Endothelial Cell–Specific Liver Kinase B1 Deletion Causes Endothelial Dysfunction and Hypertension in Mice In Vivo

Wencheng Zhang, Qilong Wang, Yue Wu, Cate Moriasi, Zhaoyu Liu, Xiaoyan Dai, Qiongxin Wang, Weimin Liu, Zu-Yi Yuan, Ming-Hui Zou

 

Abstract

 

Background—Liver kinase B1 (LKB1), a tumor suppressor, is a central regulator of cell polarity and energy homeostasis. The role of LKB1 in endothelial function in vivo has not been explored.

 

Methods and Results—Endothelium-specific LKB1 knockout (LKB1endo−/−) mice were generated by cross-breeding LKB1flox/flox mice with VE-Cadherin-Cre mice. LKB1endo−/− mice exhibited hypertension, cardiac hypertrophy, and impaired endothelium-dependent relaxation. LKB1endo−/− endothelial cells exhibited reduced endothelial nitric oxide synthase activity and AMP kinase (a downstream enzyme of LKB1) phosphorylation at Thr172 compared with wild-type (WT) cells. In addition, the levels of caveolin-1 were higher in the endothelial cells of LKB1endo−/− mice, and knockdown of caveolin-1 by siRNA normalized endothelial nitric oxide synthase activity. Human antigen R bound with the adenylate-uridylate-rich elements of caveolin-1 mRNA 3′ untranslated region, resulting in the increased stability of caveolin-1, and genetic knockdown of human antigen R decreased the expression of caveolin-1 in LKB1-deficient endothelial cells. Finally, adenoviral overexpression of constitutively active AMP kinase, but not green fluorescent protein, decreased caveolin-1, lowered blood pressure, and improved endothelial function in LKB1endo−/− mice in vivo.

 

Conclusions—Our findings indicate that endothelial LKB1 regulates endothelial nitric oxide synthase activity, endothelial function, and blood pressure by modulating AMP kinase–mediated caveolin-1 expression.

 

AMP-activated protein kinasescaveolin 1hypertensionnitric oxide synthase type III

　

Introduction

 

The endothelium is a monocellular layer that covers the inner surface of blood vessels. It functions as a physical barrier that separates the circulating blood from the vascular walls. In addition, endothelial cells produce a wide range of factors that regulate vascular tone, cellular adhesion, thromboresistance, smooth muscle cell proliferation, and vessel wall inflammation.1 In response to acetylcholine and other physiological stimuli, the endothelium releases nitric oxide (NO) to trigger endothelium-derived vascular relaxation. Endothelial dysfunction is associated with major cardiovascular risk factors such as aging, hyperhomocysteinemia, postmenopause, smoking, diabetes mellitus, hypercholesterolemia, and hypertension.2,3 However, a causative link between endothelial dysfunction and hypertension remains to be established.

 

Clinical Perspective on p 1439

 

NO is a free radical gas that is generated from the metabolism of L-arginine by endothelial NO synthase (eNOS) in endothelial cells. It is the most important factor for maintaining normal endothelial function.4 The essential role of eNOS-derived NO in the cardiovascular system is best demonstrated in eNOS knockout mice, which exhibit hypertension,5 increased vascular smooth muscle cell proliferation in response to vessel injury,6 increased leukocyte-endothelium interactions,7 hypercoagulability,8 and increased diet-induced atherosclerosis.9 eNOS is functionally inhibited through the binding of a 20-aa region of caveolin-1 (amino acids 82–101), called the caveolin-scaffolding domain.10 Thus, caveolin-1 acts as an “inhibitory clamp” that keeps eNOS inactive.11 The physiological significance of this regulation of eNOS by caveolin-1 is evident in caveolin-1 knockout mice, which are unable to regulate eNOS properly and therefore do not establish normal endothelium-dependent vascular tone.12,13 Although the regulation of eNOS by caveolin-1 is well established, the upstream signaling components that regulate caveolin-1 expression remain unclear.

 

Liver kinase B1 (LKB1), a serine/threonine protein kinase that was first described as a tumor suppressor gene, is mutated in Peutz-Jeghers syndrome.14,15 LKB1 is ubiquitously expressed in mammalian cells, and it is activated in a complex with 2 scaffolding proteins: STE20-related adaptor protein and mouse protein 25.16 Currently, LKB1 is implicated as a central regulator of cell polarity and energy metabolism in a variety of systems through its ability to phosphorylate and activate the AMP kinase (AMPK) family of proteins, which consists of 14 members.17 Further roles of LKB1 have been identified through the production of LKB1 knockout mouse models. Global knockouts of LKB1 are embryonically lethal with major defects in cardiovascular developments, indicating that LKB1 has important functions in the cardiovascular system.18 Mäkelä’s group cross-bred LKB1flox/flox mice with Tie1-Cre mice and discovered that the homozygous ablation of LKB1 with the Tie1 promoter results in embryonic lethality resulting from vascular disruption.19 Furthermore, the homozygous ablation of the LKB1 allele with Tie2-Cre produces no viable pups.20 Because Tie1 and Tie2 are expressed in hematopoietic lineages and neurological tissues,21,22 whether the embryonic lethality with Tie1- or Tie2-specific Cre mice is due to LKB1 deficiency in endothelial cells or in other Tie1/Tie2-expressing tissues remains unknown. Because VE-Cadherin-Cre (VE-CAD-Cre), unlike Tie1- or Tie2-Cre, is uniformly expressed in the endothelium of developing and quiescent vessels, with a very small percentage found in hematopoietic cells,23 using VE-CAD-Cre would eliminate the effects of non–endothelial cell LKB1 deletion. Thus, the aim of the present study was to examine the roles of LKB1 in maintaining endothelial function and blood pressure without the effects of nonendothelial LKB1 in endothelium-specific LKB1 knockout mice (LKB1endo−/−) generated by cross-breeding LKB1flox/flox mice with VE-CAD-Cre mice. Here, we report the endothelium-specific LKB1 deletion caused endothelial dysfunction and hypertension in vivo.

