Contributed equally
Vascular endothelial injury induced by oxidized low-density lipoprotein (ox-LDL) has been implicated in the early stages of the pathogenesis of atherosclerosis. In this study, we incubated bovine aortic endothelial cells (BAECs) with ox-LDL (100 μg/ml) in order to induce endoplasmic reticulum (ER) stress and to investigate the regulation of endothelial nitric oxide synthase (eNOS). Within 4 h of exposure, ox-LDL rapidly induced ER stress, as demonstrated by the measurements of proline-rich extensin-like receptor kinase (PERK) and glucose-regulated protein (GRP)78. ox-LDL induced the rapid dephosphorylation of eNOS at Ser1179 and a subsequent decrease in eNOS activity. This effect appeared to be highly specific as no change was observed in the levels of phosphorylated eNOS at Thr497 or eNOS protein. Of note, a simultaneous decrease was also observed in the active (phosphorylated) form of Akt (Thr308/Ser473), which has been demonstrated to phosphorylate eNOS at Ser1179. Further analysis indicated that Brefeldin A (BFA), an ER stress-inducing reagent, induced the rapid dephosphorylation of Akt and eNOS at Ser1179. 4-Phenylbutyric acid (PBA), an inhibitor of ER stress, blocked the ox-LDL-induced dephosphorylation of Akt and eNOS. Furthermore, JTX20, a lectin-like ox-LDL receptor-1 (LOX-1) blocking antibody significantly eliminated the ability of ox-LDL to mediate the dephosphorylation of eNOS and Akt. Our results indicate that the downregulation of eNOS by ox-LDL, as driven by LOX-1-mediated ER stress, is associated with the PI3K-Akt-eNOS signaling pathway.
Vascular endothelial dysfunction is implicated in the initial development of atherosclerosis (
Previous studies have demonstrated that endoplasmic reticulum (ER) stress induced by ox-LDL in human vascular cells modulates the balance between survival and apoptosis induced by ox-LDL (
In the present study, we found that ER stress is an important trigger for the Akt and eNOS downregulation induced by ox-LDL in ECs. Short-term treatment with ox-LDL immediately induced ER stress and caused the dephosphorylation of Akt and eNOS in bovine aortic ECs (BAECs) prior to changes in the expression of the eNOS and LOX-1 proteins. Of note, treatment with JTX20, a LOX-1 blocking antibody, or 4-phenylbutyric acid (PBA), a chemical chaperone facilitating the correct folding of proteins, partly rescued the dephosphorylation of AKT/eNOS during a short-term (0–4 h) ox-LDL treatment. Our findings demonstrated that ER stress preferentially regulated Akt/eNOS dephosphorylation prior to the alteration of eNOS and LOX-1 expression during the early phase of ox-LDL treatment. The present study provides new data regarding the inhibition of eNOS induced by ox-LDL through LOX-1-mediated ER stress, which may be implicated in endothelial dysfunction reported in coronary artery disease.
The cell culture materials used in the present study were obtained from Invitrogen (Carlsbad, CA, USA). BAECs and growth medium were purchased from Cell Systems Corp. (Kirkland, WA, USA). 2′,5′-ADP-Sepharose 4B was produced by Amersham Biosciences (Piscataway, NJ, USA). The antibody against eNOS was purchased from the BD Transduction Laboratories/BD Biosciences (San Jose, CA, USA). Antibodies against phosphorylated eNOS (p-eNOS; at serine 1179), Akt, phosphorylated Akt (p-Akt; at serine 473) and p-Akt (at threonine 308) were obtained from Cell Signaling Technology (Beverly, MA, USA). The antibody against p-eNOS (at threonine 497) was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Antibodies against protein phosphatase (PP)2A, glucose-regulated protein (GRP)78, p-proline-rich extensin-like receptor kinase (PERK) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3H-arginine was purchased from PerkinElmer Life Sciences (Waltham, MA, USA). The protease inhibitor tablet was purchased from Roche Applied Science (Indianapolis, IN, USA). Calmodulin, NADPH, tetrahydrobiopterin, L-arginine,
The BAECs were cultured in endothelial growth medium supplemented with 10% fetal bovine serum, 10 μg/ml human recombinant epidermal growth factor, 1 mg/l hydrocortisone, 50 μg/ml gentamicin, 50 ng/ml amphotericin B and 12 μg/ml bovine brain extracts. The BAECs between passages 4 and 10 were employed for the experiments.
