in the same regions as AhR. Although ARNT2 was expressed in the human adenoid, the expression level was significantly lower than that of ARNT1. CONCLUSION: These data suggest that the relationship between AhR and ARNTs may play an important role in the local immune response in adenoid and that ARNT1 may be more important than ARNT2 especially in adenoid tissue.

Expression of hypoxia-inducible factor-1α in human periodontal tissue.

BACKGROUND: Hypoxia-inducible factor (HIF)-1 is a key transcription factor responding to hypoxia. It is composed of an oxygen-sensitive α subunit (HIF-1α) and a constitutively expressed ß subunit. Increasing evidence indicates an essential role for HIF-1α in infection and immunity. Because inflamed periodontium is thought to be hypoxic, we hypothesize that HIF-1α is expressed and related to its upstream regulator tumor necrosis factor (TNF)-α and downstream effecter vascular endothelial growth factor (VEGF). METHODS: Human gingival biopsies were collected from advanced periodontitis sites and clinically healthy sites, and immunohistochemically examined for HIF-1α and VEGF peptides. The messenger ribonucleic acid (mRNA) and protein levels of HIF-1α, VEGF, and TNF-α in the biopsies were then assessed by reverse transcription polymerase chain reaction and Western blotting. RESULTS: HIF-1α-positive immunoreactivity was detected in the nuclei of epithelial and endothelial cells. In periodontal pockets, there was a marked increase in the proportion of fibroblast-like cells and leukocyte-like cells expressing HIF-1α. Protein levels of HIF-1α, VEGF, and TNF-α were significantly higher in periodontal pockets than in control gingival samples. The mRNA expression of VEGF and TNF-α was also increased in periodontal pockets. CONCLUSION: HIF-1α is expressed in healthy and diseased periodontium and may be related to TNF-α and VEGF function during periodontitis.

AhR/Arnt:XRE interaction: turning false negatives into true positives in the modified yeast one-hybrid assay.

Given the frequent occurrence of false negatives in yeast genetic assays, it is both interesting and practical to address the possible mechanisms of false negatives and, more important, to turn false negatives into true positives. We recently developed a modified yeast one-hybrid system (MY1H) useful for investigation of simultaneous protein-protein and protein:DNA interactions in vivo. We coexpressed the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) domains of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt)--namely NAhR and NArnt, respectively--which are known to form heterodimers and bind the cognate xenobiotic response element (XRE) sequence both in vitro and in vivo, as a positive control in the study of XRE-binding proteins in the MY1H system. However, we observed negative results, that is, no positive signal detected from binding of the NAhR/NArnt heterodimer and XRE site. We demonstrate that by increasing the copy number of XRE sites integrated into the yeast genome and using double GAL4 activation domains, the NAhR/NArnt heterodimer forms and specifically binds the cognate XRE sequence, an interaction that is now clearly detectable in the MY1H system. This methodology may be helpful in troubleshooting and correcting false negatives that arise from unproductive transcription in yeast genetic assays.

Hypoxia induces transforming growth factor-beta1 gene expression in the pulmonary artery of rats via hypoxia-inducible factor-1alpha.

The present study was undertaken to investigate the dynamic expression of hypoxia inducible factor-1alpha (HIF-1alpha) and transforming growth factor-beta1 (TGF-beta1) in hypoxia-induced pulmonary hypertension of rats. It was found that mean pulmonary arterial pressure (mPAP) increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d of hypoxia. HIF-1alpha mRNA staining was less positive in the control, hypoxia for 3 d and hypoxia for 7 d, but began to enhance significantly after 14 d of hypoxia, then remained stable. Expression of HIF-1alpha protein in the control was less positive, but was up-regulated in pulmonary arterial tunica intima of all hypoxic rats. TGF-beta1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-beta1 protein staining was less positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia, and then weakening after 14 and 21?d of hypoxia. Western blotting showed that HIF-1alpha protein levels increased significantly after 7 d of hypoxia and then remained at a high level. TGF-beta1 protein level was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia, and then decreasing after 14 and 21?d of hypoxia. Linear correlation analysis showed that HIF-1alpha mRNA, TGF-beta1 mRNA, TGF-beta1 protein were positively correlated with mPAP, vessel morphometry and right ventricular hypertrophy index. TGF-beta1 protein (tunica adventitia) was negatively correlated with HIF-1alpha mRNA. Taken together, our results suggest that changes in HIF-1alpha and TGF-beta1 expression after hypoxia play an important role in hypoxia-induced pulmonary hypertension of rats.

Hypoxia-inducible factors in the kidney.

