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Title The Role of AMPK and miR-92a in the Shear Stress Regulation of KLF2

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Author Wu, Wei

Publication Date 2010

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UNIVERSITY OF CALIFORNIA RIVERSIDE

The Role of AMPK and miR-92a in the Shear Stress Regulation of KLF2

A Dissertation submitted in partial satisfaction of the requirements for the degree of

Doctor of Philosophy

in

Cellular, Molecular and Developmental Biology

by

Wei Wu

December 2010

Dissertation Committee:

Dr. John Shyy, Chairperson Dr. Ameae Walker Dr. Kathryn Defea

Copyright by Wei Wu 2010

ii

The Dissertation of Wei Wu is approved:

------

------

------Committee Chairperson

University of California, Riverside

iii Acknowledgements

It is a pleasure to thank those who made this dissertation possible. First of all, I would like to thank CMDB program for giving me the opportunity to pursue my Ph.D degree in UCR. A hearty thanks to Dr. Anthony W. Norman, Dr. Helen Henry and the professors who taught and encouraged me in these past years.

I owe my deepest gratitude to my major professor, Dr. John Y-J. Shyy, for his guidance and support. I am also grateful to Dr. Ameae M. Walker and Dr. Kathryn

Defea for their valuable discussions and constructive suggestions. I am thankful to all of my colleagues and friends for their generous help and precious discussion on my work.

My parents and parents-in-law, thanks for your support and unconditional love.

Even though we are thousand of miles away, you were always there whenever I needed you.

This thesis would have never been possible without my loving husband Dong.

You were always with me at times I thought that it is impossible to continue, you helped me to keep things in perspective.

The material included in Chapters 2 was originally published in Arteriosclerosis,

Thrombosis, and Vascular Biology (2009; 29:1902). Thanks to the publishers for permission to reuse the material.

iv

ABSTRACT OF THE DISSERTATION

The Role of AMPK and miR-92a in the Shear Stress Regulation of KLF2

By

Wei Wu

Doctor of Philosophy, Graduate Program in Cellular, Molecular and Developmental Biology University of California, Riverside, December 2010 Dr. John Y-J Shyy, Chairperson

The vascular is essential to maintain normal vascular homostasis and its dysfunction is a hallmark of atherosclerosis development. Upregulated by athero-protective flow, the Kruppel-like factor 2 (KLF2) is a crucial integrator for maintaining multiple endothelial functions, including anti-inflammation, anti-thrombosis, vasodilatation, and anti-angiogenesis. To investigate the molecular mechanism by which KLF2 is regulated by different flow pattern, the regulation of KLF2 expression was examined at both transcriptional level and post-transcriptional level response to different flow pattern.

In the first part of the study, AMP-activated kinase (AMPK) was demonstrated to be necessary and sufficient to regulate the expression of pulsatile

v shear stress (PS)-induced KLF2 and its downstream (eNOS and ET-1). In addition,

The PS-induced phosphorylation of ERK5 and , which regulates the KLF2

expression, is AMPK-dependent in ECs. Furthermore, the phosphorylation levels of

ERK5 and MEF2, as well as the expression of KLF2, were significantly reduced in

the aorta of AMPKα2 knockout mice when compared with wild-type control mice.

These findings suggest that AMPK/ERK5/MEF2 is a functional signaling for the

regulation of KLF2 transcription.

MicroRNAs (miRNAs) are non-coding small RNAs that regulate

expression at the post-transcriptional level. In the second part, the role of miRNAs,

particularly miR-92a, was examined in the atheroprotective flow-regulated KLF2.

Fistly, it was demonstrated that KLF2 is regulated by miRNA by knocking down

Dicer with siRNA. In silico analysis predicted that miR-92a could bind to the 3’

untranslated region (3’UTR) of KLF2. Overexpression of miR-92a precursor

(pre-92a) decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase (eNOS) and thrombomodulin (TM) at mRNA and protein levels.

Subsequent studies revealed that, athero-protective laminar flow down-regulated the

level of miR-92a to induce KLF2, and the level of this flow-induced KLF2 was

reduced by pre-92a. Consistent with these findings, miR-92a level was lower in the

endothelium of athero-protective than athero-prone areas of the mouse aorta.

Furthermore, mouse carotid arteries receiving pre-92a exhibited impaired

vasodilatory response to flow. Collectively, these results suggest that

vi athero-protective flow patterns decrease the level of miR-92a, which in turn increases

KLF2 expression to maintain endothelial homeostasis.

Taken together, this study demonstrated the potency of shear stress on EC function is due to the upregulation of KLF2 at both transcriptional level and post-transcriptional level.

vii Table of Contents

Chapter 1 Introduction 1

Chapter 2 Flow Activation of AMP-activated Protein Kinase Leading to 57

The Expression of Krüppel-like Factor 2

Chapter 3 Flow-regulation of Krüppel-like Factor 2 Is Mediated by 89

MicroRNA-92a

Chapter 4 Conclusion and Perspectives 120

viii List of Figures

Fig. 1-1 The flow channel used to mimic blood flow in vitro 4

Fig. 1-2 Schematic comparison of the members of KLF family 6

Fig. 1-3 Schematic diagram of the functions of KLF2 9

Fig. 1-4 Schematic diagram of the transcriptional regulation of KLF2 in 15

endothelial cells

Fig. 1-5 The mechanism of miRNA-mediated translational repression in 23

animal cells

Fig. 1-6 Organization of the miR-17-92 cluster and its paralogs 30

Fig. 1-7 The transcriptional regulation of miR-17-92 cluster 32

Fig. 2-1 PS activates AMPK and induces KLF2 in cultured ECs 81

Fig. 2-2 AMPK regulates the PS-induced KLF2 and its target 82

Fig. 2-3 AMPK activates KLF2 via the ERK5-MEF2 pathway in 83

cultured ECs

Fig. 2-4 AMPK upregulates ERK5-MEF2 in ECs 84

Fig. 2-5 AMPK mediates KLF2 expression via the ERK5-MEF2 85

pathway in mouse models

Fig. 2-S1 Inhibition of KLF expression by Compound C is concentration 86

dependent

Fig. 2-S2 Optimization for KLF2 knockdown 87

ix Fig. 2-S3 AICAR-induced ERK5 phosphorylation is compound C 88

sensitive and MEK5 dependent

Fig. 3-1 miRNAs are involved in the regulation of KLF2 mRNA 113

Fig. 3-2 miR-92a targets KLF2 mRNA and decreases KLF2 translation 114

Fig. 3-3 Shear stress-induction of KLF2 is mediated through miR-92a 115

Fig. 3-4 PS down-regulates, but OS upregulates, miR-92a expression in 116

ECs

Fig. 3-5 miRISC regulates miR-92a 117

Fig. 3-6 miR-92a regulates endothelial function in vitro and ex vivo 118

Fig. 3-7 The expression of miR-17-92 cluster under the different flow 119

pattern

Fig. 4-1 Shear stress regulation of KLF2 131

x List of Tables

Table 1-1 The miRNAs and vascular function 27

Table 4-1 Transcription factors regulating KLF2 132

Table 4-2 Transcription factors regulating the miR-17~92 cluster 133

Table 4-3 KLF2-targeted miRNAs 134

xi List of Abbreviations

ACC: acetyl-CoA carboxylase

Ago: argonaute

AICAR: 5’-aminoimidazole-4-carboxamide ribonucleoside

AMPK: AMP-activated protein kinase

BAEC: bovine acrtic endothelial cell

Ad-AMPK-CA: Adeno-AMPK-constitutively active

DMEM: Dulbecco modified Eagle medium

DMSO: dimethyl sulfoxide

DRB: 5,6-dichloro-1-β-D-ribobenzimidazole

EC: endothelial cell

ET-1: endothelin-1 eNOS/NOS3: endothelial nitric-oxide synthase

ERK5: extracellular signal-regulated kinase 5

FISH: florescence in situ hybridization

HDAC: histone deacetylase

HEK293: human embryonic kidney 293

HUVEC: human umbilical vein endothelial cell

IP: immunoprecipitation

KLF2: Kruppel-like factor 2

xii KO: knockout

MEF2: myocyte enhancer factor-2

MEK5: MAPK/extracellular signal-regulated kinase kinases miRISC: miRNA induced silencing complex

NO: nitric oxide

OS: oscillatory shear flow

PS: pulsatile laminar flow

TM: thrombomodulin

3’-UTR: 3’ untranlated region

xiii Chapter 1

Introduction

1. Atherosclerosis and endothelial dysfunction

The vascular endothelium, located at the interface between the circulating blood

and the vessel wall, is extensively involved in vascular homeostasis such as the balance

between vasodilation and vasoconstriction, inhibition and stimulation of smooth

muscle cell proliferation and migration, and thrombogenesis and fibrinolysis1.

