The flavonoid quercetin induces hypoxia-inducible factor-1α (HIF-1α) 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-1α (HIF-1α) in normoxia, but its mechanism of action has not been determined. In this study we characterized the induction of HIF-1α 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-1α 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-1α 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-1α and inhibits cell proliferation predominantly by decreasing the concentration of intracellular iron through chelation.     

Induction of hypoxia inducible factor (HIF-1α) in rat kidneys by iron chelation with the hydroxypyridinone, CP94

Hypoxia inducible factor (HIF-1α) is a master regulator of tissue adaptive responses to hypoxia whose stability is controlled by an iron containing prolyl hydroxylase domain (PHD) protein. A catalytic redox cycle in the PHD's iron center that results in the formation of a ferryl (Fe(+4)) intermediate has been reported to be responsible for the hydroxylation and subsequent degradation of HIF-1α under normoxia. We show that induction of HIF-1α in rat kidneys can be achieved by iron reduction by the hydroxypyridin-4 one (CP94), an iron chelator administered intraperitoneally in rats. The extent of HIF protein stabilization as well as the expression of HIF target genes, including erythropoietin (EPO), in kidney tissues was comparable to those induced by known inhibitors of the PHD enzyme, such as desferrioxamine (DFO) and cobalt chloride (CoCl(2)). In human kidney cells and in vitro PHD activity assay, we were able to show that the HIF-1α protein can be stabilized by addition of CP94. This appears to inactivate PHD; and thus prevents the hydroxylation of HIF-1α. In conclusion, we have identified the inhibition of iron-binding pocket of PHD as an underlying mechanism of HIF induction in vivo and in vitro by a bidentate hydroxypyridinone.     

Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450k in rat kidney cortex: I. Characteristics of the hydroxylase system

Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450_K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450_K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450_K follow the same pattern of inactivation with increasing temperature. The apparent K_m for the induced hydroxylase was 7.7 μm and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450_K-dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10^?5m), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450_K-dependent aryl hydroxylase activity which differs from that present in the control rat.     

Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD)

Cyst expansion in polycystic kidney disease (PKD) results in localized hypoxia in the kidney that may activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α and autophagy, a form of programmed cell repair, are induced by hypoxia. The purposes were to determine HIF-1α expression and autophagy in rat and mouse models of PKD. HIF-1α was detected by electrochemiluminescence. Autophagy was visualized by electron microscopy (EM). LC3 and beclin-1, markers of autophagy, were detected by immunoblotting. Eight-week-old male heterozygous (Cy/+) and 4-wk-old homozygous (Cy/Cy) Han:SPRD rats, 4-wk-old cpk mice, and 112-day-old Pkd2WS25/- mice with a mutation in the Pkd2 gene were studied. HIF-1α was significantly increased in massive Cy/Cy and cpk kidneys and not smaller Cy/+ and Pkd2WS25/- kidneys. On EM, features of autophagy were seen in wild-type (+/+), Cy/+, and cpk kidneys: autophagosomes, mitophagy, and autolysosomes. Specifically, autophagosomes were found on EM in the tubular cells lining the cysts in cpk mice. The increase in LC3-II, a marker of autophagosome production and beclin, a regulator of autophagy, in Cy/Cy and cpk kidneys, followed the same pattern of increase as HIF-1α. To determine the role of HIF-1α in cyst formation and/or growth, Cy/+ rats, Cy/Cy rats, and cpk mice were treated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). 2ME2 had no significant effect on kidney volume or cyst volume density. In summary, HIF-1α is highly expressed in the late stages of PKD and is associated with an increase in LC3-II and beclin-1. The first demonstration of autophagosomes in PKD kidneys is reported. Inhibition of HIF-1α did not have a therapeutic effect.     

Basal expression of copper transporter 1 in intestinal epithelial cells is regulated by hypoxia-inducible factor 2α

Hypoxia, via stabilization of HIF2α, regulates the expression of the intestinal iron transporters DMT1 and ferroportin. Here we investigated whether the intestinal copper importer Ctr1 was also regulated by hypoxia. Copper uptake and Ctr1 mRNA expression were significantly increased in Caco-2 cells exposed to hypoxia. To determine whether HIF2α was involved in regulation of Ctr1 expression, we employed three models of HIF2α knockdown (chemical suppression of HIF2α translation in Caco-2 cells; HIF2α-siRNA-treated HuTu80 cells; HIF2α-intestinal knockout mice); Ctr1 mRNA expression was decreased in all three models under normoxic conditions. HIF2α translational inhibitor did not alter Ctr1 expression under hypoxic conditions. We conclude that basal expression of Ctr1 is regulated by HIF2α; however, the induction by hypoxia is a HIF2α-independent event.     