 

Methods

 

Animals

 

Mice expressing the Cre recombinase under control of the VE-Cadherin promoter/enhancer (VE-CAD-Cre) were purchased from Jackson Laboratory.23 LKB1-floxed (LKB1flox/flox) mice made by the DePinho laboratory were purchased from the National Cancer Institute.24 To generate endothelium-specific LKB1endo−/− mice, LKB1flox/flox mice were cross-bred with VE-CAD-Cre mice. Polymerase chain reaction–based genotyping was performed, as described previously.25 The animals were housed in a controlled environment (20±2°C, 12-hour/12-hour light/dark cycle), where they were maintained on a standard chow diet with free access to water. Male mice at 3 months of age were subjected to endothelial function analyses and blood pressure measurements. For adenoviral injections, wild-type (WT) or LKB1endo−/− mice received tail vein injections of 100 μL adenoviral vectors that expressed green fluorescent protein (GFP) or constitutively active (CA) AMPK (4×1010 viral particles). The animal protocol was reviewed and approved by the Animal Care and Use Committee at the University of Oklahoma Health Sciences Center.

 

Reagents

 

Antibodies to caveolin-1, phospho-AMPKα Thr172, AMPKα, histone H3, eNOS, and phospho-eNOS Ser1177 were from Cell Signaling Technology (Danvers, MA). Antibodies to LKB1 and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–human antigen R (HuR) was purchased from Millipore (Temecula, CA). Anti-CD31 was from Abcam (Cambridge, UK). FuGene HD transfection reagent was from Roche Applied Science (Indianapolis, IN).

 

Measurement of Blood Pressure

 

Blood pressure was measured with the radiotelemetry technique, as described previously.26 Mice were anesthetized with a ketamine and xylazine mixture (70:6 mg/kg, intraperitoneal injection). The left common carotid artery was exposed, and a catheter (PE10 tubing) was inserted. The body of the telemetry transmitter unit (TA11PA-C10; Data Sciences International, St. Paul, MN) was then inserted under the skin, and the signal was received and recorded by the data acquisition program (Dataquest ART 3.1; Data Sciences International).

 

Measurement of Vessel Tension in Mice

 

Aortas or mesenteric arteries were immersed in Krebs bicarbonate buffer (118 mmol/L NaCl, 4.7 mmol/L KCl, 25 mmol/L NaHCO3, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 2.5 mmol/L CaCl2, and 5 mmol/L glucose) and then suspended by 2 tungsten wires mounted in a vessel myograph system (Danish Myotechnologies, Aarhus, Denmark). After undergoing an equilibration period, rings were treated with phenylephrine for induction of contraction. To study vasodilator and L-nitro arginine methyl ester (L-NAME) responses, the rings were preconstricted with phenylephrine, and acetylcholine (10−10–10–4 mol/L), sodium nitroprusside (10−10–10–5 mol/L), or L-NAME (100 μmol/L) was injected at the plateau of the phenylephrine-induced contraction.

 

Assays of eNOS Activity

 

eNOS activity was detected by measuring the conversion of [3H] L-arginine to [3H] L-citrulline with the eNOS assay kit (Cayman) according to the manufacturer’s instructions. Protein concentration was determined with the BCA Assay Kit (Pierce Chemical Co, Rockford, IL). eNOS activity was expressed as pmol L-citrulline per mg protein per minute. All samples were assayed in triplicate.

 

RNA Immunoprecipitation Assays

 

The Magna RIP kit (catalog No. 17-700, Millipore) was used for RNA immunoprecipitation assays according to the manufacturer’s instructions. Briefly, whole-cell lysates were incubated at 4°C overnight with magnetic protein A/G beads previously precoated with 5 μg of either rabbit IgG or HuR antibody (Millipore). Beads were washed and incubated with proteinase K buffer (30 minutes at 55°C), followed by RNA isolation from the immunoprecipitates and subsequently cDNA synthesis. Polymerase chain reaction was performed by use of the following primers: caveolin-1: forward, 5′-CTACAAGCCCAACAACAAGGC-3′; and reverse, 5′-AGGAAGCTCTTGATGCACGGT-3′; and cyclooxygenase-2: forward, 5′-AACCGCATTGCCTCTGAAT-3′; and reverse, 5′-CATGTTCCAGGAGGATGGAG-3′.

