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-Lindaubut not hypoxia-inducible factor 2alpha-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.
Highlights
Of interactions between sense and antisense mRNA transcripts are poorly understood
We were interested in assessing whether sONE could be up-regulated in endothelial cells by stimuli that are known to decrease the stability of endothelial nitric-oxide synthase (eNOS) mRNA
We found that eNOS steady-state mRNA levels modestly increased with short term exposure to DFO (1– 4 h) but dramatically decreased after 24 h of DFO treatment. eNOS levels declined to ϳ30 and 20% of the mRNA levels of control HUVEC following 24- and 48-h treatment with DFO, respectively
Summary
Of interactions between sense and antisense mRNA transcripts are poorly understood. Several studies have implicated a role for antisense-mediated regulation of the sense transcript of S/AS pairs [2,3,4]. The molecular mechanisms responsible for the long half-life of the eNOS transcript in normoxic endothelial cells are not fully understood but are mediated, at least in part, by protein binding to the 3Ј-UTR of the eNOS mRNA (14 –16). SONE functionally regulates eNOS expression during hypoxia, since RNA interference-mediated ablation of sONE transcripts resulted in increased levels of eNOS mRNA and protein in the setting of hypoxia. To our knowledge, this is the first report of an antisense RNA playing a functional role in regulating gene expression in response to cellular stimulation
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