Arg972 insulin receptor substrate-1 inhibits endothelial nitric oxide synthase expression in human endothelial cells by upregulating microRNA-155
- Authors:
- Published online on: April 21, 2015 https://doi.org/10.3892/ijmm.2015.2192
- Pages: 239-248
Abstract
Introduction
Nitric oxide (NO) generated and released by endothelial NO synthase (eNOS) exerts multiple beneficial effects on blood vessels and plays a critical role in maintaining cardiovascular homeostasis (1). The dysregulation of NO synthesis attributable to the abnormal activity and/or the expression of eNOS is considered to be a major contributor to the pathogenesis of cardiovascular diseases, such as hypertension and atherosclerosis (2,3). Although the expression of eNOS is regulated at multiple levels, increasing evidence has suggested that post-transcriptional regulation plays an important role in the control of eNOS expression. It has been demonstrated that eNOS expression is mainly regulated through the modification of eNOS mRNA expression through the 3′-untranslated region (3′-UTR) of eNOS mRNA (4–8).
Diabetes is associated with an increased risk of cardiovascular disease due to multiple pathophysiological links between insulin resistance, obesity, hypertension and atherogenic dyslipidemia (9–11). One common characteristic of these conditions is endothelial dysfunction, caused by a relative deficiency of NO (12). Insulin has vasodilator effects, which depend on the production of NO in the vascular endothelium (13,14). Key signaling molecules mediating metabolic actions of insulin, such as the insulin receptor tyrosine kinase, phosphatidylinositol-3 kinase (PI3K) and Akt, are also necessary for insulin to stimulate the production of NO in human vascular endothelial cells (15,16). Thus, a physiological role of insulin may be to couple the regulation of hemodynamic homeostasis with metabolic homeostasis (17).
Previous studies have reported a common polymorphism in the insulin receptor substrate-1 (IRS-1) gene, in which a Gly/Arg substitution occurs at codon 972 (Arg972) (18,19). The presence of Arg972 IRS-1 is associated with an impaired IRS-1 ability to activate PI3K, leading to the diminished activity of insulin (18,19). In the present study, we investigated the association between Arg972 IRS-1 and eNOS expression/activity in a relatively large sample of human subjects and primary cultures of human endothelial cells.
Materials and methods
Subjects
Between November 2012 and October 2013, 832 subjects randomly selected from a total of 2,672 Han Chinese subjects in the database of the physical examination center at Guangdong Provincial People’s Hospital, Guangzhou, China were recruited in the present study. Blood samples were drawn from all subjects within 72 h of enrollment. The characteristics of the subjects are presented in Table I. The present study conforms to the Declaration of Helsinki and was approved by the Ethics Committee of Guangdong Provincial People’s Hospital (for the collection of patient blood samples, approval reference number: H40125; and for obtaining umbilical vein endothelial cells, approval reference number: H40126). Written informed consent was obtained from all subjects prior to enrollment.
Plasmids and reagents
A fragment of human genomic DNA containing the entire coding sequence of IRS-1 was cloned and ligated into the pcDNA3.1 expression vector as described in a previous study (20). The Arg972 IRS-1 expression vector was constructed as previously described (21). SuperFect and HiPerFect transfection reagents were purchased from Qiagen (Valencia, CA, USA). TRIzol reagent and SYBR-Green Master Mix were purchased from Invitrogen (Carlsbad, CA, USA) and PE Applied Biosystems (Foster City, CA, USA), respectively. Anti-eNOS (49G3; #9586), anti-phospho-eNOS (Ser1177; #9570) and anti-β-actin (8H10D10; #3700) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The eNOS human ELISA kit (#ab166866) was purchased from Abcam (Cambridge, MA, USA). The nitrite/nitrate colorimetric assay kit (#780001) was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). The selective PI3K inhibitor, BKM120 (#sc-364437A), and anti-IRS-1 (C-20; #sc-559), anti-Akt (5C10; #sc-81434), anti-phospho-Akt (Ser473; #sc-101629) and anti- von Willebrand factor (vWF) (1.B.690; #sc-73268) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mature miR-155 mimic (5′-UUAAUGCUAAUC GUGAUAGGGGUCCCUAUCACGAUUAGCAUUAAUU-3′) was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Antagomir-155 (#410078-00; miRCURY LNA™ microRNA power inhibitor) was purchased from Exiqon (Woburn, MA, USA). Endothelial cell growth medium (#211-500) was purchased from Cell Applications, Inc. (San Diego, CA, USA). G418, insulin and all other chemicals of reagent grade were purchased from Sigma (St. Louis, MO, USA).