 

Statistical Analysis

 

Data are presented as mean±SEM, and the results were analyzed with GraphPad Prism (GraphPad Software Inc, San Diego, CA). After the normal distribution was confirmed with the Kolmogorov-Smirnov test, statistical comparisons were made with the Student t test for unpaired data and ANOVA followed by the Bonferroni post hoc test when appropriate. Assuming a=0.5, b=0.90, and a ratio of expected effect size to expected standard deviation of 1.20, at least 10 samples per group were required. Groups were considered to be significantly different at values of P<0.05.

 

A detailed description of other methods used in this study can be found in the online-only Data Supplement.

 

Results

 

Generation and Analysis of LKB1endo−/− Mice

 

To elucidate the biological significance of LKB1 expression in vascular endothelial cells in vivo, VE-CAD-Cre mice were cross-bred with LKB1flox/flox mice to generate VE-CAD-Cre/LKB1flox/+ mice. These were further intercrossed to obtain VE-CAD-Cre/LKB1flox/flox mice (referred to as LKB1endo−/−; Figure 1A). The littermate LKB1flox/flox/Cre− mice were used as WT controls.

 

　

　

Figure 1.

Generation and characterization of LKB1endo−/− mice. A, Schematic diagram of the transgenic mice used to generate LKB1endo−/− mice. B, Genotyping analysis of 4-week-old mice. VE-Cadherin-Cre (VE-CAD-Cre)/LKB1flox/+ mice were interbred. A total of 851 mice from 40 litters were genotyped. C, Immunostaining to determine LKB1 (red) and CD31 (green) localization in aortic sections from LKB1endo−/− and wild-type (WT) mice. Scale bar, 50 μm. D, Western blot analysis to detect liver kinase B1 (LKB1) expression in WT and LKB1-deficient mouse lung endothelial cells (n=4). KO indicates knockout. *P<0.05 vs WT.

A total of 851 mice were generated from 40 litters of crosses between VE-CAD-Cre/LKB1flox/+ mice. Analysis of the resulting genotypes revealed a lower mendelian frequency for LKB1endo−/− mice (5.6% shown versus 25.0% expected; Figure 1B). Other LKB1endo−/− mice died before birth; this was consistent with previous findings.19 LKB1endo−/− mice that survived into adulthood were indistinguishable from their control littermates and were used for subsequent experiments.

 

To confirm endothelium-specific LKB1 deletion in LKB1endo−/− mice, immunofluorescence was used to detect LKB1 expression in the aortas that were isolated from these mice. Endothelial cells were detected via CD31 staining. LKB1 was expressed in the endothelium and smooth muscle of aortas from WT mice but not in the endothelium from LKB1endo−/− mice (Figure 1C). To confirm the efficiency of LKB1 knockout in endothelial cells, we isolated mouse lung endothelial cells from the mice. LKB1 was barely detectable in mouse lung endothelial cells from LKB1endo−/− mice, thus confirming effective LKB1 deletion in LKB1endo−/− mice (Figure 1D).

 

Elevated Blood Pressure and Cardiac Hypertrophy in LKB1endo−/− Mice

 

To characterize the phenotypes of LKB1endo−/− mice, we evaluated blood pressure using the radiotelemetry technique. As shown in Figure 2A, both the diastolic blood pressure (112±11 versus 83±8 mm Hg) and systolic blood pressure (148±8 versus 117±9 mm Hg) are significantly higher in LKB1endo−/− mice compared with their WT littermates. Consequently, LKB1endo−/− mice had higher mean blood pressure (125±8 mm Hg) than WT mice (94±8 mm Hg). Similarly, the ratios of heart weight to body weight in LKB1endo−/− mice were significantly greater than those in their WT littermates (Figure 2B and 2C). Morphometric analysis of the hearts from LKB1endo−/− mice showed that both the left and right ventricular walls were enlarged (Figure 2D), which was consistent with a hypertrophic response. Collectively, these results show that LKB1 deficiency in the endothelium resulted in elevated blood pressure and cardiac hypertrophy.

 

　

Figure 2.

Elevated blood pressure and cardiac hypertrophy in LKB1endo−/− mice. A, Blood pressure from male wild-type (WT) and LKB1endo−/− mice at 3 months was measured with the radiotelemetry technique (n=10). B, Appearance of hearts from WT and LKB1endo−/− mice. C, Weight of various organs relative to overall body weight in WT and LKB1endo−/− mice (n=5). D, Hearts from WT and LKB1endo−/− mice were stained with hematoxylin and eosin. Scale bar, 1 mm. dBP indicates diastolic blood pressure; KO, knockout; LKB1, liver kinase B1; mBP, mean blood pressure; and sBP, systolic blood pressure. *P<0.05 vs WT.