Endothelial dysfunction is an early marker for atherosclerosis2, which can be initiated

by various risk factors, including elevated level of inflammatory cytokines,

hypercholesterolemia, and hyperhomocysteinemia, diabetes, hypertension, disturbed

flow patterns, and tobacco smoking3. These risk factors increase the endothelial

permeability and recruitment of the circulating monocytes/macrophages that engulf the

oxidized low-density lipoprotein (ox-LDL), fats, calcium, and fibrin within the artery

wall, which causes atherogenesis4. These plaques gradually occlude the arteries, and

then decrease blood flow and vascular functions. The enlarged plaque may rapture

and quickly obstruct the flow of blood, which results in myocardial infarction and

stroke.

Shear stress, the tangential component of the hemodynamic forces resulting from

blood flow, is the most physiologically relevant stimulus that maintains endothelial

cell (EC) homeostasis and prevents the development of atherosclerosis5. The transcription factor Krüppel-like factor 2 (KLF2) is a critical regulator for endothelial lineage development and multiple vascular functions6, 7. The purpose of my study is

1 to understand the molecular mechanism by which shear stress upregulates KLF2

expression and then maintains the protective effect on ECs. In chapter 2, I describe

whether AMP-activated protein kinase (AMPK) mediates shear stress-induced

expression of KLF2. Experiments with the cultured ECs and mouse vessel wall

demonstrated the requirement of AMPK for the activation of extracellular

signal-regulated kinase 5/myocyte enhancer factor 2 (ERK5/MEF2) signaling

pathway and the expression of KLF2. In chapter 3, I examined the role of miRNA,

particularly miR-92a, in the atheroprotective flow-regulated KLF2. Evidence

collected both in vitro and in vivo suggests that atheroprotective flow decreases the

level of miR-92a, which in turn increases KLF2 expression to maintain endothelial

homeostasis. Taken together, these findings indicate that the potency of shear stress on

EC function is due to the regulation of KLF2 at both transcriptional and

post-transcriptional levels.

2. Blood flow, shear stress, and Endothelial function

The concept that “physical exercise involving increase of cardic output, and

hence increased shear rate, might retard the development of atheroma” was first

proposed 40 years ago8. In the following decades, pathophysiological studies of

human patients and various animal models clearly indicated that atherosclerotic

lesions tend to occur in the curvatures, bifurcation, and branches of the vessels, which

are termed the “atheroprone region”. In contrast, lesions develop much less in the

unbranched straight parts of the arteries, known as the “atheroprotected region”9-12.

In the atheroprone region, the blood flow rate is reduced and can reverse direction during the cardiac cycle, which is called “oscillatory flow” or “disturbed flow”. In the

2 straight part of arteries, the rate of flow changes during the cardiac cycle, but flow is

always in the forward direction and is called “laminar flow” or “pulsatile flow”13, 14.

Shear stress, the tangential component of hemodynamic forces resulting from blood flow, is an important regulator of endothelial functions15. ECs in the

atheroprone region experience unsteady and low mean shear stress, which stimulates

proatherogenic responses, such as high levels of cell turnover, inflammation, permeability, thrombosis and oxidative stress12, 13. In contrast, ECs in the

atheroprotected region experience unidirectional, high mean shear stress. Laminar

shear stress maintains the quiescent phenotype of ECs and exerts vascular protective

effects, such as anti-inflammation, anti-oxidation, anti-thrombosis, and promotion of

vasodilation16.

To investigate the mechanism by which shear stress modulates endothelial

functions, I used the flow channel system to mimic the different flow patterns in vitro

(Fig. 1-1)17. The laminar flow (with steady, laminar shear stress at 12 dyn/cm2) is the most simplified flow pattern to assess the effects of atheroprotective flow in endothelial biology. To mimic more physiological conditions, pulsatile shear flow

(PS) or oscillatory flow (OS) can also be generated by this system. A sinusoidal component with a frequency of 1 Hz (mimicking the pulse in the human body) can be introduced into the system by connecting a reciprocating syringe pump to the circulating system. In my study, PS or OS was applied to ECs as 12±4 dyn/cm2 or

0±4 dyn/cm2 respectively.

3

Fig. 1-1. The flow channel system used to mimic blood flow in vitro. A confluent monolayer of ECs is seeded onto the glass slide and sealed within the chamber and a gasket creates a space for flow. The flow rate is controlled by the height difference of two reservoirs. A reciprocating syringe pump is connected to the circulating system to introduce a sinusoidal component (frequency=1 Hz) onto the shear stress. (Adapted from Chien, 200717)

The impaired endothelium-dependent vasodilation, which is mediated by nitric

oxide (NO), is the hallmark of endothelial dysfunction and a contributor to

atherosclerosis18-20. By using the flow channel system and animal models, many

groups, including ours, have shown that laminar shear stress is an important

physiological stimulus enhancing endothelial functions, in part because of the

regulation of endothelial NO synthase (eNOS) and thereby NO production. At the

transcriptional level, the prolonged shear stress can upregulate eNOS expression in

cultured ECs and in intact arteries18, 21. At the post-translational level, shear stress can

4 cause eNOS phosphorylation at Ser-1177 and Ser-633 through multiple kinases, such

as protein kinase A (PKA), protein kinase B (AKT) and AMPK22-25. eNOS can also be

activated by an SIRT1-induced deacetylation response to shear stress26.

3. KLF2, the integrator of multiple endothelial functions

Shear stress is well known to regulate eNOS activity at th e post-translation level

(such as phosphorylation, acetylation). However, how shear stress regulates the

expression of eNOS in the long term is unclear. Over the last several years, many

studies have suggested that Kruppel-like transcription factor (KLF) is a crucial

regulator of endothelial function. Notably, KLF2 is shear inducible and could bind

to the eNOS and regulate its transcription response to shear stress27. In this

section, I summarize the role of KLF2 in endothelial function and the regulation of

KLF2 transcription.

3.1 KLF family

“Krüppel”, the German word for “cripple”, is an important transcription factor regulating the development of Drosophila28, 29. The protein is named

because Drosophila embryos lacking the protein Krüppel have altered anterior

abdominal and thoracic segments where the legs should develop from, which results

in death30. The nomenclature of the mammalian Krüppel-like factor (KLF) family is

based on the homology to the DNA-binding domain of the Drosophila Krüppel. The

first mammalian Krüppel, erythroid Krüppel-like factor (EKLF/KLF1) was identified

5 in red blood cells in 199331. It was demonstrated to play an important role in

ß-globin gene synthesis and erythrocyte development32, 33. To date, 17 mammalian

Krüppels have been identified and designated on the basis of the chronological order of discovery (ie, KLF 1-17). KLFs play key roles in regulating cellular processes in many distinct cell types, as shown by gene knockout studies34, 35. Three members of the KLF family are expressed in ECs: KLF2, , and KLF6.

Figure. 1-2. Schematic comparison of the members of KLF family. The blue region is the activation domain and the purple region is the repression domain. C-terminal is the domain. (Adapted from Jain, 20076)

KLFs are a subclass of the zinc finger family of DNA-binding transcription factors35. The C terminus of KLF protein contains 3 Cys2/His2 zinc finger motifs, which could bind to the conscious sequence "CACCC" or the "GT box" in the promoter region of the target genes36. The inter-finger-space sequence contains a highly conserved 7-aa sequence, TGEKP(Y/F)X. Although KLFs have similar zinc finger domains in the C terminus, their N termini are highly diverse. Transactivation

6 of KLFs to the targets is mediated via their various activation or repression domains in

the N terminus (Fig. 1-2).

3.2 The identification and characterization of KLF2

KLF2, a 354-aa protein, was first cloned by the Lingrel group in 199537. It was

initially termed as lung Krüppel-like factor (LKLF) because it is highly expressed in

lung tissues. Human KLF2 maps to 10p13.1 and has greater than 85%

homology to the mouse gene. Furthermore, the critical regions of the promoter are

also conserved across species, including exact identity of a 75-bp sequence38. The

N-terminal regulatory region contains a transcriptional activation domain (1-110 aa)

and an inhibitory domain (110-267 aa)38, 39. The transcriptional activation domain

can interact with a coactivator (such as p300/CBP), and the complex is able to induce

chromatin remodeling40. The inhibitory domain interacts with the WW

domain–containing E3 ubiquitin protein ligase 1 (WWP1), thus resulting in

ubiquitination and proteasomal degradation of KLF238, 41.