Human umbilical vein endothelial cell (HUVEC) culture
Umbilical cords were obtained from mothers delivering at Guangdong Provincial People’s Hospital. Primary HUVEC cultures were obtained as previously described (22). Following perfusion of the umbilical cords with 0.1% collagenase at 37°C, the HUVECs were grown on 0.2% gelatin-coated tissue culture plates in endothelial cell growth medium supplemented with 20% FBS and endothelial cell growth supplement (Cell Applications, Inc.). In all the experiments, the cells were used between the third and the fifth passages. Commercial HUVECs (#200K-05n) were purchased from Cell Applications, Inc. for the stable transfection of IRS-1 and Arg972 IRS-1. We confirmed by sequencing that the commercial HUVECs expressed homozygous wild-type (WT) IRS-1 (GG genotype).
Genotyping and determination of eNOS and nitrite/nitrate levels
The Arg972 IRS-1 polymorphism was identified by sequencing. Plasma eNOS and nitrite/nitrate levels were respectively determined with an eNOS human ELISA kit (Abcam) and a nitrite/nitrate colorimetric assay kit (Cayman Chemical) according to the manufacturer’s instructions. Nitrite/nitrate levels in the HUVEC culture media were normalized to the total proteins of HUVECs.
Transfection
Plasmid constructs were transfected into the HUVECs using SuperFect transfection reagent (Qiagen) according to the manufacturer’s instructions. Pools of stable transfectants of IRS-1 and Arg972 IRS-1 were generated through selection with G418 (800 μg/ml) as per the manufacturer’s instructions. Mature miR-155 mimic or antagomir-155 was transfected into the HUVECs using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions.
Reverse transcription-quantitative RT-qPCR
Total RNA from the HUVECs was prepared using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen) or microRNA TaqMan kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR (qPCR) was carried out on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems), using the fluorescent dye SYBR-Green Master Mix (PE Applied Biosystems) as per the manufacturer’s instructions. The results were normalized to those of the housekeeping gene β-actin in the same sample. The primers used were as follows: eNOS, 5′-GTGGCTGTCTGCATGGACCT-3′ (forward) and 5′-CCACGATGGTGACTTTGGCT-3′ (reverse); β-actin, 5′-AGCCTCGCCTTTGCCGA-3′ (forward) and 5′-CTGGTGCCTGGGGCG-3′ (reverse). Stem-loop RT-PCR (TaqMan MicroRNA assays; P/N: 000448 for miR-125a, P/N: 000449 for miR-125b, P/N: 000521 for miR-218, P/N: 000451 for miR-126, P/N: 002623 for miR-155, P/N: 000397 for miR-21, P/N: 000524 for miR-221 and P/N: 000525 for miR-222; Applied Biosystems) was used to quantify the miRNAs according to the manufacturer’s instructions. Small unclear RNA U6 was used as an internal control with the following primers: forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The reverse primer of U6 was used for reverse transcription. Each experiment was repeated 3 times in duplicate.
Western blot analysis
Western blot analysis was carried out as previously described (23). The HUVECs were lysed in 250 μl of 2X SDS loading buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromphenol blue and 5% 2-mercaptoethanol) and incubated at 95°C for 10 min. Equal amounts of protein (100 μg) for each sample were separated by 8–15% SDS-polyacrylamide gel and blotted onto polyvinylidene difluoride microporous membranes (Millipore, Billerica, MA, USA). The membranes were incubated for 1 h with a 1:1,000 dilution of primary antibody, and then washed and revealed using secondary antibodies with horseradish peroxidase conjugate (1:5,000, 1 h). Peroxidase was revealed using a GE Healthcare ECL kit (GE Healthcare, Shanghai, China).
