Expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in pituitary tumours

The present study was performed to investigate HIF-1alpha (hypoxia-inducible factor-1alpha) expression in a large number of immunohistochemically and ultrastructurally characterized surgically removed pituitary tumours. The potential relation of HIF-1alpha with outcome variables as well as the presence of HIF-1alpha expression in the tumours treated with dopamine agonists and octreotide, a long-acting somatostatin analogue was also investigated. HIF-1alpha immunoreactivity was confined to the nucleoplasm whereas the nucleoli were unconspicuous. The distribution of HIF-1alpha was evident in the tumours whereas normal adenohypophysial cells showed no HIF-1alpha staining. HIF-1alpha expression was detected not only in the tumour cells but also in endothelial cells lining the blood vessels within the tumour. ACTH producing adenomas showed the lowest level of HIF-1alpha expression whereas pituitary carcinomas and GH producing adenomas had the highest counts. The statistical study demonstrated no significant correlation between HIF-1alpha expression, patient age, gender, tumour, size, invasiveness, cell proliferation rate and vascularity. These results suggest that the behaviour of pituitary tumours does not primarily depend of HIF-1alpha expression. Our study demonstrated an increase HIF-1alpha expression in bromocriptine treated PRL producing pituitary adenomas compared with untreated tumours but no increase in octreotide treated tumours.     

The flavonoid quercetin induces hypoxia-inducible factor-1alpha (HIF-1alpha) and inhibits cell proliferation by depleting intracellular iron

Quercetin, a flavonoid with anti-oxidant, metal chelating, kinase modulating and anti-proliferative properties, can induce hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia, but its mechanism of action has not been determined. In this study we characterized the induction of HIF-1alpha and the inhibition of cell proliferation caused by quercetin in HeLa and ASM (airway smooth muscle) cells and examined the effect of iron on these processes. Furthermore, we investigated the relevance of the intracellular levels of quercetin to HIF-1alpha expression and cell proliferation. Our data demonstrate that quercetin depletes intracellular calcein-chelatable iron and that supplying additional iron from extracellular or intracellular pools abrogates the induction of HIF-1alpha by quercetin. Moreover, addition of iron reverses the quercetin-induced inhibition of DNA synthesis, cell proliferation and cycle progression, but to different extents, depending on cell type. We propose that quercetin stabilises HIF-1alpha and inhibits cell proliferation predominantly by decreasing the concentration of intracellular iron through chelation.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

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.     

Spermidine/spermine N(1)-acetyltransferase-1 binds to hypoxia-inducible factor-1alpha (HIF-1alpha) and RACK1 and promotes ubiquitination and degradation of HIF-1alpha

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of oxygen homeostasis that controls the expression of genes encoding proteins that play key roles in angiogenesis, erythropoiesis, and glucose/energy metabolism. The stability of the HIF-1alpha subunit is regulated by ubiquitination and proteasomal degradation. In aerobic cells, O(2)-dependent prolyl hydroxylation of HIF-1alpha is required for binding of the von Hippel-Lindau tumor suppressor protein VHL, which then recruits the Elongin C ubiquitin-ligase complex. SSAT2 (spermidine/spermine N-acetyltransferase-2) binds to HIF-1alpha and promotes its ubiquitination/degradation by stabilizing the interaction of VHL and Elongin C. Treatment of cells with heat shock protein HSP90 inhibitors induces the degradation of HIF-1alpha even under hypoxic conditions. HSP90 competes with RACK1 for binding to HIF-1alpha, and HSP90 inhibition leads to increased binding of RACK1, which recruits the Elongin C ubiquitin-ligase complex to HIF-1alpha in an O(2)-independent manner. In this work, we demonstrate that SSAT1, which shares 46% amino acid identity with SSAT2, also binds to HIF-1alpha and promotes its ubiquitination/degradation. However, in contrast to SSAT2, SSAT1 acts by stabilizing the interaction of HIF-1alpha with RACK1. Thus, the paralogs SSAT1 and SSAT2 play complementary roles in promoting O(2)-independent and O(2)-dependent degradation of HIF-1alpha.     

Cobalt chloride-induced estrogen receptor alpha down-regulation involves hypoxia-inducible factor-1alpha in MCF-7 human breast cancer cells

The estrogen receptor (ER) is down-regulated under hypoxia via a proteasome-dependent pathway. We studied the mechanism of ERalpha degradation under hypoxic mimetic conditions. Cobalt chloride-induced ERalpha down-regulation was dependent on the expression of newly synthesized protein(s), one possibility of which was hypoxia-inducible factor-1alpha (HIF-1alpha). To examine the role of HIF-1alpha expression in ERalpha down-regulation under hypoxic-mimetic conditions, we used a constitutively active form of HIF-1alpha, HIF-1alpha/herpes simplex viral protein 16 (VP16), constructed by replacing the transactivation domain of HIF-1alpha with that of VP16. Western blot analysis revealed that HIF-1alpha/VP16 down-regulated ERalpha in a dose-dependent manner via a proteasome-dependent pathway. The kinase pathway inhibitors PD98059, U0126, wortmannin, and SB203580 did not affect the down-regulation. A mammalian two-hybrid screen and immunoprecipitation assays indicated that ERalpha interacted with HIF-1alpha physically. These results suggest that ERalpha down-regulation under hypoxia involves protein-protein interactions between the ERalpha and HIF-1alpha.     

A prolyl-hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, induces haem oxygenase-1 expression in human cells through a mechanism independent of hypoxia-inducible factor-1alpha

Hypoxia-inducible factor (HIF)-1 is important for cellular homeostasis under hypoxia. Expression of haem oxygenase-1 (HO-1), an essential enzyme in haem catabolism, varies under hypoxia, depending on cell types. Here, we studied the role of HIF-1alpha, a component of HIF-1, in the regulation of HO-1 expression using three human cell lines: HeLa cervical cancer, and ARPE-19 and D407 retinal pigment epithelial cells. Under hypoxia (1% O(2)), the expression of HO-1 mRNA was decreased in HeLa cells, increased in D407 cells, and unchanged in ARPE-19 cells, while HIF-1alpha protein was accumulated in these cell lines. Thus, HIF-1alpha is unlikely to function as a key regulator for HO-1 expression under hypoxia. We then used ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl hydroxylases, to accumulate HIF-1alpha protein under normoxia. Treatment with EDHB (250-500 microM) increased HIF-1alpha protein levels in HeLa and D407 cells, but not in ARPE-19 cells, whereas EDHB at lower concentrations (50-100 microM) consistently induced HO-1 mRNA expression (about 20-fold) in these three cell lines. Moreover, EDHB increased the HO-1 gene promoter activity via the enhancer that lacks a HIF-1-binding site. In conclusion, the signals evoked by hypoxia and after EDHB treatment differentially regulate HO-1 mRNA expression through HIF-1alpha-independent mechanisms.