The cells were harvested and lysed on ice for 30 min in a modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 50 mM NaF, 1 mM Na3VO4, 5 mM sodium pyrophosphate and a protease inhibitor tablet). The cell lysates were centrifuged at 14,000 × g for 15 min, and the supernatant was recovered. The total protein concentration was determined using BCA protein assay reagent (Pierce Biotechnology, Inc., Rockford, IL, USA). The lysates were denatured by boiling in SDS sample buffer. The proteins were separated through gradient SDS/PAGE on a 4–20% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA) with a semi-dry transfer cell (Bio-Rad, Hercules, CA, USA). After being blocked with 3% BSA in TBS for 1 h, the membranes were probed with the appropriate primary antibodies. Membrane-bound primary antibodies were detected using secondary antibodies conjugated with horseradish peroxidase. The western blots were then visualized through enhanced chemiluminescence detection reagents (Sigma) according to the instructions provided by the manufacturer. The protein bands were quantified by scanning with the Bio-Rad GelDoc™ XR and ChemiDoc™ XRS systems and analyzed with Quantity One 1-D analysis software version 4.6.3.
The effect of ox-LDL on the eNOS-mediated metabolism of 3H-arginine to 3H-citrulline was determined, and the assay was performed under apparent
Cells were harvested and lysed on ice for 30 min in lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM Na3VO4, 5 mM sodium pyrophosphate and a protease inhibitor tablet). The cell lysates were centrifuged at 14,000 × g for 15 min, and the supernatants were recovered. The supernatants, which contained equal quantities of protein, were incubated with 2′,5′-ADP-Sepharose 4B resins (50 μl in a 50% slurry) overnight at 4°C as previously described (
Data are expressed as the means ± SD and were analyzed using SPSS 10.0 statistical software (SPSS Inc., Chicago, IL, USA). A one-way ANOVA, followed by LSD post hoc tests were employed to determine the significance of differences among the groups (P-values <0.01 and <0.05, respectively, were considered to indicate statistically significant differences).
LOX-1 has been demonstrated to be a major endothelial receptor for ox-LDL in ECs. Therefore, we investigated whether LOX-1 expression is altered following treatment with ox-LDL. The incubation of BAECs with ox-LDL (100 μg/ml) for 8 to 24 h increased the expression of LOX-1 compared with that of the control (P<0.01). These results are consistent with those of previous studies (
Previous studies have indicated that long-term treatment with ox-LDL (from 12 to 24 h) induces endothelial dysfunction by upregulating the expression of LOX-1 and inhibiting the activity of eNOS. In this study, eNOS expression was also found to be decreased following the treatment of BAECs with ox-LDL for 12 h (
As ox-LDL can trigger ER stress in ECs, and ER stress is characterized by the activation of ER stress sensors, which leads to UPR induction, we also investigated alterations in ER stress sensors in BAECs. The incubation of BAECs with ox-LDL (100 μg/ml) induced the time-dependent activation of ER stress sensors, as assessed by the phosphorylation of PERK. This activation was particularly noticeable immediately after 2 h following treatment with ox-LDL (
Taken together, these data indicate that short-term treatment with ox-LDL immediately activates ER stress and induces eNOS dephosphorylation in BAECs prior to the alteration of total eNOS and LOX-1 expression.
Akt has been described as a kinase responsible for the phosphorylation of eNOS at Ser1179 under various conditions. Therefore, we investigated whether Akt activity is altered by a short-term treatment with ox-LDL and if so, whether this change is associated with the changes in the phosphorylation of eNOS at Ser1179. Our results revealed that Akt was activated, as demonstrated by the phosphorylation of its Thr308 and Ser473 residues. As depicted in
Both Akt and eNOS at Ser1179 have been reported to be dephosphorylated by PP2A (
Akt and PP2A have been reported to be associated with eNOS (
To further understand the mechanisms through which treatment with ox-LDL leads to the loss of Akt phosphorylation and eNOS phosphorylation at active sites, we investigated whether ER stress affects eNOS phosphorylation in BAECs. We treated the BAECs with an ER stress-inducing reagent, Brefeldin A (BFA; which inhibits ER-Golgi transport, at a concentration of 5 μg/ml), to induce UPR signaling. As assessed by western blot analysis, further evaluation of the phosphorylation of eNOS indicated that BFA induced the rapid dephosphorylation of eNOS at Ser1179 (
To explore the possible role of Akt in ER stress, we then examined Akt phosphorylation in the stressed cells by western blot analysis. We determined that the basal level of p-Akt was gradually dephosphorylated upon exposure to ER stress in the BAECs. We observed a slight decrease in the level of phosphorylation at 2 h, and Akt at Thr308 and Ser473 was significantly dephosphorylated at 4 h in response to BFA (5 μg/ml). Conversely, the expression level of total Akt protein was not affected by ER stress (data not shown), indicating that the downregulation of Akt phosphorylation was not induced by the downregulation total Akt protein.
