The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells

Abstract

Endothelial cell insulin resistance contributes to the development of vascular complications in diabetes. Hypoxia-inducible factors (HIFs) modulate insulin sensitivity, and we have previously shown that a negative regulator of HIF activity, CREB-binding protein/p300 (CBP/p300) interacting transactivator-2 (CITED2), is increased in the vasculature of people with type 2 diabetes. Therefore, we examined whether CITED2 regulates endothelial insulin sensitivity. In endothelial cells isolated from mice with a "floxed" mutation in the Cited2 gene, loss of CITED2 markedly enhanced insulin-stimulated Akt phosphorylation without altering extracellular signal-related kinase 1/2 (ERK1/2) phosphorylation. Similarly, insulin-stimulated Akt phosphorylation was increased in aortas of mice with endothelial-specific deletion of CITED2. Consistent with these observations, loss of CITED2 in endothelial cells increased insulin-stimulated endothelial nitric oxide synthase phosphorylation, Vegfa expression, and cell proliferation. Endothelial cells lacking CITED2 exhibited an increase in insulin receptor substrate (IRS)-2 protein, a key mediator of the insulin signaling cascade, whereas IRS-1 was unchanged. Conversely, overexpression of CITED2 in endothelial cells decreased IRS-2 protein by 55% without altering IRS-1, resulting in impaired insulin-stimulated Akt phosphorylation and Vegfa expression. Overexpression of HIF-2α significantly increased activity of the Irs2 promoter, and coexpression of CITED2 abolished this increase. Moreover, chromatin immunoprecipitation (ChIP) showed that loss of CITED2 increased occupancy of p300, a key component of the HIF transcriptional complex, on the Irs2 promoter. Together, these results show that CITED2 selectively inhibits endothelial insulin signaling and action through the phosphoinositide 3-kinase (PI3K)/Akt pathway via repression of HIF-dependent IRS-2 expression. CITED2 is thus a promising target to improve endothelial insulin sensitivity and prevent the vascular complications of diabetes.NEW & NOTEWORTHY Endothelial cell insulin resistance is a major contributor to the development of diabetic complications. In this study, we have shown that CITED2, a transcriptional coregulator, inhibits endothelial insulin signaling through the PI3K/Akt pathway via repression of HIF-dependent IRS-2 expression, and that deletion of CITED2 enhances insulin signaling. Thus, CITED2 represents a novel and promising target to improve insulin sensitivity in endothelial cells and prevent vascular complications in diabetes.

Keywords: endothelium; hypoxia-inducible factor; insulin resistance; insulin signaling.

Graphical abstract

Figure 1.

CITED2 selectively inhibits insulin signaling in endothelial cells in vitro and in vivo. A–E: lung endothelial cells were isolated from Cited2^fl/fl mice and treated with adenovirus expressing cre recombinase (Ad-Cre) or GFP only (Ad-GFP), thereby creating CITED2 knockout (CITED2 KO) and control cultures from the same cell isolation. A: CITED2 expression was measured in cell lysate by Western blotting. B: cell cultures were serum-starved overnight and treated with 10 nM insulin for 5 min. Protein expression was measured in cell lysate by Western blotting. C–E: quantitative analysis of insulin-stimulated phosphorylation of insulin receptor-β (INSRβ), Akt, and ERK based on densitometry of 3–4 independent experiments and normalization to the baseline phosphorylation in control cells. F–G: Cdh5-cre Cited2^fl/fl and Cited2^fl/fl control mice were injected intravenously with 5 U insulin or vehicle. F: after 5 min, the aorta was isolated and flash-frozen. Levels of Akt, phosphorylated Akt, and β-actin were measured by Western blotting of tissue lysate. G: quantitative analysis of insulin-stimulated Akt phosphorylation based on densitometry of 5 pairs of animals. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Tukey’s multiple-comparisons test. CITED2, CBP/p300 interacting transactivator-2; ERK, extracellular signal-related kinase; GFP, green fluorescent protein.

Figure 2.

CITED2 impairs insulin action on endothelial cells. A: control and CITED2 KO lung endothelial cell cultures were serum-starved overnight and treated with 10 nM insulin for 5 min. Insulin-stimulated eNOS phosphorylation was measured in cell lysate by Western blotting. B: quantitative analysis of insulin-stimulated eNOS phosphorylation based on densitometry of 4 independent experiments and normalization to the baseline phosphorylation in control cells. C: cell cultures were serum-starved overnight and treated with 10 nM insulin for 4 h. Vegfa expression was measured by real-time PCR and normalized to expression of the housekeeping gene Rplp0 in 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, 2-way ANOVA followed by Tukey’s multiple-comparisons test. D: cell cultures were serum-starved for 16 h, treated with 10 nM insulin for 16 h and then labeled with EdU for an additional 4 h. EdU-labeled cells were stained with Alexa Fluor 647 (AF647) using Molecular Probes Click-iT procedure and analyzed by flow cytometry. E: insulin-stimulated EdU incorporation relative to baseline EdU incorporation for each cell type was determined in 3 independent experiments. **P < 0.01, Student’s t test. CITED2, CBP/p300 interacting transactivator-2; CITED2 KO, CBP/p300 interacting transactivator-2 knockout; eNOS, endothelial nitric oxide synthase; GFP, green fluorescent protein.