Immunofluorescence staining
The cells were washed twice with 10 mmol/l sodium phosphate (pH 7.4), 0.14 mol/l NaCl (PBS) and subsequently fixed for 10 min at room temperature with methanol. After fixation, the cells were washed twice with PBS and incubated for 1 h with anti-vWF antibody (Santa Cruz Biotechnology) (1:500 dilution). Subsequently, the coverslips were washed twice with PBS and incubated for 1 h with FITC-conjugated seconday antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), embedded in mounting medium, and analyzed under a fluorescence microscope (Axioplan 2; Carl Zeiss, Oberkochen, Germany).
IRS-1-associated PI3K activity assay
IRS-1-associated PI3K activity was determined as previously described (24). Briefly, 700 μg of total protein were immunoprecipitated with anti-IRS-1 antibody (Santa Cruz Biotechnology), and kinase activity was detected by the appearance of radiolabeled 32P-labeled phosphatidylinositol 3-phosphate ([32P]PI-3-P) following thin-layer chromatography (TLC) as previously described (25). Autoradiographic signals were quantified using NIH Image software version 1.63.
Statistical analysis
Statistical analyses were carried out using SPSS 15.0 software (IBM, Chicago, IL, USA). All continuous variable values are expressed as the means ± SD. Comparisons of the means between 2 independent groups were performed using the Student’s t-tests. A stepwise multi-linear regression model was used to determine which variables explained unique variance in plasma eNOS levels. All variables listed in Table I were tested in the multi-linear regression model. Comparisons of the means among multiple groups were performed using one-way ANOVA followed by post hoc pairwise comparisons using Tukey’s tests. Categorical variables were compared using χ2 tests. A value of p=0.05 was considered to indicate a statistically significant difference in the present study.
Results
Association between Arg972 IRS-1 and plasma eNOS and nitrite/nitrate levels in human subjects
The dysregulation of NO synthesis attributable to the abnormal expression and activity of eNOS is considered to be a major contributor to the pathogenesis of vascular diseases (2,3). The results from one of our pilot in vitro studies suggested that Arg972 IRS-1 exerts significant regulatory effects on eNOS expression in human endothelial cells. In the present study, we first genotyped 832 randomly selected human subjects for the Arg972 IRS-1 polymorphism and determined their plasma eNOS and nitrite/nitrate levels. As shown in Table I, 76.8% of the total subjects carried the homozygous WT IRS-1 (GG genotype), 21.2% carried the heterozygous Arg972 IRS-1 (GA genotype) and only 2% carried the homozygous Arg972 IRS-1 (AA genotype). The A allele frequency was found to be 12.6% in our study population. Multivariate regression analysis revealed that after adjustment for all variables except for the plasma nitrite/nitrate level (which highly correlated with the plasma eNOS level) listed in Table I, the copy number of the Arg972 IRS-1 A allele, age, body mass index (BMI), smoking and blood fasting glucose levels were independent predictors of the plasma eNOS levels, collectively explaining 50.8% of the total variance (Table II). Analyses of the subject characteristics by the Arg972 IRS-1 genotypes revealed that the Arg972 IRS-1 heterozygous and homozygous carriers had significantly higher blood pressure and a prevalence of hyperlipidemia, coronary heart disease and type 2 diabetes mellitus (T2DM), but lower levels of plasma eNOS and nitrite/nitrate than the homozygous WT IRS-1 carriers (Table III). These findings indicate that Arg972 IRS-1 is associated with decreased eNOS expression and activity in the human body.