Previous studies have indicated that phosphorylated PERK (p-PERK) and GRP78 are induced in response to ER stress (
To investigate the hypothesis that the downregulation of eNOS phosphorylation is associated with the Akt signaling pathway through the induction of ER stress, the BAECs were treated with ox-LDL in the presence or absence of PBA in order to inhibit ER stress. The induction of p-PERK and GRP78 by ox-LDL was significantly reduced (P<0.01) in the BAECs pre-treated with PBA (10 mM) for 4 h (
We then investigated the hypothesis that the downregulation of eNOS phosphorylation is associated with the Akt signaling pathway through the induction of ER stress by LOX-1 (
ox-LDL has been demonstrated to be an important pathogenic factor in the formation of atherosclerotic plaque. Endothelial dysfunction is the initial change that occurs in the vascular wall during the course of atheroma formation. LOX-1 has been identified as the major receptor for ox-LDL in ECs. As one of the key intrinsic molecules, LOX-1 has been reported to induce endothelial dysfunction after being triggered by ox-LDL (
In the present study, we examined the effects of LOX-1 in eNOS expression and activity in ox-LDL-treated BAECs. Our results demonstrated that ox-LDL did not upregulate the expression of LOX-1 until after 4 h (
LOX-1 is a 50 kDa type II transmembrane glycoprotein comprising 273 amino acids (
Therefore, we investigated the hypothesis that changes in Akt activity may account for the alteration of eNOS phosphorylation at Ser1179 following short-term treatment with ox-LDL. Akt has been reported to be activated following its phosphorylation at the Thr308 and Ser473 residues. As depicted in
ER stress has been demonstrated to be induced by ox-LDL in human vascular cells (
The levels of ox-LDL-induced ER stress and the UPR can be assessed by the phosphorylation of an ER stress sensor (p-PERK) and the expression of ER-resident chaperones (GRP78) (
Taken together, these data demonstrated that short-term treatment with ox-LDL immediately induced ER stress and caused the dephosphorylation of eNOS in BAECs prior to the alteration of total eNOS and expression of LOX-1. Based on these data, we performed a series of experiments to investigate these cellular pathways.
A previous study indicated that the inactivation of Akt is involved in ER stress-mediated signaling. As demonstrated, Akt was gradually inactivated in response to exposure to ER stress (
This finding led us to investigate whether the inhibition of eNOS activity induced by ER stress mediated by ox-LDL is associated with LOX-1 in BAECs. Accordingly, we treated BAECs with ox-LDL in the presence or absence of the anti-LOX-1 blocking antibody, JTX20, and subsequently examined the level of eNOS phosphorylation. The JTX20 antibody reversed the ox-LDL-induced increase in p-PERK and GRP78 expression. JYX20 also significantly reduced the ox-LDL-induced eNOS and Akt dephosphorylation observed in the BAECs (
In conlcusion, although long-term treatment with ox-LDL upregulated LOX-1 expression and downregulated eNOS expression, thereby mediating eNOS activity, our results also indicated that eNOS was downregulated through dephosphorylation at Ser1179 through a rapid regulatory mechanism associated with the Akt signaling pathway. This mechanism was induced by LOX-1-triggered ER stress during the short-term treatment of BAECs with ox-LDL, and the mechanism involved was independent of the stimulation of LOX-1 expression and independent of changes in eNOS enzyme levels.
This study was partly supported by grants from the National Natural Science Foundation of China (grant nos. 30770446, 31000471 and 31171027), the Scientific Research Foundation for Returned Overseas Chinese Scholars, the State Education Ministry, the Hubei Province Natural Science Foundation (grant no. 2006S2153), the Fundamental Research Funds for the Central Universities, HUST (grant no. 2010JC055, 2010MS082 and 2011TS010) and the Scientific and Technological Project in Hubei Province (grant no. 2007AA301B31-2).
(A and B) Effects of oxidized low-density lipoprotein (ox-LDL) (100 μg/ml) on the expression of lectin-like ox-LDL receptor-1 (LOX-1) and endothelial nitric oxide synthase (eNOS) and on the phosphorylation status of eNOS at Ser1179 and Thr497. (C) The activity of phosphorylated eNOS (p-NOS) was assayed by measuring the conversion of [3H]-arginine to [3H]-citrulline. Results are expressed as pmol citrulline/mg protein/min. The data are presented as the means ± SE (**P<0.01 vs. control, n=5). Representative blots are depicted from 3 independent experiments.
