Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis
Goutam Ghosh, … , Yan Zeng, Sundaram Ramakrishnan
Goutam Ghosh, … , Yan Zeng, Sundaram Ramakrishnan
Published October 25, 2010
Citation Information: J Clin Invest. 2010;120(11):4141-4154. https://doi.org/10.1172/JCI42980.
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Research Article

Hypoxia-induced microRNA-424 expression in human endothelial cells regulates HIF-α isoforms and promotes angiogenesis

Abstract

Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.

Authors

Goutam Ghosh, Indira V. Subramanian, Neeta Adhikari, Xiaoxiao Zhang, Hemant P. Joshi, David Basi, Y.S. Chandrashekhar, Jennifer L. Hall, Sabita Roy, Yan Zeng, Sundaram Ramakrishnan

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Figure 2

miR-424 stabilizes HIF-1α and HIF-2α and increases transcription of HIF-1α/HIF-2α target genes.

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miR-424 stabilizes HIF-1α and HIF-2α and increases transcription of HIF-...
(A) Top: Western blot of HIF-1α in the whole-cell lysates of HUVECs. Normoxia (N; lane 1) and hypoxia (H; lane 2) after 24 hours, and miR-control (lane 3) and miR-424 under normoxia (lane 4) after 48 hours. Bottom: Western blot of HIF-2α. miR-c, miR-control. (B) Cellular distribution of HIF-1α in HUVECs transfected with either miR-control or miR-424 under normoxia. HUVECs exposed to hypoxia were used as a positive control. Arrowheads indicate nuclear accumulation of HIF-1α. Scale bar: 10 μm. (C) Western blot of HIF-1α and CUL2 in HUVECs after knockdown of miR-424 under hypoxia. HUVECs treated with control morpholino under normoxia (lanes 1 and 2), hypoxia (lanes 3 and 4), and miR-424–specific morpholino under hypoxia (lanes 5 and 6) are shown. (D) HUVECs were cotransfected with either miR-control or miR-424 and a VEGF-luciferase reporter construct under normoxia, or were transfected only with reporter construct and maintained under hypoxia (24 hours). Renilla luciferase levels were used as an internal control. (E and F) HUVECs were transfected with either an miR-control or miR-424 construct and maintained under normoxia. GLUT1, EPO, and fibronectin transcript levels were determined by q-PCR. Values represent mean ± SD. n = 3. *P ≤ 0.05; **P ≤ 0.02.