Partial HIF-1alpha deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia

Chronic hypoxia depolarizes and reduces K+ current in pulmonary arterial smooth muscle cells (PASMCs). Our laboratory previously demonstrated that hypoxia-inducible factor-1 (HIF-1) contributed to the development of hypoxic pulmonary hypertension. In this study, electrophysiological parameters were measured in PASMCs isolated from intrapulmonary arteries of mice with one null allele at the Hif1a locus encoding HIF-1alpha [Hif1a(+/-)] and from their wild-type [Hif1a(+/+)] littermates after 3 wk in air or 10% O2. Hematocrit and right ventricular wall and left ventricle plus septum weights were measured. Capacitance, K+ current, and membrane potential were measured with whole cell patch clamp. Similar to our laboratory's previous results, hypoxia-induced right ventricular hypertrophy and polycythemia were blunted in Hif1a(+/-) mice. Hypoxia increased PASMC capacitance in Hif1a(+/+) mice but not in Hif1a(+/-) mice. Chronic hypoxia depolarized and reduced K+ current density in PASMCs from Hif1a(+/+) mice. In PASMCs from hypoxic Hif1a(+/-) mice, no reduction in K+ current density was observed, and depolarization was significantly blunted. Thus partial deficiency of HIF-1alpha is sufficient to impair hypoxia-induced depolarization, reduction of K+ current density, and PASMC hypertrophy.     

HIF-1alpha, HIF-2alpha and VHL mRNA expression in different cell lines during hypoxia

Hypoxia inducible factor-1alpha (HIF-1alpha) mRNA expression is significantly decreased under hypoxia in different cell lines exposed directly to hypoxia or treated with dimethyloxalylglycine which mimics hypoxic effects under normoxic conditions. However, the decreased expression of HIF-1alpha mRNA is accompanied by an increase of HIF-1alpha protein (pHIF-1alpha) level as well as by overexpression of known HIF-dependent genes (VEGF, Glut1, PFKFB-3 and PFKFB-4) under hypoxic conditions or with the use of dimethyloxalylglycine. Expression of HIF-1alpha mRNA also depends on iron because desferrioxamine and cobalt chloride produce similar to hypoxia effects on the levels of this mRNA. It was shown that HIF-1alpha mRNA expression did not change significantly in some cell lines (SKBR3, MDA-MB468 and BT549) under hypoxia. However, in these cell lines hypoxia decreases expression of HIF-2alpha mRNA, another member of HIF-alpha gene family, as a result of cell specific regulation of HIF-alpha genes under hypoxia. Moreover, hypoxia slightly induces expression of PFKFB-4 mRNA in SKBR3, MDA-MB468 and BT549 as compared to other cell lines where this effect of hypoxia was much stronger and adaptation to hypoxia is controlled by HIF-1alpha. Hypoxia slightly reduces expression of tumor suppressor VHL which targets HIF-1alpha for ubiquitination. Thus, our results clearly demonstrated down regulation of HIF-1alpha or HIF-2alpha in different cell lines by hypoxia.     

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.     

Hypoxia-inducible factor 1alpha (HIF-1alpha)-mediated hypoxia increases BACE1 expression and beta-amyloid generation

The incidence of Alzheimer disease (AD) and vascular dementia is greatly increased following cerebral ischemia and stroke in which hypoxic conditions occur in affected brain areas. beta-Amyloid peptide (Abeta), which is derived from the beta-amyloid precursor protein (APP) by sequential proteolytic cleavages from beta-secretase (BACE1) and presenilin-1 (PS1)/gamma-secretase, is widely believed to trigger a cascade of pathological events culminating in AD and vascular dementia. However, a direct molecular link between hypoxic insults and APP processing has yet to be established. Here, we demonstrate that acute hypoxia increases the expression and the enzymatic activity of BACE1 by up-regulating the level of BACE1 mRNA, resulting in increases in the APP C-terminal fragment-beta (betaCTF) and Abeta. Hypoxia has no effect on the level of PS1, APP, and tumor necrosis factor-alpha-converting enzyme (TACE, an enzyme known to cleave APP at the alpha-secretase cleavage site). Sequence analysis, mutagenesis, and gel shift studies revealed binding of HIF-1 to the BACE1 promoter. Overexpression of HIF-1alpha increases BACE1 mRNA and protein level, whereas down-regulation of HIF-1alpha reduced the level of BACE1. Hypoxic treatment fails to further potentiate the stimulatory effect of HIF-1alpha overexpression on BACE1 expression, suggesting that hypoxic induction of BACE1 expression is primarily mediated by HIF-1alpha. Finally, we observed significant reduction in BACE1 protein levels in the hippocampus and the cortex of HIF-1alpha conditional knock-out mice. Our results demonstrate an important role for hypoxia/HIF-1alpha in modulating the amyloidogenic processing of APP and provide a molecular mechanism for increased incidence of AD following cerebral ischemic and stroke injuries.     