Induction of endoplasmic reticulum (ER) stress by oxidized low-density lipoprotein (ox-LDL) in bovine aortic endothelial cells (BAECs). (A) Time course of the ER stress sensors, phosphorylated proline-rich extensin-like receptor kinase (p-PERK) and glucose-regulated protein (GRP)78, in BAECs treated with ox-LDL (100 μg/ml). Western blot analysis was performed on total protein extracts using β-actin protein levels as the control. (B) Quantitative densitometry analyses of p-PERK and GRP78 in the presence of ox-LDL. Data are presented as the means ± SE (**P<0.01 vs. control, n=4).
(A) Changes in Akt, protein phosphatase (PP)2A and PP1 in oxidized low-density lipoprotein (ox-LDL)-treated bovine aortic endothelial cells (BAECs) measured by western blot analysis. Effects of ox-LDL (100 μg/ml) on Akt phosphorylation at Thr308 and Ser473 in BAECs. As the phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1179 decreased upon ox-LDL treatment, the phosphorylated form of Akt at Thr308 and Ser473 was also reduced in a time-dependant manner. However, the Akt protein levels depicted did not change according to the results presented here. (B) Effects of ox-LDL (100 μg/ml) on the expression of PP2A and PP1 in BAECs after treatment for 0, 2 and 4 h. No significant change was observed in the levels of PP2A and PP1 in the BAECs before and after ox-LDL treatment. (B and C) Representative blots are presented from 3 independent experiments. (B) Effects of ox-LDL on the association of PP1, PP2A and Akt with eNOS. After 2 or 4 h of treatment of ox-LDL (200 μg/ml), eNOS in the BAECs was pulled down with the 2′,5′-ADP-Sepharose beads, and eNOS-associated proteins were measured by western blot analysis with the corresponding antibodies. Consistent with the results obtained with the cell lysates, ox-LDL treatment decreased the phosphorylation of Akt (at Ser473 and Thr308) and eNOS (at Ser1179). However, in the samples containing equal amounts of eNOS input, the levels of PP1, PP2A and Akt associated with eNOS were not significantly altered by ox-LDL treatment. All the data presented are representative of 3 independent experiments. (C) Quantitative densitometry analyses of Akt phosphorylation at Thr308 and Ser473 associated with eNOS. Data are presented as the means ± SE (*,&,#P<0.05; **,&&,##P<0.01 vs. the levels observed prior to treatment with ox-LDL, n=3).
(A and B) Effects of the endoplasmic reticulum (ER) stress-inducing reagent, Brefeldin A (BFA), on endothelial nitric oxide synthase (eNOS) phosphorylation in bovine aortic endothelial cells (BAECs). (A) eNOS phosphorylation at Ser1179 decreased as early as 0.5 h, as phosphorylated proline-rich extensin-like receptor kinase (p-PERK) and glucose-regulated protein (GRP)78 expression increased in the BAECs treated with BFA (5 μg/ml). (B) Quantitative densitometry analyses of the data in (A) were conducted. Data are presented as the means ± SE. (C) Effects of BFA (an inducer of ER stress) and insulin-like growth factor-1 (IGF-1) (an activator of PI3K) on the status of Akt and eNOS phosphorylation in BAECs. The levels of phosphorylated Akt and eNOS were determined by western blot analysis at the indicated times after stimulation with 5 μg/ml of BFA. Selected wells were also stimulated simultaneously with 100 ng/ml of IGF-1 and 5 μg/ml of BFA, as depicted in the figure. All the experiments were performed 4 times, and similar results were obtained.
The chemical chaperone, 4-phenylbutyric acid (PBA), reduces endoplasmic reticulum (ER) stress and prevents the loss of Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in bovine aortic endothelial cells (BAECs). The expression of (A) eNOS, (B) Akt and (C) ER stress sensors in BAECs pre-treated with PBA (10 mM) for 14 h. Representative blots are presented from 3 independent experiments. ox-LDL, oxidized low-density lipoprotein.
The anti-LOX-1 blocking antibody, JTX20, reduces endoplasmic reticulum (ER) stress and prevents the loss of Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in bovine aortic endothelial cells (BAECs). The expression of (A) eNOS, (B) Akt and (C) ER stress sensors in BAECs pre-treated with JTX20 (40 μg/ml) for 30 min. Representative blots are presented from 3 independent experiments. ox-LDL, oxidized low-density lipoprotein; LOX-1, lectin-like ox-LDL receptor-1.