Endothelial Deletion of LKB1 Impairs eNOS-Dependent Vasodilation

 

The vascular contractile response is an important peripheral component of the blood pressure regulatory system. Given the increased blood pressure in LKB1endo−/− mice, we examined the effect of endothelial LKB1 deletion on vascular function. As shown in Figure 3A, there is no significant change in the phenylephrine (10–6 mol/L)-induced contraction force between the aortas from WT (6.46±0.72 mN) and LKB1endo−/− (6.28±1.27 mN) mice. When phenylephrine-induced contractions reached a plateau, increasing doses of acetylcholine (10−10 to 10–4 mol/L) were added. Acetylcholine-induced relaxations in WT aortas were concentration dependent. However, acetylcholine-induced relaxations were significantly reduced in LKB1endo−/− aortas (Figure 3B). Interestingly, acetylcholine elicited similar responses in the 2 strains when the aortas were pretreated with an eNOS inhibitor (L-NAME, 10–4 mol/L) for 30 minutes (Figure 3C). These results indicate that the impaired vasorelaxation in the LKB1endo−/− aorta is eNOS mediated. In contrast, relaxations caused by sodium nitroprusside, which is an endothelium-independent agent, did not significantly differ between WT and LKB1endo−/− mice (Figure 3D), indicating that LKB1 deficiency affects endothelial function only in LKB1endo−/− mice. Using mesenteric vessels to further confirm this phenotype, we found that vessels from LKB1endo−/− mice were less responsive to acetylcholine (Figure 3E) and that pretreatment with L-NAME elicited similar levels of responsiveness (Figure 3F). Basal NO release in LKB1endo−/− mice was also reduced as depicted in the trace (Figure 3G). Vessels were preconstricted with a submaximal dose of phenylephrine (10–7 mol/L), and L-NAME (10–4 mol/L) was added at the peak of constriction to remove endogenous NO, which caused further constriction of the WT vessels (3.53±0.76 mN). However, constriction of the vessels in response to L-NAME was significantly attenuated in LKB1endo−/− mice (1.10±0.46 mN; Figure 3H). Overall, these data suggest that endothelial deletion of LKB1 impairs eNOS-dependent vasodilation.

 

　

Figure 3.

Endothelial deletion of liver kinase B1 (LKB1) impairs endothelial nitric oxide synthase (eNOS)–dependent vasodilation. A, Phenylephrine-induced contraction force in aortic rings from LKB1endo−/− and wild-type (WT) mice. Vascular reactivity of LKB1endo−/− and WT aortic rings to acetylcholine (ACh) without (B) or with (C) L-nitro arginine methyl ester (L-NAME) pretreatment (100 μmol/L) for 30 minutes. D, Vascular reactivity of LKB1endo−/− and WT aortic rings to sodium nitroprusside (SNP). Vascular reactivity of LKB1endo−/− and WT mesenteric vessels to ACh without (E) or with (F) L-NAME pretreatment. Aortic rings from LKB1endo−/− and WT mice were contracted with phenylephrine (10–7 mol/L) and then incubated with L-NAME (100 μmol/L) to remove basal nitric oxide synthesis. A representative contraction curve is shown (G), and the force (H) was quantified. Data are mean±SEM; n=5 animals and 2 aortic rings per animal. KO indicates knockout; and LKB1, liver kinase B1. *P<0.05 vs WT.

Decreased eNOS Activity in LKB1endo−/− Mice

 

To measure eNOS activity in lung tissues, we monitored the conversion of [3H] arginine to citrulline. The activity of eNOS was decreased in LKB1endo−/− lung extracts (0.34±0.08 pmol L-citrulline per mg protein per minute) compared with WT (1.24±0.20 pmol L-citrulline per mg protein per minute; Figure 4A). To understand how LKB1 regulates eNOS activity in endothelial cells, the protein levels of phosphorylated eNOS at Ser1177 and total eNOS were assessed via Western blot analysis. There were no changes in the levels of phosphorylated eNOS and total eNOS between WT and LKB1endo−/− endothelial cells, suggesting that LKB1 regulates eNOS activity through alternative signaling pathways (Figure 4B).

　

 

Figure 4.

Decreased endothelial nitric oxide synthase (eNOS) activity in LKB1endo−/− mice. A, eNOS activity from wild-type (WT) or LKB1endo−/− lung extracts was measured according to [3H] arginine–to–citrulline conversion (n=5). *P<0.05 vs WT. Western blot detection of phosphorylated eNOS at Ser1177 (B) and caveolin-1 (C) in mouse lung endothelial cells (MLECs) from WT or LKB1endo−/− mice (n=4). *P<0.05 vs WT. D, eNOS activity was monitored in WT or liver kinase B1 (LKB1)–deficient MLECs transfected with control (CTR) siRNA or caveolin-1 siRNA (n=4). *P<0.05 vs WT+CTR siRNA. KO indicates knockout; and p, phosphorylated.

Caveolin-1 can bind to eNOS and inhibit its bioactivity by holding it in an inactive conformation.27,28 Importantly, LKB1 deletion significantly increased caveolin-1 levels (Figure 4C). Although eNOS activity was much lower in LKB1endo−/− endothelial cells, siRNA knockdown of caveolin-1 significantly increased eNOS activity in both WT and LKB1endo−/− endothelial cells (Figure 4D). These data suggest that increased caveolin-1 contributes to the decrease in eNOS activity in LKB1endo−/− endothelial cells.