3.3 KLF2 animal model

In the embryo of wild-type mice, the expression of KLF2 is initially noted at

embryonic day 7(E7), then decreased at day 11, and reactivated at day 1537. Thus,

KLF2 expression is developmentally regulated. KLF2 knockout mice exhibit abnormal blood vessel formation because of insufficient smooth muscle cell recruitment, thus resulting in embryonic hemorrhage and death between day 12.5 and

7 14.537, 42. These mice also display abnormal lung , which supports an

essential role for this factor in lung development42, 43. The endothelial-specific

KLF2 knockout mouse is also embryonic lethal because of heart failure in association

with reduced vessel tone but not vascular defects44. The viable hemizygous KLF2

knockout mouse (KLF2+/-) does not have an apparent endothelial phenotype45.

However, when crossed to apolipoprotein E (ApoE)-deficient mice, KLF2+/- mice show aggravated atherosclerosis development, possibly through increased foam-cell formation. These animal models showed that KLF2 plays an important role during development and maintains EC functions.

3.4 KLF2 regulates EC functions

In 2002, Dekker et al. first demonstrated that knockdown of KLF2 prevented

flow-mediated induction of eNOS and flow-mediated reduction of endothelin-1, which is the first important evidence of KLF2 regulating flow-mediated effects in

ECs46. The recent transcriptome analysis showed that KLF2 governs 70% of the

shear stress-regulated gene sets47. Emerging evidence indicates that KLF2 is a critical

integrator of multiple endothelial functions (anti-inflammation, anti-thrombosis,

anti-angiogenesis, vascular tone, blood vessel development) that are resistant to

atherogenesis (Fig. 1-3)6, 7. For example, KLF2 inhibits NFκB-dependent vascular

cell adhesion molecule 1 (VCAM-1) and E-selectin expression response to

inflammatory cytokines, which in turn decreases the attachment of immune cells and

rolling to ECs27. KLF2 can also strongly inhibit transforming growth factor (TGF)-β

8 signaling by decreasing the levels of phosphorylated nuclear activating transcription factor 2 (ATF2)48. Parmar et al. found that induction of several vasodilatory factors that depend on KLF2 include the flow-mediated upregulation of eNOS, argininosuccinate synthetase (ASS), and C-natriuretic peptide (CNP), as well as the downregulation of endothelin-1 (ET-1) and VCAM-17. KLF2 has potent anti-angiogenic effects and keeps ECs quiescent by inhibiting the expression of the key VEGF (VEGFR2) and then reducing EC proliferation and migration49.

Fig.1-3. Schematic diagram of the functions of KLF2. (Adapted from Jain, 201050)

9 3.5 The regulation of KLF2 transcription

3.5.1 Flow regulation of KLF2

As discussed above, laminar blood flow is thought to be benefit to the endothelium. The disturbed flow patterns are thought to contribute to atherogenesis in the atheroprone regions. However, the exact mechanism by which laminar flow imposes atheroprotective effects remains poorly understood.

Using a gene profiling approach, the Horrevoets group first demonstrated that

KLF2 is induced by prolonged laminar flow in cultured ECs46. Furthermore, this

group showed with in situ hybridization that KLF2 is highly expressed in the linear

segments of the vessel and is decreased at branch points, which are the more

atheroprone regions of the vasculature. Parmar et al. demonstrated the

flow-dependent expression of endothelial KLF2 in vivo using the sih zebrafish mutant,

which lacks blood flow7. In vitro and in vivo experiments with vascular ECs indicate

that KLF2 exhibits sustained induction under laminar shear stress. Furthermore, Wang

et al. reported that pulsatile flow induced sustained expression of KLF2, but

oscillatory flow caused prolonged suppression after a transient induction in ECs51.

3.5.2 MEF2, an important transcription factor of KLF2

Several groups used promoter analysis to elucidate the mechanism of flow-mediated induction of KLF2. In 2004, Huddleson et al. identified a 62-bp shear stress-responsive region in the KLF2 promoter that contains a 30-bp (-138 to

10 -108 bp) tripartite palindrome motif52. Deletion analysis revealed that with lack of

this region, KLF2 expression would not be upregulated by shear stress. The authors

further found a consensus myocyte enhancing factor 2 (MEF2)-binding site (ctaaatttag) in this region. Chromtin immunoprecipitation (ChIP) assays revealed that both

MEF2A and MEF2C bind to this region of the KLF2 promoter in ECs7.

MEF2 factors are members of the MADS box (MCM1, Agamous, Deficiens,

Serum response factor) family of transcription factors that bind to A/T-rich

sequences53, 54. MEF2 was initially identified as a transcription factor that activates the expression of skeletal and cardiac muscle structural genes55. Recently, MEF2

was found to be required for EC proliferation and viability and involved in the

pathogenic mechanisms associated with endothelial disorders56 For example,

mutations in the human mef2a gene have been observed in patients with inherited

coronary artery disease56, 57. -knockout mouse is lethal, with profound cardiac

and vascular defects due to the impaired endothelial integrity and permeability58.

3.5.3 The regulation of MEF2 transactivation

The identification of MEF2 factors as regulators of KLF2 expression provided an

immediate link to flow. ERK5 (also known as big mitogen-activated protein kinase

1) was identified as a highly flow-induced factor by the Berk group, in

199959. MEF2 is one of the best-characterized targets of ERK560, 61. ERK5 is

capable of greatly enhancing the transactivation activity of MEF2C by phosphorylating MEF2C at Ser 387 and Thr 300 and MEF2A at Ser 355, Thr 312 and

11 Thr 319 both in vitro and in vivo62. Yeast 2-hybrid screening and

co-immunoprecipitation also showed that ERK5 and MEF2C interact with each

other61.

The connection between KLF2 and this pathway was confirmed by consequent

work. Loss-of-function studies performed by the Winoto group showed that ERK5

is essential for embryonic KLF2 expression and that ERK5 drives KLF2 transcription by activating MEF2 transcription factors63. Consistent with this

observation, Parmar et al. showed that overexpression of a dominant-negative MEF2

or mutant MEK5 (an upstream activator of ERK5) prevented flow-mediated induction

of KLF2 expression in endothelial cells7. These results indicated that the

mechanisms linking shear stress and KLF2 expression involve activation of a

MEK5/ERK5/MEF2 pathway.

3.5.4 Other transcription factors

In addition to MEF2, several transcription factors (TFs) that bind to the KLF2

promoter by using DNA affinity chromatography and mass spectrometry were

identified64. These factors include p300/CBP-associated factor (PCAF), heterogeneous

nuclear ribonucleoprotein D (hnRNP D), and nucleolin. Furthermore, these were required for the shear induction of KLF2 by chromatin immunoprecipitation and

gel-shift analysis. Shear stress regulates the binding of this complex through a

phosphoinositide-3 kinase (PI3K)-dependent/Akt-independent pathway65.

12 3.6 Other stimuli regulates KLF2 expression

Considering its beneficial effects to the endothelium, KLF2 is an ideal target for drug development for cardiovascular disease. Recently, several medicines and chemicals have been shown to improve EC function by regulating KLF2 expression.

3.6.1 Statins

Statins act to reduce plasma cholesterol levels by inhibiting

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and constitute the

most widely prescribed class of drugs for reducing morbidity and mortality associated

with cardiovascular disease. Interestingly, many of the benefits of statins may

extend beyond their lipid-lowering effects66-68. The pleiotropic effects include the

improvement of endothelial function, inhibition of vascular inflammation and

oxidation, and stabilization of atherosclerotic plaques. At the cellular level, similar to shear stress, statins have been shown to induce eNOS phosphorylation and also upregulate eNOS in endothelium69.

Because of much overlap between the beneficial cellular effects of statins and

shear stress, several groups studied whether KLF2 mediates statin-induced effect in

ECs. Indeed, multiple statins could induce KLF2 expression70-72, which suggested that

statins induce KLF2 expression by inhibiting a Rho pathway. Promoter deletion

and mutational analysis identified that the MEF2-binding site of KLF2 promoter is

13 also required for statin-mediated KLF2 induction73. Furthermore, KLF2-knockdown

experiments demonstrated that KLF2 is required for statin-mediated induction of

eNOS and TM mRNA and protein levels72. These data strongly implicate KLF2 as a

novel nuclear mediator of statin effects in ECs.

3.6.2 Resveratrol

Resveratrol (3,5,4′-trihydroxystilbene), a natural polyphenol and phytoalexin,

has a wide range of beneficial effects on the cardiovascular system74. The

NAD+-dependent deacetylase Sirtuin 1 (SIRT1), activated by resveratrol, mediates the effect of anti-inflammatory, vasodilatory, and anti-thrombotic properties to the vasculature75. Resveratrol and SIRT1 activators improved endothelial

dysfunction and reduced vascular inflammation in mice76. Previous studies have

demonstrated that endothelial-specific overexpression of SIRT1 improves endothelial

function and decreases atherosclerosis in ApoE-null mice77. Recently, the Garcia

group demonstrated that resveratrol could induce the expression of KLF2 in cultured

HUVECs. This induction depended on SIRT1 and sequentially the MEK5/MEF2

pathway but not ERK578.

