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.
View: Text | PDF
Research Article Article has an altmetric score of 12

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

×

Figure 7

Hypoxia-induced expression of PU.1 is regulated by RUNX-1 (AML1) and C/EBPα.

Options: View larger image (or click on image) Download as PowerPoint
Hypoxia-induced expression of PU.1 is regulated by RUNX-1 (AML1) and C/E...
(A) HUVECs were transfected with a construct containing the miR-424 promoter, including PU.1 response element and luciferase. Luciferase activity was determined under normoxia (bar 1) or hypoxia (bar 2). Cells were cotransfected with RUNX-1 expression vector, normoxia (bar 3), or hypoxia (bar 4). The effect of RUNX-1–specific shRNAs under hypoxia was determined (bars 5–7). (B) Western blot of RUNX-1 in whole-cell lysates from HUVECs cultured under normoxia or hypoxia. (C) Top: Schematic diagram of luciferase reporter constructs. Bottom: Luciferase reporter activity was measured after cotransfecting HUVECs with the construct containing the PU.1-URE-promoter or PU.1-URE-construct lacking the proximal –68-bp C/EBPα binding site. Cells were cultured under normoxia or hypoxia in the presence and absence of RUNX-1–specific shRNA. Cotransfected Renilla luciferase construct was used as an internal control. Binding sites for RUNX-1, R; C/EBPα, C; PU.1, P. (D) Western blot showing increase in C/EBPα levels in HUVECs under hypoxia. (E) Luciferase reporter activity (mean ± SD) in HUVECs transfected with PU.1-URE-promoter-luciferase (schematic diagram, top) under normoxia or hypoxia in the presence and absence of shRNA to C/EBPα. (F) Luciferase reporter activity showing the importance of the C/EBPα binding site at –68 bp of PU.1. HUVECs were transfected with PU.1-URE promoter luciferase construct (left) or a mutant construct lacking the C/EBPα binding site at –68 bp (right) under hypoxia. Values represent mean ± SD. *P < 0.05; ***P ≤ 0.001. n = 3.