Tissue hypoxia not only occurs under pathological conditions but is also an important microenvironmental factor that is critical for normal embryonic development. Hypoxia-inducible factors HIF-1 and HIF-2 are oxygen-sensitive basic helix-loop-helix transcription factors, which regulate biological processes that facilitate both oxygen delivery and cellular adaptation to oxygen deprivation. HIFs consist of an oxygen-sensitive alpha-subunit, HIF-alpha, and a constitutively expressed beta-subunit, HIF-beta, and regulate the expression of genes that are involved in energy metabolism, angiogenesis, erythropoiesis and iron metabolism, cell proliferation, apoptosis, and other biological processes. Under conditions of normal Po(2), HIF-alpha is hydroxylated and targeted for rapid proteasomal degradation by the von Hippel-Lindau (VHL) E3-ubiquitin ligase. When cells experience hypoxia, HIF-alpha is stabilized and either dimerizes with HIF-beta in the nucleus to form transcriptionally active HIF, executing the canonical hypoxia response, or it physically interacts with unrelated proteins, thereby enabling convergence of HIF oxygen sensing with other signaling pathways. In the normal, fully developed kidney, HIF-1alpha is expressed in most cell types, whereas HIF-2alpha is mainly found in renal interstitial fibroblast-like cells and endothelial cells. This review summarizes some of the most recent advances in the HIF field and discusses their relevance to renal development, normal kidney function and disease.

Calcium signaling stimulates translation of HIF-alpha during hypoxia.

Hypoxia-inducible factors (HIFs) are ubiquitous transcription factors that mediate adaptation to hypoxia by inducing specific sets of target genes. It is well accepted that hypoxia induces accumulation and activity of HIFs by causing stabilization of their alpha subunits. We have demonstrated that hypoxia stimulates translation of HIF-1alpha and -2alpha proteins by distributing HIF-alpha mRNAs to larger polysome fractions. This requires influx of extracellular calcium, stimulation of classical protein kinase C-alpha (cPKC-alpha), and the activity of mammalian target of rapamycin, mTOR. The translational component contributes to approximately 40-50% of HIF-alpha proteins accumulation after 3 h of 1% O2. Hypoxia also inhibits general protein synthesis and mTOR activity; however, cPKC-alpha inhibitors or rapamycin reduce mTOR activity and total protein synthesis beyond the effects of hypoxia alone. These data show that during general inhibition of protein synthesis by hypoxia, cap-mediated translation of selected mRNAs is induced through the mTOR pathway. We propose that calcium-induced activation of cPKC-alpha hypoxia partially protects an activity of mTOR from hypoxic inhibition. These results provide an important physiologic insight into the mechanism by which hypoxia-stimulated influx of calcium selectively induces the translation of mRNAs necessary for adaptation to hypoxia under conditions repressing general protein synthesis.

Experimental hypoxia and embryonic angiogenesis.

We examined the role of hypoxia and HIF factors in embryonic angiogenesis and correlated the degree of hypoxia with the level of HIF and VEGF expression and blood vessel formation. Quail eggs were incubated in normoxic and hypoxic (16% O(2)) conditions. Tissue hypoxia marker, pimonidazol hydrochloride, was applied in vivo for 1 hr and detected in sections with Hypoxyprobe-1 Ab. VEGF and HIF expression was detected by in situ hybridization. HIF-1alpha protein was detected in sections and by Western blot. Endothelial cells were visualized with QH-1 antibody. Hypoxic regions were detected even in normoxic control embryos, mainly in brain, neural tube, branchial arches, limb primordia, and mesonephros. The expression patterns of HIF-1alpha and HIF-1beta factors followed, in general, the Hypoxyprobe-1 marked regions. HIF-2alpha was predominantly expressed in endothelial cells. Diffuse VEGF expression was detected in hypoxic areas of neural tube, myocardium, digestive tube, and most prominently in mesonephros. Growing capillaries were directed to areas of VEGF positivity. Hypoxic regions in hypoxic embryos were larger and stained more intensely. VEGF and HIF-1 factors were proportionately elevated in Hypoxyprobe-1 marked regions without being expressed at new sites and were followed by increased angiogenesis. Our results demonstrate that normal embryonic vascular development involves the HIF-VEGF regulatory cascade. Experimentally increasing the level of hypoxia to a moderate level resulted in over-expression of HIF-1 factors and VEGF followed by an increase in the density of developing vessels. These data indicate that embryonic angiogenesis is responsive to environmental oxygen tension and, therefore, is not entirely genetically controlled.

[Modification and application of recombinant yeast bioassay for measuring the AhR ligand activity].

A recombinant yeast bioassay, a yeast Saccharomyces cerevisiae, in which the human AhR and ARNT complex are coexpressed, is one of the methods to screen the active AhR agonist. In this study, the original agonist test was modified. The exposure time was reduced from 18 to 8 hours when experiment was under the following conditions: (1) the yeast was cultured in 0.2% glucose medium for 24 hours; (2) chemical exposure was carried in 2% galactose medium in glass tube. Finally, the AhR acitivity of hexachlorobenzene and pentachlorobenzene were assessed, and their toxicity equivalent factors were found to be 0.018629 and 0.000294, respectively.