3.6.3 Angiopoietin-1

Angiopoietin-1 (Ang1) is a ligand for the endothelium-specific receptor tyrosine

kinase Tie-279. Ang1/Tie2 signaling promotes vascular formation during

development and physiological and pathological angiogenesis but maintains the

14 quiescence of mature blood vessels in normal adult vasculature by enhancing vascular

integrity and endothelial survival80. In the presence of cell–cell contact,

Ang1-induced KLF2 expression was demonstrated by microarray analysis81. The

following study showed that Ang1/Tie2 signal stimulates transcriptional activity of

MEF2 through a PI3K/AKT pathway to induce KLF2 expression, thereby contributing to vascular quiescence82.

Fig. 1-4. Schematic diagram of the transcriptional regulation of KLF2 in endothelial cells. (Adapted from Jain, 201050)

3.6.4 Cytokine-mediated Inhibition

An important observation is that many proinflammatory stimuli inhibit KLF2 expression in ECs. Kumar et al. determined that TNF inhibition of KLF2 is mediated by both the NF- B and histone deacetylase (HDAC) pathways83. A combination of promoter deletion and mutational analysis, coimmunoprecipitation and

15 chromatin immunoprecipitation assays revealed that HDAC4/5 and p65 (a component of NF- B) can form a tri-mer complex with MEF2 factors on the KLF2 promoter and can inhibit the ability of MEF2 to induce KLF2 expression. This work suggests that endothelial activation by inflammatory cytokines is potentially mediated by reduction in KLF2 activity, thus leading to unopposed NF- B activity and its subsequent deleterious effects.

4. AMPK, an energy sensor in vascular biology

AMPK has long been recognized as a metabolic switch that senses and regulates

the cellular energy homeostasis84. Mammalian AMPK is a trimeric enzyme

composing a catalytic α subunit and regulatory β and γ subunits. AMPK activation

requires the phosphorylation of Thr172 at the catalytic α-subunit by an upstream

AMPK kinase85. Under several physiological and pathological conditions, such as

exercise, hypoxia, and nutrient depletion, an increased AMP-to-ATP ratio rapidly

activates AMPK by LKB1 (encoded by the Peutz-Jegher tumor suppressor gene)86.

AMPK can also be activated by CaMKK with changes in the intracellular level of

Ca2+, which is independent of elevated AMP/ATP ratio87, 88. Such an activation

“switches on” ATP-generating catabolic processes such as glycolysis and fatty acid

oxidation but “switches off” ATP-consuming anabolic pathways, such as fatty acid

and protein synthesis, to restore the energy balance in the cell86, 89, 90. Recent studies

indicated that AMPK, by activating eNOS and leading to the NO production, may

also play an important role in vascular biology. Zhang et al. demonstrated for the

16 first time that shear stress activates AMPK in ECs, then induced not only eNOS

phosphorylation at Ser 1179 but also eNOS expression with increased NO release25. A more recent study in our lab showed that AMPK also can phosphorylate eNOS at

Ser-633 constantly in response to laminar shear stress91. Furthermore, several

widely used pharmacological agents, including statins and metformin, have been

shown to activate AMPK-eNOS signaling in ECs92, 93. Considering the critical role of

AMPK and KLF2 in EC function, I hypothesize that AMPK may mediate shear

stress-induced KLF2/eNOS expression. This hypothesis was tested in the first

project of this dissertation. The experimental approaches and results are presented in

Chapter 2.

5. Flow regulates KLF2 at the post-transcriptional level via miRNAs

Although both atheroprotective flow and statins could induce KLF2 transcription

to a similar level, flow is much more potent to enhance the levels of the KLF2

downstream targets eNOS and TM71. One of the explanations is that shear stress,

but not statins, increases the stability of KLF2 mRNA, thus resulting in increased

KLF2 protein expression and concomitant strong induction of KLF2 downstream

targets. The molecular basis of such an increased stability of KLF2 mRNA remains

elusive.

Recently, microRNAs (miRNAs) have emerged as important regulators at the

posttranscriptional level. MiRNAs are short (20–23 nt), endogenous, single-stranded

RNA molecules that regulate gene expression by degradation of mRNA and

17 repression of translation94. In humans, miRNAs are predicted to control the activity

of more than 60% of all protein-coding genes and affect many cellular pathways, including development, cancer and cardiovascular disease95. In this section, I

summarize the miRNA biogenesis, the repression mechanism of miRNAs on targets,

and the function of argonaute (Ago) proteins in the miRNA-induced silencing

complex (miRISC). Specifically, I discuss the regulation and function of the

miR-17~92 cluster.

5.1 miRNA Biogenesis

According to the genomic organization and the regulation of transcription,

miRNAs are separated into intronic and intergenic miRNAs96. Intronic miRNAs are in the intron of encoding genes, which are expressed with their host genes, spliced out of the transcript and further processed into mature miRNAs97. Intergenic miRNAs

are in the non-coding or the intron region of a given gene but in the opposite

orientation to the gene transcript96. These miRNAs have their own promoter region

and are transcribed independently. In animals, miRNAs are frequently transcribed

together as polycistronic primary transcripts that are processed into multiple

individual mature miRNAs98. The genomic organization of these miRNA clusters is

often highly conserved, which suggests an important role for coordinated regulation

and function.

18 Most mammalian miRNAs are transcribed by RNA polymerase II (pol II),

which generate a primary miRNA (pri-miRNA) transcript that contains one or more hairpin structures, each composed of a stem and a terminal loop99, 100. In the nucleus,

the pri-miRNA is cleaved into a 70-nt hairpin-structured precursor (pre-miRNA) by

Drasha (an RNase III enzyme) and its cofactor DGCR8101. Pre-miRNAs, which fold

into stem-loop structures, is recognized by exportin 5 (Exp5) (the nuclear export factor) and then trafficked from the nucleus to the cytoplasm102. In the cytoplasm,

pre-miRNA hairpin is recognized by Dicer, another RNase III-like endonuclease, and

cut the loop end off, thus creating an 18- to 24-nt RNA duplex103. Subsequently, the

miRNA duplex is incorporated into the miRNA-induced silencing complex (miRISC), the final effector104. Once loaded, one strand of the duplex, which is relatively

thermodynamically stable, remains as a mature miRNA (the guide strand or miRNA),

whereas the other strand (the passenger strand or miRNA*) is degraded. Typically, the

miRNA strand, whose 5’ end is less stably base-paired, will be more frequently

chosen as the guide.

5.2 Principles of target reorganization in animals

Once the miRISC is assembled, the miRNA guides the complex to its target by

base-pairing with the target mRNA. In plants, miRNAs bind to a generally perfectly

complementary site in either the coding or 3’-untranslated regions (UTRs) of the

target mRNA104. However, most animal miRNAs bind to multiple, partially

complementary sites in the 3’-UTR of targets105. The most stringent requirement is a

19 perfect match of the nucleotides 2–8 in the 5’ end of the miRNA, which represent the

“seed sequence”. In addition, an A residue across position 1 of the miRNA and an A

or a U across position 9 improve miRNA activity, although they do not need to

base-pair with miRNA nucleotides. Complementarity of the miRNA 3’ half is quite

relaxed, though it stabilizes the interaction. Generally, miRNA–mRNA duplexes

contain mismatches and bulges in the central region, which prevent endonucleolytic

cleavage of mRNA by an RNAi mechanism.106 Furthermore, an AU-rich

neighbourhood, the position at each end of 3’ UTR and longer 3′ UTRs of mRNA

increase the chance of miRNA binding. These factors can make the 3′ UTR regions less structured and hence more accessible to miRISC recognition. In addition, multiple sites for the same or different miRNAs are generally required for effective repression. When miRNAs are tethered by binding to the closed sites (within 10-40 nt), they tend to act cooperatively. Therefore, their effect exceeds that expected from the independent contributions of two single sites.107

5.3 Repression mechanism

The molecular mechanism underlying miRNA-mediated silencing is still not

entirely clear. A large number of studies showed that miRNAs inhibit translation or

destabilize mRNA by deadenylation and degradation. In the following section, I

introduce these repression mechanisms.

20 5.3.1 mRNA cleavage mediated by Ago2-associated RISC

Both the siRNAs and miRNAs, when perfectly base-paired to their target mRNA,

could direct cleavage of the target mRNA. This cleavage is a result of the ‘‘Slicer’’

activity in the RISC. Argonate protein, which contains the signature PAZ and PIWI

domains, is the most important and best-characterized components of miRISC.108

Structural studies revealed that miRNAs can be limited between the PAZ and PIWI domains, thus positioning the target mRNA closed to the catalytic center.