Differences in hydroxylation and binding of Notch and HIF-1α demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1)

FIH-1, factor inhibiting hypoxia-inducible factor-1 (HIF-1), regulates oxygen sensing by hydroxylating an asparagine within HIF-α. It also hydroxylates asparagines in many proteins containing ankyrin repeats, including Notch1–3, p105 and IκBα. Relative binding affinity and hydroxylation rate are crucial determinants of substrate selection and modification. We determined the contributions of substrate sequence composition and length and of oxygen concentration to the FIH-1-binding and/or hydroxylation of Notch1–4 and compared them with those for HIF-1α. We also demonstrated hydroxylation of two asparagines in Notch2 and 3, corresponding to Sites 1 and 2 of Notch1, by mass spectrometry for the first time.Our data demonstrate that substrate length has a much greater influence on FIH-1-dependent hydroxylation of Notch than of HIF-1α, predominantly through binding affinity rather than maximal reaction velocity. The K_m value of FIH-1 for Notch1, <0.2 μM, is at least 250-fold lower than that of 50 μM for HIF-1α. Site 1 of Notch1–3 appeared the preferred site of FIH-1 hydroxylation in these substrates. Interestingly, binding of Notch4 to FIH-1 was observed with an affinity almost 10-fold lower than for Notch1–3, but no hydroxylation was detected. Importantly, we demonstrate that the K_m of FIH-1 for oxygen at the preferred Site 1 of Notch1–3, 10–19 μM, is an order of magnitude lower than that for Site 2 or HIF-1α. Hence, at least during in vitro hydroxylation, Notch is likely to become efficiently hydroxylated by FIH-1 even under relatively severe hypoxic conditions, where HIF-1α hydroxylation would be reduced.     

Carvedilol prevents cardiac hypertrophy and overexpression of hypoxia-inducible factor-1α and vascular endothelial growth factor in pressure-overloaded rat heart

Summary The use of -blockers has emerged as a beneficial treatment for cardiac hypertrophy. Hypoxia-inducible factor-1 (HIF-1) is tightly regulated in the ventricular myocardium. However, the expression of HIF-1 in cardiac hypertrophy due to pressure overload and after treatment with -blocker is little known. To evaluate the effect of carvedilol on both myocardial HIF-1 expression and cardiac hypertrophy, infra-renal aortic banding was performed for 4 weeks in adult Sprague-Dawley rats to induce cardiac hypertrophy. Carvedilol at 50 mg/kg body weight per day after surgery was given. Heart weight and the ratio of heart weight and body weight increased significantly after aortic banding for 4 weeks in the absence of drug treatment. Mean arterial pressure increased from 80 ± 9 mmHg in the sham group to 94 ±5 mmHg (p < 0.001) in the banding group. Echocardiography showed concentric hypertrophy after aortic banding. Mean arterial pressure decreased after treatment with carvedilol. The increased wall thickness and heart weight was reversed to normal by carvedilol. Western blot showed that HIF-1, vascular endothelial growth factor (VEGF) and brain natriuretic peptide (BNP) proteins were up-regulated and nerve growth factor- (NGF-) down-regulated in the banding group. Treatment with valsartan, doxazosin, or N-acetylcysteine did not significantly affect HIF-1 and VEGF proteins expression in the banding groups. Real-time polymerase chain reaction showed that mRNA of HIF-1, VEGF and BNP increased and mRNA of NGF- decreased in the banding group. Treatment with carvedilol reversed both protein and mRNA of HIF-1, VEGF, BNP, and NGF- to the baseline values. Increased immunohistochemical labeling of HIF-1, VEGF, and BNP in the ventricular myocardium was observed in the banding group and carvedilol again normalized the labeling. In conclusion, HIF-1, VEGF, and BNP mRNA and protein expression were up-regulated, while NGF- mRNA and protein was downregulated in the rat model of pressure-overloaded cardiac hypertrophy. Treatment with carvedilol is associated with a reversal of abnormal regulation of HIF-1,VEGF, BNP, and NGF- in the hypertrophic myocardium.