 | Table IIMultivariate regression analysis of independent predictors of plasma endothelial nitric oxide synthase (eNOS) level. |
| Table IIICharacteristics of study subjects by Arg972 insulin receptor substrate-1 (IRS-1) genotypes. |
eNOS expression/activity and eNOS activation/phosphorylation in HUVECs expressing homozygous WT IRS-1 or heterozygous Arg972 IRS-1
To further examine the association between Arg972 IRS-1 and eNOS expression/activity, we used HUVEC cultures established from delivering mothers expressing homozygous WT IRS-1 (GG genotype) or heterozygous Arg972 IRS-1 (GA genotype). HUVEC cultures expressing homozygous Arg972 IRS-1 (AA genotype) were not included for the following reasons: i) the low frequency of homozygous Arg972 IRS-1 in this study (Han Chinese) population (Table I); ii) heterozygous Arg972 IRS-1 carriers showed significantly decreased plasma eNOS and nitrite/nitrate levels compared to the homozygous WT IRS-1 carriers (Table III). Based on a two-sided p=0.05, power=0.80 and an effect size=1.6 (based on data from our pilot in vitro study), a sample size of 8 was calculated for the comparison of eNOS expression between 2 HUVEC groups (26). As shown in Fig. 1A, the majority of the cultured HUVECs (~90%) displayed typical endothelial morphology and immunofluorescence staining for the endothelial cell marker, vWF. Without insulin stimulation, the HUVECs showed no significant differences in eNOS expression and nitrite/nitrate levels; we examined different methods of insulin stimulation, and found that treatment with 10−8 M of insulin for 24 h had the strongest stimulatory effects on eNOS expression and nitrite/nitrate levels in the HUVECs (data not shown). Thus, in all subsequent experiments in the present study, the HUVECs were pre-stimulated with 10−8 M of insulin for 24 h. Following insulin stimulation, HUVECs expressing heterozygous Arg972 IRS-1 (GA group) showed a 38% decrease in the eNOS mRNA level compared with those expressing homozygous WT IRS-1 (GG group) (Fig. 1B). Western blot analyses revealed that compared with the GG group of HUVECs, the GA group showed a 42% decrease in the eNOS protein level, and a 58% decrease in eNOS phosphorylation at serine 1177 (Ser1177), which is required for the insulin-stimulated activation of eNOS activity (Fig. 1C) (15). In addition, IRS-1-associated PI3K activity and eNOS activity (measured by the nitrite/nitrate level in the cell culture media) in the GA group decreased by approximately 30 and 70%, respectively (Fig. 1D).
miRNA levels in HUVECs expressing homozygous WT IRS-1 or heterozygous Arg972 IRS-1
A recent study demonstrated that eNOS expression can be regulated by microRNAs (miRNAs or miRs) (27). Thus, in the HUVECs, we examined the expression levels of miRNAs reportedly involved in regulating vascular tone (miR-125a, miR-125b, miR-218, miR-126, miR-155 and miR-21) (28) or eNOS expression in endothelial cells (miR-155, miR-221 and miR-222) (27), particularly miR-155, which has been shown to inhibit eNOS expression by directly targeting the 3′-untranslated region (3′-UTR) of eNOS mRNA (27). Following insulin stimulation, the expression level of miR-155 in the GA (heterozygous Arg972 IRS-1) group of HUVECs was increased approximately 2.5-fold compared with that in the GG (homozygous WT IRS-1) group of HUVECs, while there were no significant group differences observed in the expression level of the other miRNAs (Fig. 2).