 

To investigate whether the association of eNOS with caveolin-1 can blunt AMPK-induced eNOS phosphorylation, human umbilical vein endothelial cells were transfected with control siRNA or caveolin-1 siRNA. Forty-eight hours after transfection, human umbilical vein endothelial cells were incubated with AICAR (2 mmol/L) for 30 minutes. As shown in Figure I in the online-only Data Supplement, AICAR induces eNOS phosphorylation at Ser1177 at similar levels in both control siRNA– and caveolin-1 siRNA–transfected human umbilical vein endothelial cells. This suggests that the association of eNOS with caveolin-1 does not alter the ability of AMPK to phosphorylate eNOS at Ser1177.

 

Increased Expression of Caveolin-1 in the Endothelium of LKB1endo−/− Mice

 

Compared with WT mice, caveolin-1 expression was significantly increased in the endothelium of LKB1endo−/− mice (Figure 5A). Moreover, LKB1endo−/− endothelial cells exhibited a 1.86-fold increase in caveolin-1 mRNA levels (Figure 5B). In LKB1endo−/− lung extracts, increased coimmunoprecipitation of caveolin-1 and eNOS was observed (Figure 5C and 5D), thus confirming that they associate with each other. These data suggest that LKB1 deletion in the endothelium results in the aberrant overexpression of caveolin-1 and the association of caveolin-1 with eNOS.

 

　

Figure 5.

Increased expression of caveolin-1 in the endothelium of LKB1endo−/− mice. A, Immunohistochemical staining for caveolin-1 in aortas from wild-type (WT) and LKB1endo−/− mice. Scale bar, 50 μm. B, Reverse transcription–polymerase chain reaction to detect caveolin-1 mRNA levels in mouse lung endothelial cells from WT and LKB1endo−/− mice (n=4). Caveolin-1 (C) or endothelial nitric oxide synthase (eNOS; D) was immunoprecipitated (IP) from WT or LKB1endo−/− lung lysates, and eNOS or caveolin-1 in the immunoprecipitates was detected by Western blot (n=5). IB indicates immunoblotting; KO, knockout; and LKB1, liver kinase B1. *P<0.05 vs WT.

Caveolin-1 Is a Target of HuR

 

To better understand the regulation of caveolin-1 expression by LKB1, we examined its 5′ untranslated region (UTR) and 3′ UTR to assess whether caveolin-1 may be a target of posttranscriptional modification. We identified 4 conserved adenylate-uridylate–rich elements in the 3′ UTR of caveolin-1 mRNA (Figure 6A). HuR is an RNA-binding protein that regulates the stability of adenylate-uridylate–rich element–containing transcripts, and AMPK activation can inhibit HuR translocation from the nucleus to the cytosol.29 Therefore, we evaluated the effect of LKB1 deletion on HuR translocation. As depicted in Figure 6B, LKB1endo−/− endothelial cells have elevated cytoplasmic HuR levels, although LKB1 deletion does not alter total HuR levels (Figure II in the online-only Data Supplement).

 

　

Figure 6.

Caveolin-1 is a target of human antigen R (HuR). A, Schematic representations of predicted adenylate-uridylate–rich elements (AREs) in the 3′ untranslated region (UTR) of caveolin-1 mRNA are depicted, along with the RNA probes used in the RNA pull-down assay. B, Cytoplasmic and nuclear fractions were isolated from wild-type (WT) and liver kinase B1 (LKB1)–deficient endothelial cells, followed by immunoblotting to detect HuR (n=4). *P<0.05 vs WT cytosol. C, RNA immunoprecipitation was carried out with the anti-HuR antibody or control IgG. Cyclooxygenase-2 was used as the positive control in this assay. Lanes 1 and 5, no template polymerase chain reaction control; lanes 2 and 6, IgG RNA immunoprecipitation; lanes 3 and 7, Anti-HuR RNA immunoprecipitation; and lanes 4 and 8, 10% input. D, Proteins that were pulled down with biotinylated RNA probes corresponding to different AREs in the 3′ UTR of caveolin-1 mRNA, 3′ UTR of Myc (positive control), or 3′ UTR of GAPDH (negative control) were visualized by immunoblotting using the HuR antibody. E, Endothelial cells were transfected with LacZ or HuR plasmid and then treated with actinomycin D (5 μg/mL). The levels of caveolin-1 mRNA were determined by reverse transcription–polymerase chain reaction (n=4). F, Mouse lung endothelial cells from WT and LKB1endo−/− mice were transfected with control or HuR-specific siRNA for 48 hours, followed by Western blot analysis to detect caveolin-1 (n=4). CR indicates coding region; KO, knockout. *P<0.05 vs WT+CTR siRNA.

The ability of HuR to bind with the 3′ UTR of caveolin-1 mRNA was also assessed. RNA immunoprecipitation with either an anti-HuR antibody or control IgG showed that HuR binds to caveolin-1 mRNA (Figure 6C). Cyclooxygenase-2, a known target of HuR, was used as a positive control in this assay. To map out the HuR binding site in the 3′ UTR of caveolin-1 mRNA, we used an RNA pull-down assay in which biotinylated RNA probes that corresponded to the fragments depicted in Figure 6A were incubated with endothelial cell extracts. GAPDH 3′ UTR and Myc 3′ UTR probes were used as the negative and positive controls, respectively. HuR was pulled down with the probe that contained the second adenylate-uridylate–rich element (Figure 6D), suggesting that the second adenylate-uridylate–rich element within the 3′ UTR of caveolin-1 is necessary for its association with HuR. Next, we examined the effects of HuR on the stability of endogenous caveolin-1 mRNA. Endothelial cells were transfected with HuR or LacZ plasmids and then treated with actinomycin D, a transcriptional inhibitor. The half-life of caveolin-1 mRNA was increased from 6.5 to 10 hours after HuR transfections (Figure 6E), indicating that HuR enhances caveolin-1 mRNA stability.