Figure 3.

CITED2 represses IRS-2 expression in endothelial cells. A–E: lung endothelial cells were isolated from Cited2^fl/fl mice and treated with adenovirus expressing Cre recombinase (Ad-Cre) or control adenovirus expressing GFP only (Ad-GFP), creating CITED2 KO and control cultures from the same cell isolation. A–B: expression of Irs1 and Irs2 was measured by real-time PCR and normalized to the housekeeping gene Rplp0 and expression in control cells. C: expression of IRS-1 and IRS-2 was measured by Western blotting. D–E: quantitative analysis of IRS-1 and IRS-2 expression based on densitometry of 3 independent experiments and normalized to expression in control cells. F–H: MS1 endothelial cells were cultured with lentivirus expressing FLAG-CITED2 for 12 h, and lentiviral medium was then replaced with complete medium for 48 h. Stably transduced MS1 cells were selected with puromycin (0.1%) for 72 h. F: expression of IRS-1 and IRS-2 was measured by Western blotting. G–H: quantitative analysis of IRS-1 and IRS-2 expression based on densitometry of 3 independent experiments and normalized to expression in control cells. *P < 0.05, ** P < 0.01, Student’s t test. CITED2, CBP/p300 interacting transactivator-2; CITED2 KO, CBP/p300 interacting transactivator-2 knockout; eNOS, endothelial nitric oxide synthase; IRS, insulin receptor substrate.

Figure 4.

CITED2 overexpression recapitulates endothelial insulin resistance. A–C: MS1 endothelial cells were cultured with lentivirus expressing FLAG-CITED2 for 12 h, and then, lentiviral medium was replaced with complete medium for 48 h. Stably transduced MS1 cells were selected with puromycin (0.1%) for 72 h. A: control and FLAG-CITED2-overexpressing cell cultures were serum-starved overnight and treated with 10 nM insulin for 5 min. Protein levels were measured in cell lysate by Western blotting. B: quantitative analysis of insulin-stimulated phosphorylation of Akt based on densitometry of 4 independent experiments. C: control and FLAG-CITED2-overexpressing cultures were serum-starved overnight and treated with 10 nM insulin for 4 h. Vegfa expression was measured by real-time PCR of 3 independent experiments, and relative Vegfa mRNA expression was determined by normalization to the housekeeping gene Rplp0. *P < 0.05, **P < 0.01, 2-way ANOVA followed by Tukey’s multiple-comparisons test. CITED2, CBP/p300 interacting transactivator-2.

Figure 5.

CITED2 limits HIF-2α-mediated transcription of Irs2. A: MS1 endothelial cells transduced with lentivirus expressing FLAG-CITED2 were cotransfected with Renilla luciferase and normoxia-stable pcDNA3.1 mHIF-2α MYC P405A/P530V/N851A (HIF2α-DNA3.1), Irs2 promoter reporter (Irs2-pGL3), or empty vector control (pGL3, pcDNA3.1) plasmids. After 24 h, cells were lysed and luminescent signal measured with the Dual Luciferase Reporter Gene Assay kit. B: CITED2 KO and control endothelial cells were cross-linked, lysed, and sonicated to produce 200–1,000-bp chromatin fragments as analyzed by agarose gel electrophoresis. Immunoprecipitations were performed with 10 μg of p300 antibody (NB100-616, Novus Biologicals) or mouse IgG. Relative enrichment of chromatin was determined by real-time PCR. C: CITED2 inhibition of the p300/CBP-HIF-2α transcriptional complex decreases Irs2 transcription and IRS-2 expression, leading to impaired insulin signaling in endothelial cells. **P < 0.01, Student’s t test. ***P < 0.001, ****P < 0.0001, 1-way ANOVA followed by Tukey’s multiple-comparisons test. CBP, CREB-binding protein; CITED2, CBP/p300 interacting transactivator-2; ChIP, chromatin immunoprecipitation; HIF, hypoxia-inducible factor; HRE, hypoxia-response element; INSR, insulin receptor.