eNOS expression/activity and eNOS activation/phosphorylation in HUVECs overexpressing IRS-1 or Arg972 IRS-1 in the presence or absence of miR-155 or antagomir-155
All the above findings suggested that Arg972 IRS-1 decreases eNOS expression and activity in endothelial cells by upregulating miR-155. Thus, we examined eNOS expression/activity and miR-155 expression levels in HUVECs stably overexpressing IRS-1 or Arg972 IRS-1 in the presence or absence of miR-155 or a functional inhibitor of miR-155 (antagomir-155). As shown in Fig. 3A, compared with the controls, the stable overexpression of IRS-1 in the insulin-stimulated HUVECs decreased the miR-155 expression level by approximately 60%; this effect was reversed by transfection with mature miR-155 mimic or by treatment with the selective PI3K inhibitor, BKM120 (50 μM), for 60 min. On the other hand, the stable overexpression of Arg972 IRS-1 increased the miR-155 expression level by 1.55-fold; this effect was reversed by transfection with antagomir-155. Notably, antagomir-155 decreased the miR-155 expression level, possibly by inhibiting the quantitative RT-PCR reaction by competing with the reverse transcription (RT) primer to bind miR-155 during reverse transcription. As shown in Fig. 3B and C, the overexpression of IRS-1 in the HUVECs increased eNOS expression by approximately 1.5-fold; this effect was reversed by transfection with mature miR-155 mimic or treatment with BKM120. On the other hand, the overexpression of Arg972 IRS-1 decreased eNOS expression by approximately 50%, which was reversed by transfection with antagomir-155. Transfection with mature miR-155 mimic or treatment with BKM120 alone decreased eNOS expression by approximately 80 and 70%, respectively, while transfection with antagomir-155 alone increased eNOS expression by over 1.6-fold. In addition, mature miR-155 mimic, antagomir-155 and BKM120 showed no significant effect on IRS-1 expression in the HUVECs (Fig. 3C), indicating that their effects on eNOS expression are not mediated by altering the expression of IRS-1. Taken together, these findings indicate that Arg972 IRS-1 inhibits eNOS expression in endothelial cells by upregulating miR-155. Of note, the overexpression of IRS-1 and Arg972 IRS-1 increased and decreased eNOS phosphorylation at Ser1177 by 4.4-fold and 75%, respectively; these effects were much more pronounced than their effects on eNOS expression and were only partially reversed by miR-155 and antagomir-155, respectively (Fig. 3C). By contrast, the effects of mature miR-155 mimic and antagomir-155 on eNOS phosphorylation at Ser1177 were similar to those on eNOS expression (Fig. 3C).
Arg972 IRS-1 is reportedly associated with impaired insulin/IRS-1 signaling to activate the PI3K/Akt pathway, which is required to activate eNOS activity by phosphorylating eNOS at Ser1177 (15). Thus, we examined IRS-1-associated PI3K activity, Akt activation/phosphorylation and eNOS activity (measured by the nitrite/nitrate level in the cell culture media) in the HUVECs stably overexpressing IRS-1 or Arg972 IRS-1 in the presence or absence of miR-155 or antagomir-155.
IRS-1-associated PI3K activity, Akt activation/phosphorylation and eNOS activity in HUVECs overexpressing IRS-1 or Arg972 IRS-1 in the presence or absence of miR-155 or antagomir-155
As shown in Fig. 4A, compared with the controls, the stable overexpression of IRS-1 in the insulin-stimulated HUVECs increased the IRS-1-associated PI3K activity by 2-fold, which was abolished by treatment with BKM120, but not by transfection with mature miR-155 mimic. On the other hand, the stable overexpression of Arg972 IRS-1 decreased the IRS-1-associated PI3K activity by approximately 40%, which was not significantly altered by transfection antagomir-155. Mature miR-155 mimic and antagomir-155 showed no significant effects on the IRS-1-associated PI3K activity. A similar data trend was observed with Akt phosphorylation at serine 473 (ser473) (Fig. 4B), which is required for the activation of Akt by PI3K (29). As shown in Fig. 4C, the overexpression of IRS-1 and Arg972 IRS-1 increased and decreased the nitrite/nitrate level by 3.9-fold and 70%, respectively; these effects were much more pronounced than their effects on eNOS expression (Fig. 3C) and were only partially reversed by transfection with miR-155 and antagomir-155, respectively. By contrast, the effects of mature miR-155 mimic and antagomir-155 on the nitrite/nitrate level were similar to those on eNOS expression (Fig. 3C). Taken together, our findings indicate that under insulin stimulation, Arg972 IRS-1 inhibits eNOS activity by two different means: i) by downregulating eNOS expression through the upregulation of miR-155; and ii) by inhibiting eNOS activation through the decrease in the PI3K-mediated phosphorylation of eNOS at Ser1177.
Discussion
The dysregulation of NO synthesis attributable to the abnormal expression/activity of eNOS is considered to be a major feature of insulin-resistant states (30,31), as well as an essential contributor to the pathogenesis of cardiovascular diseases (2,3). Arg972 IRS-1 is reportedly associated with impaired IRS-1 ability to activate PI3K, leading to insulin resistance (18,19). In the present study, to the best of our knowledge, we provide the first evidence that Arg972 IRS-1 inhibits eNOS expression in human endothelial cells by upregulating miR-155.