 

Interestingly, HuR knockdown with siRNA suppressed caveolin-1 expression in WT and LKB1endo−/− cells (Figure 6F). This suggests that HuR binds to and stabilizes endogenous caveolin-1 mRNA transcripts, thus resulting in elevated caveolin-1 protein levels.

 

LKB1 Regulates Caveolin-1 Expression in an AMPK-Dependent Manner

 

Because AMPK is a well-characterized substrate of LKB1, we monitored the phosphorylation of AMPKα (Thr172) in mouse lung endothelial cells from WT or LKB1endo−/− mice. Phosphorylated AMPK was significantly decreased in LKB1endo−/− cells (Figure 7A). To determine whether the regulation of caveolin-1 by LKB1 was mediated through AMPK, mouse lung endothelial cells were treated with AICAR or compound C (an AMPK inhibitor) for 48 hours. AMPK activation with AICAR decreased caveolin-1 expression, whereas AMPK inhibition with compound C increased the levels of caveolin-1 (Figure 7B).

 

　

Figure 7.

Liver kinase B1 (LKB1) regulates caveolin-1 expression in an AMP kinase (AMPK)-dependent manner. A, Western blot analysis of phosphorylated (p) AMPKα (Thr172) in mouse lung endothelial cells (MLECs) from wild-type (WT) or LKB1endo−/− mice. *P<0.05 vs WT. B, WT MLECs were treated with AICAR (2 mmol/L) or compound C (CpC; 20 μmol/L) for 48 hours, followed by Western blot analysis (n=4). *P<0.05 vs mock. C, Immunostaining for caveolin-1 in aortas from WT, AMPKα1−/−, and AMPKα2−/− mice. Scale bar, 50 μm. D, MLECs were transfected with control (CTR) siRNA or AMPKα1/2-specific siRNA, followed by RNA immunoprecipitation (IP) with the anti–human antigen R (HuR) antibody or control IgG. Caveolin-1 mRNA levels in each immunoprecipitate (IP) were determined by reverse transcription–polymerase chain reaction. Results are presented as the relative enrichment in HuR RNA IP compared with IgG RNA IP. n=4. *P<0.05 vs CTR siRNA IgG; #P<0.05 vs CTR siRNA HuR. E, MLECs from AMPKα1−/− and AMPKα2−/− mice were subjected to Western blot analysis to detect AMPKα levels (n=4). *P<0.05 vs WT. Western blot analysis of caveolin-1 levels (F) and endothelial nitric oxide synthase (eNOS) activity measurement (G) in MLECs from WT, AMPKα−/−, and LKB1endo−/− mice (n=4). *P<0.05 vs WT. H, WT or LKB1-deficient MLECs were infected with constitutively active (CA) AMPK or the control green fluorescent protein (GFP) virus for 48 hours, followed by Western blot analysis (n=4). *P<0.05 vs WT+GFP. KO indicates knockout.

To further confirm the inverse relationship of caveolin-1 and AMPK, immunohistochemical staining was used to evaluate the levels of caveolin-1 in the aortas from AMPKα1−/− or AMPKα2−/− mice. Caveolin-1 levels were markedly elevated in the endothelial cell layers in the aortas of AMPKα1−/− or AMPKα2−/− mice (Figure 7C).

 

To examine the effect of AMPK on HuR binding with caveolin-1 mRNA, AMPKα was silenced with AMPKα-specific siRNA, followed by RNA immunoprecipitation with IgG or the HuR antibody. Caveolin-1 mRNA was significantly enriched in the HuR immunoprecipitate relative to the IgG control, and AMPKα knockdown further increased HuR enrichment with caveolin-1 mRNA (Figure 7D). These results indicate that AMPK regulates caveolin-1 expression through HuR.

 

To confirm the role of AMPK in the regulation of caveolin-1 and eNOS activity, eNOS expression and activity were assayed in lung endothelial cells isolated from AMPKα1−/− or AMPKα2−/− mice. Although AMPKα1 was the main isoform in the endothelium (Figure 7E), the levels of caveolin-1 in endothelial cells from either AMPKα1−/− or AMPKα2−/− mice were markedly higher than those in WT mice (Figure 7F). Consistently, eNOS activities in AMPKα1−/− or AMPKα2−/− endothelial cells were significantly lower than those in WT mice (Figure 7G). As expected, endothelial cells from LKB1endo−/− mice exhibited higher levels of caveolin-1. eNOS activity in LKB1endo−/− mice was consistently markedly lower than that from AMPKα1−/− or AMPKα2−/− mice (Figure 7F and 7G).