Mammals contain 4 AGO proteins, Ago1 to Ago4. Although all human Ago proteins bind both miRNAs and siRNAs, only Ago2-containing complexes can cleave mRNA targets109. The mammalian miR-196 has a near-perfect complementary

sequence in the 3′ UTR of the Hoxb8 mRNA and leads to direct mRNA cleavage and

degradation of the target mRNA110. All the other mammalian miRNAs are believed to

base-pair to their targets at partially complementary sites and inhibit protein

accumulation.

5.3.2 Repression of translation

At the beginning of the mammalian miRNA study, many reports showed that

most of miRNAs are partially complementary to their multiple target mRNAs, thus

resulting in the inhibition of protein accumulation without strongly affecting mRNAs.

The repression of translation is the predominant effect of miRNA silencing.

miRNAs could repress the initiation of translation by recruiting proteins that

interfere with the translational initiation factor eIF4E–eIF4G interaction or bind

21 directly to the m7GpppN cap, which inhibits the assembly of the 40S initiation

complex and the joining of the 60S subunit107, 108. In addition, miRNAs also could

decelerate translation elongation or induce the ribosome drop off to terminate the

translation early. Some studies also showed that the synthesized protein could be

rapidly degraded by proteases recruited by miRISC111.

5.3.3 Compartmentalization of miRNA repression

P-bodies are now known to be temporary sites of storage for repressed mRNAs in

yeast and mammals112, 113. AGO proteins, miRNAs and mRNAs repressed by

miRNAs are all enriched in P-bodies, which suggests that P-bodies function in miRNA repression and in the fate of repressed mRNAs. Within the P-bodies, miRNA targets are sequestered from translational machinery and are subjected to mRNA degradation. This repression is reversible depending on different environment and stimuli. For example, the endogenous cationic amino acid transporter 1

(CAT-1) mRNA, a target of miR-122, localizes to P-bodies when translation is repressed but not when it is reversed by stress.114

5.3.4 mRNA deadenylation and decay

Although initial studies suggested that the levels of mRNAs remain mostly

unchanged with the miRNA effect, more recent work has demonstrated that miRNAs

can induce pronounced target mRNA degradation115. Likewise, microarray studies

of mRNA levels in cells and tissues with altered miRNA levels revealed marked

22 changes in the abundance of dozens of validated or predicted miRNA targets, consistent with a role for miRNAs in mRNA destabilization105.

Fig. 1-5. The mechanism of miRNA-mediated translational repression in animal cells. The miRISC–mRNA interaction can lead to several modes of direct translational repression. (a) Initiation block: The recruitment of 40S and/or 60S ribosomes near the 5′ cap of mRNA is inhibited. (b) Ribosomal dropoff: The 40S/60S ribosomes are dissociated from mRNA. (c) Stalled elongation: The 40S/60S ribosomes are prohibited from joining during the elongation process. (d) The indirect translational repression by mRNA degradation and sequestration in P-bodies. (Adapted from Sun, 2010116) In eukaryotes, mRNA degradation has two pathways, each of which is initiated by a gradual shortening of the mRNA poly(A) tail. The mRNA body can then be degraded by progressive 3′→5′ decay, which is catalyzed by the exosome or by the removal of the 5’ cap followed by 5′→3′ degradation, which is catalyzed by the exonuclease117. These decay machinery components are enriched in P-bodies, where miRISC is stored. For example, the P-body protein GW182, which is a key factor

23 that marks mRNAs for decay, could interact with Ago1 in mammals and worms.

GW182 depletion leads to an upregulation of many mRNA targets that are also

upregulated by Ago1 depletion.118 Depletion of the components of the CCR4–NOT deadenylating complex prevents the decay-promoting activity of GW182, which suggests that GW182 recruits CCR4–NOT to repressed mRNAs.

5.3.5 Translation repression, mRNA destabilization, both?

The exact molecular basis underlying miRNA-mediated gene silencing is not entirely clear. Many examples showed endogenous mammalian mRNAs that are regulated by miRNAs at the level of translation without change in target mRNA levels. Many examples show that the target mRNA levels were altered. In this context, translational repression may be the primary event, with any reduction in mRNA levels a consequence of translational repression and subsequent relocalization to P-Bodies. In one of the most recent studies, ribosome profiling was used to measure the overall miRNA effects on protein production and compare these to simultaneously measured effects on mRNA levels.119 The lowered mRNA levels account for most (≥84%) of the decreased protein production. These results indicated that destabilization of target mRNAs is the predominant reason for reduced protein output.

Whether Ago1 or Ago2 is more important for miRNA function in mammals is still unclear. Among the Ago family members, Ago1 mediates the miRNA-induced translational inhibition and deadenylation and 5'→3′ decay. Neither of the cases

24 requires a perfect match between miRNAs and the 3’ UTR of the targeted mRNAs.

However, Ago2 modulates mRNA degradation, a process that requires a near-perfect

complementary sequence in the 3′ UTR of the target.117, 118 Recently, a chaperone

model suggested that Ago1 facilitates the association of the guide strand of the

miRNA with the target mRNA and then Ago2 is recruited to the miRNA–mRNA

complex.120 Even with no perfect match between the miRNA and the target mRNA,

Ago2 still could incorporate to miRISC and degrade mRNA. In the chapter 3, I

describe the function of Ago1 and Ago2 in regulation.

6. miRNA and vascular biology

6.1 The role of miRNAs in endothelial cells

The importance of miRNAs in endothelial function was first revealed by

disrupting the function of Dicer, a key enzyme for miRNA biogenesis121, 122.

Dicer1-null embryonic stem (ES) cells are unviable, and the homozygous Dicer knockout mouse is early embryonic lethal123, 124. Dicer hypomorphic mice have

defects in vascular remodeling during development or ovary angiogenesis125.

EC-specific deletion of Dicer in mice provided direct in vivo evidence that endothelial miRNAs are required for postnatal angiogenesis in response to angiogenic stimuli, including VEGF, tumors, limb ischemia, and wound healing121. Knockdown of Dicer

in vitro in human ECs results in decreased angiogenesis and EC proliferation and impaired morphogenesis121. These situations are due to upregulation of

25 thromobospondin-1 (Tsp-1), an inhibitor of angiogenesis, as well as other key

regulators of endothelial function in ECs125. Tsp-1 is a predicted target of the let-7

family and miR-17-92 cluster. The transfection of the miR-17-92 cluster rescued the

Tsp-1 expression and EC proliferation.

miRNA profiling revealed about 40 miRNAs that are highly expressed in human

umbilical vein ECs (HUVECs), including miR-221/222, miR-21, the let-7 family, the

miR-17-92 cluster and miR-126121, 122, 126. These miRNAs provide a new layer of

information on the regulation of multiple EC functions (see Table 1-1). For example,

miR-126 is the only EC-specific expressed miRNA and the first vascular miRNA to

be knocked out in mice127. The deletion of miR-126 caused leaky vessels and partial

embryonic lethality because of loss of vascular integrity and defective angiogenesis128.

MiR-126 null mice also showed reduced survival and defective neovascularization after myocardial infarction. The proangiogenic function of miR-126 is mediated by promoting mitogen-activated protein kinase (MAPK) and PI3K signaling in response to VEGF, through targeting negative regulators of these signaling pathways, including

Spred-1 and PI3K regulatory subunit2 (PI3KR2)129. Besides Spred-1 and PI3KR2,

miR-126 also targets vascular cell adhesion protein 1 (VCAM-1), thereby regulating the adhesion of leukocytes to the endothelium, which suggests a role of miR-126 in vascular inflammation128.

26 Table 1-1. The miRNAs and vascular function

Vascular function miRNAs Targets Ref. miR-17-92 Tsp1, CTGF 130, 131 miR-27a ZBTB10, Tsp1 132 miR-130a GAX, HOXA5 133 miR-378 Fus-1 134 Angiogenesis miR-210 EPHA3, eNOS 135 miR-221, miR-222 c-kit, eNOS 136, 137 Let-7f 136 miR-27b 136 miR-126 SPRED1 126 Vasoconstriction Intron-based miRNAs eNOS 138-140 Vascular integrity miR-126 SPRED1 128 Cell adhesion miR-126 VCAM1 141 Inflammation Cell migration mIR-126 prostein 142 Tumor invasion Pro-injury Cell proliferation miR-21 Bcl-2, PTEN 131 and survival miR-155 AT1R 143 Hypertension miR-208 144 Plasma cholesterol miR-122 HMGCR 145

6.2 Shear stress and miRNAs

The endothelial function response to shear stress is regulated by transcription and

signaling networks and also miRNAs. Recently, shear-regulated miRNA profiles of

ECs were reported by 3 different groups. Wang et al. compared the miRNA profile of HUVECs under 24-hr PS to that of the static condition146. Eight miRNAs were

upregulated (including the miR-23b cluster) and 13 miRNAs were downregulated

(including miR-17–92, miR-16, and miR-221 clusters) in ECs in response to PS.

