Background: Functional relationships between the microRNA and cellular hypoxia response pathways are unknown. Results: Dicer is down-regulated in chronic hypoxia; this mechanism maintains the induction of hypoxia-inducible factor-␣ subunits and hypoxia-responsive genes. Conclusion: Loss of Dicer-dependent microRNA regulation is important for maintaining the concerted cellular response to hypoxia. Significance: Altogether, we provide a newer perspective into the post-transcriptional pathways that regulate the cellular hypoxic response.

Endothelial-derived nitric oxide (NO) is classically viewed as a regulator of vasomotor tone. NO plays an important role in regulating O(2) delivery through paracrine control of vasomotor tone locally and cardiovascular and respiratory responses centrally. Very soon after the cloning and functional characterization of the endothelial nitric oxide synthase (eNOS), studies on the interaction between O(2) and NO made the paradoxical finding that hypoxia led to decreases in eNOS expression and function. Why would decreases in O(2) content in tissues elicit a loss of a potent endothelial-derived vasodilator? We now know that restricting our view of NO as a regulator of vasomotor tone or blood pressure limited deeper levels of mechanistic insight. Exciting new studies indicate that functional interactions between NO and O(2) exhibit profound complexity and are relevant to diseases states, especially those associated with hypoxia in tissues. NOS isoforms catalytically require O(2). Hypoxia regulates steady-state expression of the mRNA and protein abundance of the NOS enzymes. Animals genetically deficient in NOS isoforms have perturbations in their ability to adapt to changes in O(2) supply or demand. Most interestingly, the intracellular pathways for O(2) sensing that evolved to ensure an appropriate balance of O(2) delivery and utilization intersect with NO signaling networks. Recent studies demonstrate that hypoxia-inducible factor (HIF) stabilization and transcriptional activity is achieved through two parallel pathways: (1) a decrease in O(2)-dependent prolyl hydroxylation of HIF and (2) S-nitrosylation of HIF pathway components. Recent findings support a role for S-nitrosothiols as hypoxia-mimetics in certain biological and/or disease settings, such as living at high altitude, exposure to small molecules that can bind NO, or anemia.

Human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Posttranscriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially under hypoxic conditions. Here, we show that the human eNOS 3= untranslated region (3= UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and RNA electrophoretic mobility shift assays (EMSAs) revealed the formation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1)-containing RNP complexes at these 3=-UTR elements. Knockdown of hnRNP E1 decreased eNOS mRNA half-life, mRNA levels, and protein expression. Significantly, these stabilizing RNP complexes protect eNOS mRNA from the inhibitory effects of its antisense transcript sONE and 3=-UTR-targeting small interfering RNAs (siRNAs), as well as microRNAs, specifically, hsa-miR-765, which targets eNOS mRNA stability determinants. Hypoxia disrupts hnRNP E1/eNOS 3=-UTR interactions via increased Akt-mediated serine phosphorylation (including serine 43) and increased nuclear localization of hnRNP E1. These mechanisms account, at least in part, for the decrease in eNOS mRNA stability under hypoxic conditions. Thus, the stabilization of human eNOS mRNA by hnRNP E1-containing RNP complexes serves as a key protective mechanism against the posttranscriptional inhibitory effects of antisense RNA and microRNAs under basal conditions but is disrupted under hypoxic conditions.