 

The effect of adenoviral overexpression of CA-AMPK on the expression of caveolin-1 in endothelial cells from LKB1endo−/− mice was also determined. As shown in Figure 7H, adenoviral overexpression of CA-AMPK, but not the virus-encoded GFP, suppresses caveolin-1 expression in LKB1endo−/− endothelial cells (Figure 7H).

 

Administration of CA-AMPK Alleviates Endothelial Dysfunction and Lowers High Blood Pressure in LKB1endo−/− Mice

 

The administration of CA-AMPK increased the phosphorylation of AMPKα (Thr172) in the vascular endothelium of WT and LKB1endo−/− mice (Figure 8A). A decrease in the mean blood pressure of LKB1endo−/− mice was observed after 3 days, whereas the adenoviral administration of GFP had no effect (Figure 8B). The administration of CA-AMPK significantly lowered the mean blood pressure of LKB1endo−/− mice (102±8 mm Hg for LKB1endo−/−+CA-AMPK versus 124±7 mm Hg for LKB1endo−/−+GFP; Figure 8C). Similarly, CA-AMPK administration decreased the mean blood pressure of WT mice to 82±8 mm Hg compared with 95±11 mm Hg in GFP-treated WT mice (Figure 8B and 8C). Acetylcholine-induced endothelial relaxation responses in both LKB1endo−/− and WT mice were also significantly improved with CA-AMPK administration (Figure 8D). Our data indicate that AMPK plays an important role in the LKB1-mediated regulation of endothelial function and blood pressure.

　

 

Figure 8.

Administration of constitutively active (CA) AMP kinase (CA-AMPK) alleviates endothelial dysfunction and lowers high blood pressure in LKB1endo−/− mice. A, Wild-type (WT) or LKB1endo−/− mice received tail vein injections of 100 μL adenoviral solutions that expressed green fluorescent protein (GFP) or CA-AMPK (4×1010 viral particles; n=6 in each group). After 7 days, immunohistochemical staining for phosphorylated (p) AMPKα (Thr172) in aortas was performed. Scale bar, 50 μm. Blood pressure from WT and LKB1endo−/− mice after adenoviral injections was monitored by the radiotelemetry technique (B), and mean blood pressure (mBP) was quantified after 7 days of injection (C). *P<0.05 vs WT+GFP; #P<0.05 vs LKB1endo−/−+GFP. D, Acetylcholine (ACh)-induced vascular reactivity of aortic rings from LKB1endo−/− and WT mice after 7 days of injection (n=6). *P<0.05 vs WT+GFP; #P<0.05 vs LKB1endo−/−+GFP. E, Diagram for liver kinase B1 (LKB1)–regulated endothelial nitric oxide (NO) synthase (eNOS) activity and endothelial function through caveolin-1. HuR indicates human antigen R; and KO, knockout.

　

Discussion

 

This study is the first to show that endothelium-specific LKB1 deletion decreases eNOS activity and impairs endothelial function, resulting in hypertension and cardiac hypertrophy. In LKB1endo−/− endothelial cells, knockdown of caveolin-1 with siRNA normalized eNOS activity, genetic knockdown of HuR decreased caveolin-1 levels, and overexpression of HuR increased the mRNA stability of caveolin-1. Finally, adenoviral overexpression of CA-AMPK decreased caveolin-1, lowered blood pressure, and improved endothelial function in LKB1endo−/− mice in vivo. We conclude that LKB1 plays an essential role in mediating the expression of caveolin-1 via AMPK to maintain normal endothelial function and blood pressure (Figure 8E).

 

To research the roles of LKB1 in the endothelium in vivo, several groups have attempted to develop endothelial cell–specific knockout mice by cross-breeding LKB1flox/flox mice with Tie1- or Tie2-Cre mice. However, the homozygous ablation of the LKB1 allele with Tie1- or Tie2-Cre produces no viable pups because of vascular defects.19,20 This can be explained by the broad expression of Tie1/Tie2-Cre in hematopoietic lineages, neuronal populations, and the mesoderm.21,22 On the other hand, VE-CAD-Cre is more specifically expressed in endothelial cells.23 Therefore, it appears to be a better model than Tie1 or Tie2 for studying the functions of essential genes in endothelial cells. Indeed, a recent study reported that specifically deleting vascular endothelial growth factor in endothelial cells with VE-CAD-Cre yields viable adult mice, although most of these mice die at various stages.30 By cross-breeding LKB1flox/flox mice with VE-CAD-Cre mice, we were able to obtain sufficient numbers of LKB1endo−/− mice, although the ratio of LKB1flox/flox:VE-CAD-Cre+ was only 5.6%. However, the reasons for the differences in viability between LKB1-VE-CAD-Cre mice and LKB1-Tie1/Tie2-Cre mice are unknown and warrant further investigation.