Intermittent hypoxia changes HIF-1alpha phosphorylation pattern in endothelial cells: unravelling of a new PKA-dependent regulation of HIF-1alpha

Vascularized tumors are exposed to intermittent hypoxia, that is, hypoxia followed by periods of reoxygenation. Abnormal structure and dysfunction of tumor blood vessels are responsible for these conditions. These repeated short periods of hypoxia concern tumor cells as well as endothelial cells. However, the effects of intermittent hypoxia are poorly understood. The aim of this study was to investigate the effects of intermittent hypoxia on endothelial cells and particularly on HIF-1alpha, a central actor in adaptive response to hypoxia. For that, endothelial cells were exposed to four repeated cycles of 1-h hypoxia followed by 30 min of reoxygenation. We showed that repeated cycles of hypoxia/reoxygenation induced a modification in HIF-l alpha phosphorylation pattern: a progressive increase in HIF-1alpha phosphorylated form was observed during the hypoxic periods. Activation of p42/p44, Akt and PKA was observed in parallel. PKA was shown to be involved in the phosphorylation of HIF-lalpha under intermittent hypoxia, while p42/p44 and Akt were not. As HIF-1 activity is often associated with enhanced cell survival, a better knowledge of the effects of intermittent hypoxia on endothelial cells and the highlight of particular mechanisms induced by intermittent hypoxia are essential to understand the behavior of endothelial cells during neo-angiogenesis.     

The polypyrimidine tract-binding protein stimulates HIF-1alpha IRES-mediated translation during hypoxia

When oxygen supply is restricted, protein synthesis is rapidly abrogated owing to inhibition of global translation. However, HIF-1α protein expression can persist during hypoxia, owing to an internal ribosome entry site (IRES) in the 5′-untranslated region of its mRNA. Here, we report on the molecular mechanism of HIF-1α IRES-mediated translation during oxygen deprivation. Using RNA affinity chromatography and UV-crosslinking experiments, we show that the polypyrimidine tract binding protein (PTB) can specifically interact with the HIF-1α IRES, and that this interaction is enhanced in hypoxic conditions. Overexpression of PTB enhanced HIF-1α IRES activity, whereas RNA interference-mediated downregula-tion of PTB protein expression inhibited HIF-1α IRES activity. Furthermore, hypoxia-induced stimulation of the HIF-1α IRES was reduced in cells in which PTB function was downregulated. In agreement with these results, the IRES activity of HIF-1α IRES deletion mutants that are deficient in PTB-binding could not be stimulated by oxygen deprivation. All together, our data suggest that PTB plays a stimulatory role in the IRES-mediated translation of HIF-1α when oxygen supply is limited.     

Differential degradation of photosystem I subunits under iron deficiency in rice

Rice (Oryza sativa) is one of the staple foods of the world. Iron (Fe) deficiency is a major abiotic stress factor that contributes world-wide to losses in crop yield and decline in nutritional quality. As cofactor for many enzymes and proteins, iron is an essential element. It plays a pivotal role in chlorophyll (Chl) biosynthesis, and iron deficiency may result in decreased Chl production and, thus, reduced photosynthetic capacity. Photosystem I (PSI) is a prime target of iron deficiency because of its high iron content (12 Fe per PS). To understand the protein level changes in the light-harvesting complex (LHC) of PSI (LHCI) under iron deficiency, rice seedlings were grown in Hoagland's nutrient medium with and without Fe. Chlorophyll content and photosynthetic efficiency decreased under iron deficiency. Protein gel blots probed with antibodies against the PSI core and Lhca 1-4 proteins revealed that the core subunits PsaA and PsaB remained stable under iron deficiency, whereas PsaC and PsaD decreased by about 50%, and PsaE was completely degraded. Among the LHCI subunits, Lhca1 and Lhca2 decreased by 40 and 50%, respectively, whereas Lhca3 and Lhca4 were completely degraded. We propose that the dissociation of LHCI subunits may be due to increased levels of reactive oxygen species, which is suggested by the increased activity of superoxide dismutase.