Specifically, miR-23b mediated shear-induced cell arrest by regulating the expression

27 level of and retinoblastoma (Rb) hypophosphorylation. Qin et al. reported that

35 miRNAs were significantly upregulated and 26 miRNAs were significantly

downregulated in 12-hr laminar-flow–treated HUVECs as compared with static

control cells147. Specifically, the upregulated miR-19a regulated the inhibition of EC

proliferation by targeting cyclin D response to steady laminar flow. In Weber et

al.’s study, 13 miRNAs were upregulated in HUVECs exposed to prolonged

unidirectional shear stress (USS, 24 h, 15 dyn/cm2)148. The miRNA with the greatest change was miR-21, which directly targets PTEN. HUVECs overexpressing miR-21 showed decreased apoptosis and increased eNOS phosphorylation and nitric oxide (NO ) production131.

Recently, Fang et al. reported differential endothelial miRNA expression in vivo at athero-susceptible and -protected regions of the aorta and renal arteries in normal adult swine149. The authors identified 7 downregulated and 27 upregulated miRNAs in athero-susceptible aortic arch (AA) endothelia as compared with athero-protected

dorsal descending thoracic aorta (DT). miR-10a, the miRNA most significantly

downregulated by athero-susceptible flow, despressed its targets MAP3K7 and βTRC

and then upregulated IκB/NF-κB–mediated inflammation in this area. This study is

the first to link miRNA regulation in arterial endothelium with endothelial

athero-susceptibility or -protection signatures in vivo. Interestingly, the profile in

this study also showed miR-92a is upregulated by athero-susceptible flow. This result

is consistent with my second project described in Chapter 3.

28 7. miR-17-92 cluster

The miR-17-92 cluster, the first set of miRNAs, are commonly overexpressed in human tumors and have been implicated in the control of tumor growth, survival and angiogenesis. Recently, some studies indicated that this cluster also plays an important role in the regulation of endothelial function. In the following section, I summarize the genome organization, regulation and function of this cluster.

7.1 Genome Organization of the miR-17-92 cluster

The miR-17-92 cluster is a polycistronic miRNA gene within an 800-bp region located in the third intron of a primary transcript (C13orf25) on human chromosome 13133. This cluster encodes 7 individual mature miRNAs (miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a). Both the sequences of these mature miRNAs and their organization are highly conserved in all vertebrates. The mammalian genome has two miR-17-92 cluster paralogs because of ancient gene duplications. The miR-106b-25 cluster is located on human and the miR-106a-363 cluster on the X chromosome. Each cluster contains homologous miRNAs to a subset of miR-17-92 components. According to their seed sequence, these mature miRNAs can be categorized into 4 miRNA families: the miR-17 family (miR-17, miR-20a/b, miR-106a/b, and miR-93); miR-18 family

(miR-18a/b); miR-19 family (miR-19a/b); and miR-25 family (miR-25, miR-92a and miR-363)134.

29

Fig. 1-6. Organization of the miR-17-92 cluster and its paralogs. (A) The genomic organization and primary transcript structures of the human miR-17-92, miR-106a-363, and miR-106b-25 clusters. (B) From their seed sequences, the miRNAs of these clusters can be grouped into 4 families: the miR-17 family (miR-17, miR-20a/b, miR-106a/b, and miR-93); miR-18 family (miR-18a/b); miR-19 family (miR-19a/b); and miR-25 family (miR-25, miR-92a, and miR-363). (Adapted from Mendell, 2008150)

7.2 The regulation of the miR-17-92 cluster

The miR-17-92 cluster was first identified as a potential oncogene because of its

genomic amplification and elevated expression in B-cell lymphoma cell lines151.

The following studies showed the enhanced expression of the miR-17-92 cluster in multiple hematopoitic malignancies and solid tumors152. In addition to being

expressed in cancer cells and tissue, miR-17-92 is upregulated in ischemic muscle or

the heart and induced by pro-inflammatory cytokines such as interleukin 6 (IL-6) in

ECs153.

30 Expression profile studies, miRNA functional screens, and computational

analyses demonstrated that the miR-17-92 cluster is a key component of the

oncogenic and tumor suppressor network. The first evidence came from O’Donnell,

who demonstrated that c- stimulates the transcription of the cluster in human

B-cells and chronic myeloid leukemia cells154, 155. The oncogenes c-myc and n-myc

are well known to regulate cell proliferation, growth and apoptosis, which is

consistent with the function of miR-17-92 in promoting proliferation. In addition to

myc, the family, AML1 and Cyclin D1 are targeted by miR-17 and miR-20a and

in turn can bind to the promoter region of miR-17-92, thereby establishing a negative

feedback loop (Fig. 1-7)147, 156, 157. In contrast, a recent study indicated that

could inhibit the expression of the miR-17-92 cluster and then promote apoptosis in a

colon cancer cell line under hypoxia158. p53 blocks the initiation of transcription

because its binding site is overlapped with a TATA-box within

the miR-17-92 promoter. Moreover, STAT3 could bind to the promoter region of the miR-17-92 cluster and then miR-17-5p and miR-20 target on bone morphogenetic protein receptor type II (BMPR2), which promoted cell survival in HPAEC under pulmonary hypertension or stimulated the pro-imflammatory cytokine IL-6153.

31 p53

Fig. 1-7. The transcriptional regulation of the miR-17-92 cluster. (Adapted from Dimmeler, 2009159)

7.3 MiR-92a and EC function

Although miR-17-92 is frequently overexpressed in different types of tumors, no evidence exists of the direct effect of miR-92a on cell survival in the context of cancer.

Moreover, crucial studies showing that the miR-17-92 cluster with a truncated cluster lacking miR-92a suggested that miR-92a does not necessarily augment lymphoma growth133. In ECs, overexpression of miR-92a does not change EC apoptosis and

proliferation160.

32 To date, only one group reported miR-92a function in cardiovascular system160.

Bonauer et al. demonstrated that miR-92a negatively regulates angiogenesis of ECs in vitro and in vivo by loss- and gain-of-function experiments. Since miR-92a was significantly upregulated after induction of ischemia in animal models in vivo, the authors used antigomir, the modified antasense oligo of miR-92a, to determine the therapeutic effect of miR-92a inhibition. Indeed, systemic inhibition of miR-92a improved the functional recovery in models of hind-limb ischemia and acute myocardial infarction. By using mRNA expression array, the authors found the changed expression of several genes related to endothelial function. Specifically, the intergrin subunit 5 (ITGA5) was identified as a direct target of miR-92a in ECs; miR-92a is a crucial regulator of EC functions and vessel growth by mediation of cell–matrix interaction, anti-apoptotic signaling and cell migration. Interestingly, miR-92a overexpression in HUVECs suppressed the expression of several

KLF2-regulated genes such as eNOS and TM160. The lack of miR-92a binding sites

in the 3’UTR of these genes suggests the involvement of a mechanism other than direct targeting of miR-92a. In chapter 3, the role of miR-92a in shear-regulated EC function is described.

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56 Chapter 2

Flow Activation of AMP-activated Protein Kinase Leading to The

Expression of Krüppel-like Factor 2

2.1 Abstract

Objective: Vascular endothelial cells (ECs) confer atheroprotection at locations of the arterial tree where pulsatile laminar flow (PS) exists with a high shear stress and a large net forward direction. We investigated whether the PS-induced expression of the transcription factor Krüppel-Like Factor 2 (KLF2) in cultured ECs and mouse vessel

wall is regulated by AMP-activated protein kinase (AMPK).

Methods and results: the AMPK inhibition by siRNA had a significant blocking effect on the PS-induced KLF2 expression. The induction of KLF2 by PS led to augmentation of eNOS and suppression of ET-1, which can be reversed by KLF2 siRNA. In addition, PS induced the phosphorylation of ERK5 and MEF2, which increased the KLF2 expression. These mechanotransduction events were abrogated by the blockade of AMPK. Furthermore, the phosphorylation levels of ERK5 and MEF2, as well as the expression of KLF2, were significantly reduced in the aorta of

AMPKα2 knockout mice when compared with wild-type control mice.

57 Conclusion: The flow-mediated AMPK activation is a novel KLF2 regulatory

pathway in vascular endothelium that acts via ERK5/MEF2.