Multivariate regression analysis using data from 832 human subjects revealed that Arg972 IRS-1 was an independent predictor of the human plasma eNOS level after adjustment for multiple potential confounders, which was in agreement with our results that heterozygous and homozygous Arg972 IRS-1 carriers had significantly lower levels of plasma eNOS and nitrite/nitrate than homozygous WT IRS-1 carriers. This was corroborated by our findings that HUVECs derived from delivering mothers expressing heterozygous Arg972 IRS-1 had significantly lower expression levels of eNOS and nitrite/nitrate than those expressing homozygous WT IRS-1.
A recent study demonstrated that miR-155 inhibits eNOS expression in human endothelial cells by directly targeting the eNOS mRNA (27). In the present study, the overexpression of IRS-1 and Arg972 IRS-1, respectively, upregulated and downregulated miR-155 in human endothelial cells by altering PI3K signaling. This provides a mechanistic explanation for the enhanced and inhibited eNOS expression induced by the overexpression of IRS-1 and Arg972 IRS-1, respectively. Since miR-155 and its inhibitor, antagomir-155, respectively, reversed the effects of the overexpression of IRS-1 and Arg972 IRS-1 on eNOS expression, it can be concluded that Arg972 IRS-1 inhibits eNOS expression in endothelial cells by upregulating miR-155. Although our findings indicate that Arg972 IRS-1 upregulates miR-155 in endothelial cells by impairing PI3K signaling, the mechanisms through which PI3K signaling alters the miR-155 level in endothelial cells remain unclear. We aim to elaborate on this issue in our future studies.
We noted that compared with their effects on eNOS expression, the overexpression of IRS-1 and Arg972 IRS-1 exerted much more pronounced effects on eNOS activity, which were only partially reversed by miR-155 and antagomir-155, respectively. This may be explained by the fact that Arg972 IRS-1 inhibited eNOS activity both by downregulating eNOS expression through the upregulation of miR-155, and by inhibiting eNOS activation through the impairment of the PI3K signaling-mediated phosphorylation of eNOS (Ser1177), which is required to activate eNOS (15).
Federici et al (22) reported that Arg972 IRS-1 inhibited eNOS expression in insulin-stimulated endothelial cells in vitro. In the present study, we uncovered a mechanism responsible for this phenomenon by demonstrating that Arg972 IRS-1 inhibits eNOS expression through the upregulation of miR-155 in endothelial cells. Moreover, our in vivo data from a relatively large sample of human subjects support the in vitro inhibitory effects of Arg972 IRS-1 on eNOS expression; this increases the generalizability of our findings. In addition, our findings that Arg972 IRS-1 both downregulates eNOS expression and inhibits PI3K-mediated eNOS activation/phosphorylation account for the marked inhibitory effects of Arg972 IRS-1 on eNOS activity in vitro. In view of the close association of eNOS activity/NO production with cardiovascular diseases, such as hypertension and atherosclerosis (2,3), our in vitro findings were also in line with the data on human subjects showing that heterozygous and homozygous Arg972 IRS-1 carriers had significantly higher blood pressure and a prevalence of coronary heart disease than homozygous WT IRS-1 carriers. Thus, the present study provides both in vivo and in vitro evidence supporting an important pathophysiological role for Arg972 IRS-1 in cardiovascular diseases.
In conclusion, our in vivo data demonstrate that Arg972 IRS-1 is associated with decreased plasma eNOS and nitrite/nitrate levels in human subjects. Our in vitro data demonstrate that Arg972 IRS-1 inhibits eNOS expression in human endothelial cells by upregulating miR-155 through the impairment of PI3K signaling; additionally, Arg972 IRS-1 exerts marked inhibitory effects on eNOS activity by inhibiting both eNOS expression and eNOS activation/phosphorylation through the impairment of PI3K signaling. The present study provides new insight into the pathophysiological role of Arg972 IRS-1 in cardiovascular diseases.
Acknowledgments
The present study was supported by the Guangdong Provincial Natural Science Foundation (grant nos. 12B423 and 13B175), Guangdong, P.R. China.
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