Hypoxia-inducible Expression of a Natural cis-Antisense Transcript Inhibits Endothelial Nitric-oxide Synthase

Abstract

The destabilization of endothelial nitric-oxide synthase (eNOS) mRNA in hypoxic endothelial cells may be important in the etiology of vascular diseases, such as pulmonary hypertension. Recently, an overlapping antisense transcript to eNOS/NOS3 was implicated in the post-transcriptional regulation of eNOS. We demonstrate here that expression of sONE, also known as eNOS antisense (NOS3AS) or autophagy 9-like 2 (APG9L2), is robustly induced by hypoxia or functional deficiency of von Hippel-Lindau protein. sONE is also up-regulated in the aortas of hypoxic rats. In hypoxic endothelial cells, sONE expression negatively correlates with eNOS expression. Blocking the hypoxic induction of sONE by RNA interference attenuates the fall in both eNOS RNA and protein. We provide evidence that the induction of sONE primarily involves transcript stabilization rather than increased transcriptional activity and is von Hippel-Lindau-but not hypoxia-inducible factor 2␣-dependent. We also demonstrate that sONE transcripts are enriched in the nucleus of normoxic cells and that hypoxia promotes an increase in the level of cytoplasmic and polyribosome-associated, sONE mRNA. The finding that eNOS expression can be regulated by an overlapping cis-antisense transcript in a stimulus-dependent fashion provides evidence that sense/antisense interactions may play a previously unappreciated role in vascular disease pathogenesis.

Hypoxic Repression of Endothelial Nitric-oxide Synthase Transcription Is Coupled with Eviction of Promoter Histones

“…Importantly, this primer set also amplifies pre-mRNA generated from the sONE/NOS3AS gene, which is transcribed in a tail-to-tail fashion to eNOS on the opposite DNA strand. We have recently demonstrated that NOS3AS transcription is maintained during hypoxic exposure of endothelial cells (1-6 h) (34).…”

“…Hypoxia Down-regulates eNOS Transcription-Prior studies by us and others have investigated hypoxia-mediated decreases in eNOS expression using in vitro approaches (4,34,35). We have also previously determined the effect of hypoxic exposure on the transcriptional activity of the chromatin-integrated eNOS promoter in vivo by exposing eNOS insertional promoter/reporter transgenic mice to hypoxia (8%, 48 h) and assessing reporter expression in the endothelial cells of the renal papilla (36).…”

“…We have previously demonstrated that eNOS mRNA levels decrease following longer durations (6 -24 h) of hypoxic exposure (34). Because hypoxia is known to regulate eNOS mRNA levels through both transcriptional and post-transcriptional mechanisms, RNA polymerase II (pol II) ChIP assays of the native eNOS promoter in HUVEC were used to assess the effect of hypoxia on eNOS transcription.…”

Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia

“…Total RNA extraction and real-time PCR was performed as described (8). Specific primers are indicated in Table S2 and Table S3.…”

“…For instance, hypoxia leads to decreases in endothelial NOS (eNOS) mRNA and increases nNOS mRNA expression in vascular endothelium and smooth muscle cells, respectively (8,9). Studies have demonstrated that acute anemia causes tissue hypoxia and an increase in hypoxia-inducible factor-alpha (HIF-α) and neuronal NOS (nNOS) protein levels, especially in the brain (10,11).…”