2.2 Introduction

Vascular endothelial cells (ECs) in the arterial tree are subjected to shear stress

resulting from blood flow. ECs exposed to laminar flow patterns are spared from early

lesions of atherosclerosis. In contrast, ECs at the arterial bifurcations and curvatures,

where disturbed flow patterns exist, are susceptible to the development of

atherosclerotic lesions. There is ample evidence indicating that the different patterns

of shear stress associated with laminar vs. disturbed flows play a significant role in

regulating endothelial phenotype and vascular homeostasis.

Krüppel-like factor 2 (KLF2) is a transcription factor whose expression is

flow-dependent in vitro and in vivo and has been shown to regulate various EC

functions related to inflammation, thrombosis, proliferation, and vascular tone1-3.

KLF2 belongs to the family of KLF zinc-finger transcription factors that are important regulators of cell differentiation and development4. Using microarray analysis, Dekker

et al. demonstrated that KLF2 is upregulated by prolonged laminar shear stress and

that it is expressed in ECs of atherosclerosis-resistant regions of the human aorta1.

58 Subsequent work by J. Lingrel and colleagues demonstrated that laminar shear stress

induces transcriptional activation of KLF2 in ECs5. Our previous studies showed that

KLF2 was induced by the atheroprotective flow, but suppressed by atheroprone

flow3,6. KLF2 has been implicated in mediating the anti-inflammatory effects of flow,

presumably by inhibiting pro-inflammatory transcription factors, such as ATF27, AP-1, and NFκB8, which in turn regulate gene expression.

Functioning as a “fuel gauge” in multiple organ systems, AMPK is also

important in the vessel wall. Nagata et al. showed that hypoxia activates AMPK in human umbilical vein ECs (HUVECs), as indicated by increased phosphorylation at

Thr-172 of the AMPKα subunit9. The suppression of AMPK signaling by a dominant-negative mutant of AMPK (AMPK-DN) inhibited vascular endothelial growth factor (VEGF)-enhanced EC migration and hypoxia-induced differentiation into tube-like structures. Notably, AMPK phosphorylates eNOS at Ser-1177/1179 and

thereby augments the eNOS-derived NO bioavailability10-14. We have previously demonstrated that shear stress regulates AMPK in ECs and that this may account for the NO bioavailability and cell cycle arrested in G0/G1 under steady laminar flow15,

16.

59 Based on the findings that both AMPK and KLF2 are regulated by atheroprotective flow and both have similar effects on EC biology, here we investigate whether AMPK activation is functionally linked to KLF2 expression in the vascular endothelium.

2.3 Materials and Methods

2.3.1 Cell culture and reagents

Human umbilical cord venous endothelial cells (HUVECs) were isolated from human umbilical cord veins with collagenase as previously described17. The cells were cultured on plates coated with collagen I (BD Biosciences, Bedford, MA) and maintained in medium M199 (Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum (Omega, Tarzana, CA), 25% endothelial cell growth medium (Cell

Applications, San Diego, CA), 2 mM L-glutamine, 1 mM sodium pyruvate, and 1%

penicillin/streptomycin. All cell cultures were kept in a humidified 5% CO2-95% air incubator at 37˚C. Cells within passages 2-6 were used in the experiments.

Antibodies used in this study were purchased from the following commercial sources: anti-phospho-AMPKα Thr-172, anti-AMPKα, anti-phospho-ACC Ser-79, and anti-ACC (Cell Signaling Technology, Beverly, MA); anti-phospho-MEF2

Thr-312 (Abcam, Cambridge, MA), anti-phospho-ERK5 Thr-218/Tyr-220 (Millipore,

60 Billerica, MA) anti-β actin (Sigma, St. Louis, MO): anti-α tubulin (Sigma, St. Louis,

MO). The anti-KLF2 antibody was obtained by immunization of the N-terminal region (aa 1-245) of human KLF2 fused to Fc. Serum was tested for KLF2 reactivity, and hybridoma supernatants were tested by ELISA assays. The specificity of antibody was tested by immunoblotting lysates collected from HEK293 cells transfected with cDNAs encoding KLF2, KLF4, KLF6, and KLF13. Immunoreactivity was detected only in cells transfected with KLF2. Compound C was from Calbiochem (La Jolla,

CA). The siRNA targeting KLF2 sequence [nucleotides 1482-1502

(AATTTGTACTGTCTGCGGCAT) of the cDNA (GenBank Accession No.

NM_016270)] was custom synthesized by Ambion (Austin, TX). The siRNA targeting

AMPK-α1 and AMPK-α2 catalytic-subunits (Hs_PRKAA1_5_HP, Hs_PRKAA2_6

HP Validated siRNA) were purchased from QIAGEN (Valencia, CA). A negative control siRNA (Silencer Negative Control #1) and a positive control siRNA (Silencer

FAM labeled GAPDH siRNA) for monitoring siRNA delivery efficiency were purchased from Ambion.

2.3.2 Flow experiments

A circulating flow system, previously described, was used to impose shear stress on a confluent monolayer of HUVECs seeded on a collagen I-coated glass slide

61 (75×38 mm)18. A reciprocating syringe pump was connected to the circulating system to introduce a sinusoidal component (frequency = 1 Hz) onto the shear stress. The flow system was kept at 37˚C in a temperature-controlled hood, and the circulating

medium was ventilated with humidified 5% CO2-95% air. Pulsatile shear flow (PS) was applied to cells with a shear stress of 12 ± 4 dyn/cm2.

2.3.3 RNA isolation, cDNA synthesis, and real-time PCR

Total RNA was isolated with the use of Trizol reagent (Invitrogen). Reverse transcription was carried out with 3 μg of total RNA by the Superscript II reverse transcriptase (Invitrogen). The synthesized cDNA was used to perform real-time quantitative PCR (qPCR) with the iQ SYBR Green supermix (Bio-Rad, Hercules, CA) on the iCycler real-time PCR detection system (Bio-Rad). The sequences of primer sets were: KLF2, AGACCTACACCAAGAGTTCGCATC and

CATGTGCCGTTTCATGTGCAGC; eNOS, TGGTACATGAGCACTGAGATCG and

CCACGTTGATTTCCACTGCTG; AMPK, GAATGGAAGGCTGGATGAAA and

TTCTGGTGCAGCATAGTTGG; endothelin-1 (ET-1),

TCCTCTGCTGGTTCCTGACT and CAGAAACTCCACCCCTGTGT; GAPDH,

ATGACATCAAGAAGGTGGTG and CATACCAGGAAATGAGCTTG.

62 2.3.4 Protein isolation and immunoblotting

HUVECs were washed with ice-cold PBS twice and lysed with RIPA buffer

(1% Igepal CA-630, 0.5% sodium deoxycholate, and 0.1% SDS in 1X PBS) containing 100 μg/mL phenylmethanesulfonyl fluoride (PMSF), 1 mM sodium

orthovanadate (Na3VO4), 2 mM sodium fluoride (NaF), and 150 KIU/mL aprotinin.

Proteins for in vivo experiments were isolated from the aorta harvested from control

(C57BL/6J) or AMPKα2-knockout mice. Equal amounts of denatured protein lysates

were separated on SDS polyacrylamide gels and transferred to a nitrocellulose

membrane (Bio-Rad, Hercules, CA). After 1-hr blocking with 5% bovine serum

albumin (BSA) in 1X TBST, the membrane was probed with various primary

antibodies and the appropriate secondary antibodies conjugated with horseradish

peroxidase (HRP), followed by ECL detection (GE Healthcare, Piscataway, NJ). The

density of the protein bands was quantified by using the ImageJ software (National

Institutes of Health).

2.3.5 siRNA transfection

HUVECs were transfected with siRNA using the siPORT NeoFX transfection

reagent (Ambion). The cells were incubated with the siRNA-transfection reagent

complex in 2 mL of culture media on a glass slide (for flow experiments) for 12 hr.

63 Fresh culture media were then added and replaced again at 24 hr. Twenty-four or 48

hr post-transfection, the cells were used in PS experiments.

2.3.6 Animal Experiments

All experiments were performed according to institutional protocols

(University of California) using 8-week-old male mice. After euthanization, the abdominal aorta was harvested from C57BL6J wild-type mice and AMPKα2-/- mice19

(a gift from Dr. B. Viollet in Institute Cochin, Université Paris 5). Aortas were then homogenized, and two aortic extracts were pooled to yield one sample, which was

then analyzed for protein expression and phosphorylation by immunoblotting. Three

independent experiments were performed (six animals total) for each study.

