Posttranscriptional and transcriptional regulation of endothelial nitric-oxide synthase during hypoxia: the role of microRNAs

Abstract

Understanding the cellular pathways that regulate endothelial nitric oxide (eNOS, NOS3) expression and consequently nitric oxide (NO) bioavailability during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. eNOS expression and eNOS-dependent NO cellular signaling during hypoxia promote an equilibrium of transcriptional and posttranscriptional molecular mechanisms that belong to both proapoptotic and survival pathways. Furthermore, NO bioavailability results not only from eNOS levels, but also relies on the presence of eNOS substrate and cofactors, the phosphorylation status of eNOS, and the presence of reactive oxygen species (ROS) that can inactivate eNOS. Since both NOS3 levels and these signaling pathways can also be a subject of posttranscriptional modulation by microRNAs (miRNAs), this class of short noncoding RNAs contribute another level of regulation for NO bioavailability. As miRNA antagomirs or specific target protectors could be used in therapeutic approaches to regulate NO levels, either by changing NOS3 mRNA stability or through factors governing eNOS activity, it is critical to understand their role in governing eNOS activity during hypoxa. In contrast to a large number of miRNAs reported to the change eNOS expression during hypoxia, only a few miRNAs modulate eNOS activity. Furthermore, impaired miRNA biogenesis leads to NOS3 mRNA stabilization under hypoxia. Here we discuss the recent studies that define miRNAs' role in maintaining endothelial NO bioavailability emphasizing those miRNAs that directly modulate NOS3 expression or eNOS activity.

Keywords: ER stress; Hypoxia; NO bioavailability; NOS3; Nitric oxide; eNOS; miRNA; sONE.

Fig. 1

Hypoxia induces dynamic changes in the mRNA expression profiles of the NOS3, HIF1A and HIF2A (EPAS1) in human human umbilical vein endothelial cells (HUVECs). The mRNA levels during hypoxia were monitored in qRT-PCR experiments as described in [49, 50]. The results from 2 independent experiments (n = 8) are plotted normalized to 18S rRNA levels and expressed as a fold-change compared to the normoxic control. The hypoxia had no impact on HUVECs viability as monitored with Real Time xCelligence as described in [106]. Error bars represent standard deviations. Significant changes (p < 0.05) are marked with an “*”

Fig. 2

The ER stress induced dynamic changes in the mRNA of the NOS3 that are negatively correlated with CHOP expression in human umbilical vein endothelial cells (HUVECs). The ER stress was induced with 100 μM Calpain Inhibitor I (ALLN). mRNA levels were monitored in qRT-PCR experiments as described in [106, 107]. The results from 2 independent experiments (n = 8) are plotted normalized to TBP mRNA levels and expressed as a fold-change over the untreated control. Error bars represent standard deviations. Significant changes (p < 0.05) are marked with an “*”

Fig. 3

The transcriptional and posttranscriptional influence of hypoxia on NOS3 mRNA levels. During hypoxia, HIF-1 and HIF-2 accumulate in the nucleus, where they bind to a sequence in the promoter region of NOS3 termed the hypoxia-response element (HRE), and in doing so, induce NOS3 expression. Hypoxia disrupts hnRNP E1/NOS3 3′-UTR interactions and makes this transcript susceptible to sONE and miRNA-related down-regulation. HIF-1 induces miR-155 that along with miR-765 and miR-24 destabilizes NOS3 mRNA. Furthermore, during hypoxia, miR-214 has negative effect on NOS3 expression. Hypoxia is accompanied by deregulation of ER homeostasis and HIF-1-related activation of the ER stress response. The proadaptive and proangiogenic ER stress transcription factor sXBP1 stimulates NOS3 expression transcriptionally and postranscriptionally through reduction of miR-24, miR-125 and miR-214. However, another proapoptotic ER stress transcription factor, CHOP, binds to the 5′UTR and represses transcription. The hypoxamiRs also modulate Act signaling pathway and consequently eNOS activity. During hypoxia, the changes in the expression levels of miR-155, miR-101, miR-486, miR-21 and miR-126 stimulate Akt activation, whereas miR-26 prevents Act signaling. The (+) expression profile changed during hypoxia contributes to increased expression and activity of NOS3; the (-) expression profile changed during hypoxia has a negative effect on the expression and activity of NOS3; ^(¥) depicts indirect effects on the NOS3 gene