Transcriptional adaptations to hypoxia are mediated by hypoxia-inducible factor (HIF)-1, a heterodimer of HIF-␣ and aryl hydrocarbon receptor nuclear translocator subunits. The HIF-1␣ and HIF-2␣ subunits both undergo rapid hypoxia-induced protein stabilization and bind identical target DNA sequences. When coexpressed in similar cell types, discriminating control mechanisms may exist for their regulation, explaining why HIF-1␣ and HIF-2␣ do not substitute during embryogenesis. We report that, in a human lung epithelial cell line (A549), HIF-1␣ and HIF-2␣ proteins were similarly induced by acute hypoxia (4 h, 0.5% O 2 ) at the translational or posttranslational level. However, HIF-1␣ and HIF-2␣ were differentially regulated by prolonged hypoxia (12 h, 0.5% O 2 ) since HIF-1␣ protein stimulation disappeared because of a reduction in its mRNA stability, whereas HIF-2␣ protein stimulation remained high and stable. Prolonged hypoxia also induced an increase in the quantity of natural antisense HIF-1␣ (aHIF), whose gene promoter contains several putative hypoxia response elements to which (as we confirm here) the HIF-1␣ or HIF-2␣ protein can bind. Finally, transient transfection of A549 cells by dominant-negative HIF-2␣, also acting as a dominant-negative for HIF-1␣, prevented both the decrease in the HIF-1␣ protein and the increase in the aHIF transcript. Taken together, these data indicate that, during prolonged hypoxia, HIF-␣ proteins negatively regulate HIF-1␣ expression through an increase in aHIF and destabilization of HIF-1␣ mRNA. This transregulation between HIF-1␣ and HIF-2␣ during hypoxia likely conveys target gene specificity.

Alveolar hypoxia may impair sodium-dependent alveolar fluid transport and induce pulmonary edema in rat and human lung, an effect that can be prevented by the inhalation of ␤ 2 -agonists. To investigate the mechanism of ␤ 2 -agonist-mediated stimulation of sodium transport under conditions of moderate hypoxia, we examined the effect of terbutaline on epithelial sodium channel (ENaC) expression and activity in cultured rat alveolar epithelial type II cells exposed to 3% O 2 for 24 h. Hypoxia reduced transepithelial sodium current and amiloridesensitive sodium channel activity without decreasing ENaC subunit mRNA or protein levels. The functional decrease was associated with reduced abundance of ENaC subunits (especially ␤ and ␥) in the apical membrane of hypoxic cells, as quantified by biotinylation. cAMP stimulation with terbutaline reversed the hypoxia-induced decrease in transepithelial sodium transport by stimulating sodium channel activity and markedly increased the abundance of ␤-and ␥-ENaC in the plasma membrane of hypoxic cells. The effect of terbutaline was prevented by brefeldin A, a blocker of anterograde transport. These novel results establish that hypoxiainduced inhibition of amiloride-sensitive sodium channel activity is mediated by decreased apical expression of ENaC subunits and that ␤ 2 -agonists reverse this effect by enhancing the insertion of ENaC subunits into the membrane of hypoxic alveolar epithelial cells.

To explore the possibility that vitamin D status regulates sulfate homeostasis, plasma sulfate levels, renal sulfate excretion, and the expression of the renal Na-SO 4

Proliferation of tubular epithelial cells underlies the development of cystic lesions and the subsequent impairment of renal function after renal mass reduction. The effect of HMG CoA reductase inhibitors (HRI) on cell proliferation was investigated in rat renal proximal tubular epithelial cells in primary culture. Treatment of renal tubular epithelial cells with three different HRI reduced fetal calf serum (FCS)-induced [3H]-thymidine incorporation (IC50 values were 0.7 microM, 1.7 microM, and 1.6 microM for simvastatin, lovastatin, and compactin, respectively), and lovastatin blocked BrdUrd incorporation, as assessed by immunocytochemical studies. The proliferative effect of epidermal growth factor (EGF) was similarly abolished by lovastatin. The effect of lovastatin (1 microM) was prevented by 100 microM mevalonate, 5 microM farnesyl-pyrophosphate and 5 microM geranylgeranyl-pyrophosphate (in percent of control value, 31% vs. 102%, 60%, and 82%, respectively) while cholesterol and other products of the mevalonate pathway were inactive. Immunoblot analysis showed that lovastatin decreased membrane-bound p21ras and inhibited FCS-induced c-fos and c-jun protein expression. Furthermore, electrophoretic mobility shift assay demonstrated the functional impairement of AP-1 DNA binding activity in lovastatin-treated cells. In conclusion, these results demonstrate that HRI are antiproliferative in epithelial tubule cells and that this effect is exerted, at least in part, via inhibition of the p21ras-activated and AP-1 dependent mitogenic cascade.