 

Caveolin-1 is a multifunctional scaffolding protein with multiple binding partners that are associated with cell surface caveolae.31 It has the ability to bind to and inactivate eNOS.11 The most important finding of the present study is that LKB1 deletion leads to the aberrant expression of caveolin-1, which consequently suppresses eNOS activity and results in endothelial dysfunction, likely via AMPK. Mechanistically, we found that caveolin-1 is regulated by HuR via AMPK. Indeed, a previous study29 has demonstrated that AMPK activation inhibits the translocation of HuR from the nucleus to the cytosol through regulation of importin-α1, an adaptor protein involved in nuclear import. First, AMPK triggered the acetylation of importin-α1 on Lys22, a process dependent on the acetylase activity of p300. Second, AMPK phosphorylated importin-α1 on Ser105.29 Consistently, published studies from our group and others have shown that the LKB1-AMPK signaling pathway is active in the vasculature. LKB1 in cultured endothelial cells activates AMPK in response to protein kinase Cζ activation, and the treatment of cultured endothelial cells with AICAR activates AMPK in an LKB1-dependent manner in cultured human umbilical vein endothelial cells.32,33 We consistently found that LKB1 deletion decreased the levels of phosphorylated AMPK Thr172 and increased the translocation of HuR into the cytoplasmic compartments, where HuR was bound to the 3′ UTR of caveolin-1 mRNA, thus resulting in enhanced stability. HuR is known to bind to and stabilize critical adenylate-uridylate–rich element–containing mRNAs such as plasminogen activator inhibitor-2, vascular endothelial growth factor, and p2134–36 to enhance translation. Our findings provide a new regulatory mechanism for caveolin-1 expression. Endothelial caveolin-1 plays an important role in the regulation of NO production and vascular permeability,37 atherosclerosis,38 and angiogenesis,39 thus implying a role for LKB1-mediated caveolin-1 regulation in these processes.

 

AMPK can mediate the function of eNOS via multiple mechanisms. Previous studies have demonstrated that eNOS phosphorylation and activation are AMPK-dependent in the presence of pharmacological agents and physiological stress.40–42 However, there are also reports showing that AMPK activation by hypoxia, pentobarbital, and glucose deprivation does not increase eNOS phosphorylation,43,44 suggesting a context-dependent regulation of eNOS by AMPK. In our study, LKB1 deletion in endothelial cells decreased phosphorylated AMPKα (Thr172) levels and eNOS activity, whereas phosphorylated eNOS levels remained unchanged, suggesting a novel mechanism by which AMPK regulates eNOS activity via HuR-mediated caveolin-1 expression.

 

Findings from this study may be clinically applicable in human diseases in which the expression or activity of LKB1 or AMPK is reduced. Consistently, the expression or activity of LKB1 is reduced in cardiac hypertrophy.45 Furthermore, 4-hydroxy-trans-2-nonenal, a common reactive lipid oxidant that is elevated in many human diseases, directly inhibits LKB1 by binding to Lys-97.46 In our study, we presented one mechanism that might be linked to hypertension in humans. However, patients with Peutz-Jeghers syndrome, a disease caused by LKB1 mutations, appear to be normotensive, implicating the need for total LKB1 deletion to induce the mechanism described here in mice. Moreover, the hearts from LKB1endo−/− mice developed comparable left and right ventricular hypertrophy (Figure 2D). These results raise the possibility that besides systemic hypertension, LKB1endo−/− mice may have pulmonary hypertension. Further investigation into the contribution of LKB1 to pulmonary hypertension is warranted.

 

Conclusions

 

This study identifies a molecular mechanism by which LKB1 deficiency causes endothelial dysfunction and hypertension. The loss of LKB1 contributes to increased caveolin-1 expression and the subsequent suppression of eNOS activity, thereby adversely affecting endothelial function and blood pressure regulation. Because AMPK activation can effectively attenuate the effects of LKB1 deletion, AMPK might be a therapeutic target of antihypertension therapy.

 

Sources of Funding

 

This study was supported by National Institutes of Health grants (HL074399, HL080499, HL089920, HL110488, and HL105157) and funds from the Warren Chair in Diabetes Research at the University of Oklahoma Health Science Center (all to Dr Zou). Dr Zou is a recipient of the National Established Investigator Award from the American Heart Association.

 

Disclosures

 

None.

 

Footnotes

 

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.113.004146/-/DC1.

Received May 29, 2013.

Accepted January 8, 2014.

© 2014 American Heart Association, Inc.

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CLINICAL PERSPECTIVE

 

Endothelial dysfunction is associated with many cardiovascular diseases. Liver kinase B1(LKB1), a serine/threonine protein kinase for the AMP kinase family, is a known tumor suppressor gene in Peutz-Jeghers syndrome. However, its contribution to vascular function and blood pressure homeostasis remains unknown. In the present study, we have shown that LKB1 is critically involved in vascular function. Genetic endothelium-specific deletion of LKB1 in mice resulted in impaired endothelial nitric oxide synthase–dependent vasodilation, which was associated with elevated blood pressure and cardiac hypertrophy. Mechanistically, this phenotype was attributed to the excessive translocation of human antigen R, thus increasing caveolin-1 expression, which decreased endothelial nitric oxide synthase activity. Furthermore, administration of constitutively active AMP kinase improved endothelial function and decreased blood pressure in endothelium-specific LKB1 knockout mice. These observations unveil new facets of LKB1 that may link energy metabolism with endothelial function and vascular diseases. Our novel findings support the notion that LKB1 is a potential therapeutic target for combating vascular dysfunction associated with vascular diseases such as hypertension.

 

Source from: http://circ.ahajournals.org/content/129/13/1428.full