MEF2 en face staining was performed according to protocols previously

published16. The aortas used were isolated from 10-week-old male mice. The primary antibody was rabbit anti-phospho-MEF2 antibody (Abcam, Cambridge, MA) and the

second antibody is Alexa 647-labeled goat anti-rabbit IgG (Invitrogen, San Diego,

CA). Rabbit IgG was used as negative control. The quantitative analysis was

performed using LCS Lite software (version 2.0).

64 2.3.7 Statistical analysis

All data were analyzed by Student’s t test (for paired testing between two

groups used only once) or two-way ANOVA (for testing of multiple groups). Results

are expressed as mean±SD from at least three independent experiments. P values <

0.05 were considered to be statistically significant.

2.4 Results

2.4.1 Pulsatile shear flow activates AMPK and induces KLF2 expression in ECs

We have previously shown that a laminar steady flow activates AMPK in

bovine aortic endothelial cells (BAECs)15, 16. Here, we investigate the effect of PS in

regulating AMPK activities in HUVECs. As shown in Fig. 2-1A, PS with a shear stress of 12 ± 4 dyn/cm2 and a frequency of pulsatility at 1 Hz increased the

phosphorylation of AMPK Thr172 and its target ACC Ser79 as early as 5 min. The increased phosphorylation of AMPK and ACC peaked at 1 hr and remained elevated for at least 4 hr. The elevation could lasted for 16 hr (data not shown). Thus, in contrast to the transient AMPK activation under steady flow in BAECs15, 16, the

phosphorylation of AMPK under PS was sustained in HUVECs (Fig 1A). Under the same experimental conditions, PS also caused the induction of the KLF2 gene at both mRNA and protein levels, as determined by real-time qPCR and Western blot analysis,

65 respectively. As seen in Figure 1B, consistent with the time course of PS-mediated

AMPK activation, the increase in KLF2 mRNA began 1 hr after the initiation of PS

and remained at a high level for more than 4 hr. Concomitantly, there was an increase

in KLF2 protein expression 4 hr after the initiation of PS (Figs. 1C).

2.4.2 AMPK regulates KLF2 expression in response to PS

Since both AMPK and KLF2 play important roles in flow-regulated eNOS

activities/expression, we investigated whether AMPK activation is critical for the

induction of KLF2 under PS. We inhibited AMPK activity by treating ECs with

Compound C (an AMPK antagonist) or knocked down AMPK expression using

siRNA. The levels of KLF2 expression in control and treated cells exposed to PS or

maintained under static (no flow) conditions were then assessed. As shown in Fig.

2-2A, Compound C reduced basal expression of KLF2 in static cells, and abolished

the shear-induction of KLF2 expression at 4 hr. The negative regulation of KLF2 by

Compound C was concentration-dependent (supplementary Fig. 2-S1). Similar to the

effect of Compound C, siRNA knockdown of either AMPKα1 or AMPK α2 significantly reduced the 4-hr shear-induction of KLF2, with a marginal decrease in the basal expression of KLF2 (Fig. 2-2B,C). These results confirmed that AMPK activation is critical for the induction of endothelial KLF2 in response to PS.

66 2.4.3 KLF2 regulates the eNOS and ET-1 expression in response to PS

In order to further delineate the effects of PS-induced KLF2 on its downstream

targets, we first determined the optimal siRNA concentration, which was able to

knockdown KLF2 expression in HUVECs with the efficiency of ~80% suppression

(supplementary Fig. 2-S2). We next examined the expression of two known targets

of KLF2, eNOS and ET-1. As seen in Fig. 2-2D, silencing of KLF2 expression led to

a reduced level of eNOS in static cells and also a significant decrease in eNOS

induction in ECs exposed to PS. Knockdown of KLF2 also resulted in the

abolishment of PS-suppression of ET-1, which remained at basal level in the presence

of PS (Fig. 2-2E). These results suggest that KLF2 acts as a key transcription factor

mediating the PS-dependent expression of eNOS and ET-1, two critical genes for the

maintenance of vascular homeostasis and vasomotor tone.

2.4.4 AMPK-KLF2 signaling in cultured ECs

To further elucidate the mechanism of KLF2 upregulation by AMPK,

HUVECs were treated with AICAR (an AMPK agonist), or infected with

Ad-AMPK-CA expressing a constitutively active form of AMPK. As shown in Fig.

2-3A, KLF2 expression increased significantly in ECs treated with AICAR and those infected with Ad-AMPK-CA as indicated by real-time Taqman PCR.

67 Since the MEK5/ERK5/MEF2 pathway regulates the flow-induced KLF2, and

the phosphorylation of MEF2 at Thr312 by ERK5 increases the rate of

MEF2-mediated transcription3, 20, we tested whether AICAR-induction of KLF2

mRNA is mediated by MEK5 and MEF2. As seen in Fig. 2-3B, C, the increase in

KLF2 in ECs treated with AICAR was abrogated by infection with Ad-MEK5-DN

(the dominant negative mutant of the ERK5-upstream kinase MEK) or Ad-MEF2-DN

expressing a dominant negative mutant of MEF2.

We then explored whether AMPK activation can increase the phosphorylation

of MEF2 through the MEK5/ERK5 pathway. The AICAR-induced phosphorylations

of AMPK Thr172, ERK5 Thr218, and MEF2 Thr312 were markedly decreased in ECs in which AMPKα1 was knocked down in comparison to those in cells treated with control siRNA (Fig. 2-4A). The knockdown of AMPKα2 also resulted in similar reductions (Fig. 2-4B). In consistent with these results, the AICAR-increased ERK5 phosphorylation was inhibited by Compound C (Fig. 2-S3A) and AD-MEK5-DN (Fig.

2-S3B). More importantly, Compound C treatment was able to inhibit the PS flow-increased AMPK activity and ERK5 and MEF2 phosphorylation (Fig. 2-4C).

These results established that the activation of KLF2 by AMPK is mediated through

ERK5 and MEF2 in cultured ECs.

68 2.4.5 AMPK-KLF2 signaling in vivo

To examine the role of the AMPK-KLF2 pathway in vivo, KLF2 expression

was studied in AMPK knockout mice. AMPK α2 seems to play a more important role

than AMPK α1 in fuel sensing and downstream signaling in vivo 19, 21. Although we

showed that both AMPK isoforms (α1 and α2) have similar effects on KLF2

expression in cultured endothelial cells, we studied the AMPK-KLF2 signaling in α2-/- mice due to in vivo results of α2-/- mice studies in fuel sensing and downstream

signaling are more robust 19, 21, as well as the availability of the α2-/- mice. To this end,

aortas were harvested from C57BL6 wild-type and AMPKα2-/- mice, protein lysates

were prepared and use for immunoblotting analysis to assess the expression levels of

KLF2 and its downstream target gene eNOS and the phosphorylation state of its

upstream regulators AMPK, ERK5, and MEF2. As shown in Fig. 2-5A, aortas from

AMPKα2-/- mice exhibited reduced expressions of KLF2 and eNOS when compared

to aortas from wild-type control mice. Moreover, aortas from AMPKα2-/- mice displayed a reduced phosphorylation level of AMPK, which was accompanied by decreases in ERK5 and MEF2 phoshorylations (Fig 5B). p-MEF2 en face immunostaining confirmed that the level of MEF2 phosphorylation in the thoracic endothelium of AMPKα2-/- mice was lower than that in the wild-type controls (Fig

69 5C). Collectively, these results indicate that AMPK is a regulator of KLF2 expression

in the vascular wall in vivo, and support our in vitro mechanistic studies.

2.5 Discussion

In order to elucidate the roles of wall shear stress in regulating KLF2 expression and modulating vascular homeostasis, we investigated additional mechanotransduction mechanisms by which shear stress upregulates KLF2 expression

in vascular ECs. Data collected from both in vitro and in vivo experiments revealed

that AMPK is an upstream kinase regulating the PS-induced endothelial KLF2 expression. Our results also link the AMPK regulation to the ERK5/MEF2/KLF2 pathway, thus providing a detailed mechanistic explanation of KLF2 induction by PS via AMPK, ERK5, and MEF2 signaling3.

AMPK, functioning as a cellular energy sensor, plays important roles in vascular

biology.20 Many stimuli such as hypoxia, estrogen, shear stress, adiponectin and statins can act on the vascular EC to activate AMPK, which in turn phosphorylates

eNOS Ser1177 to enhance the NO bioavailability in ECs10,14,15,22,23. KLF2, upregulated by the atheroprotective laminar shear stress, causes elevation of eNOS expression and integration of the flow-mediated endothelial atheroprotective phenotype1,3,24. The mechanism underlying the shear stress-induced KLF2 has been

70 investigated by several groups. Lingrel et al. showed the importance of a phosphoinositide-3-kinase (PI3K)-dependent/Akt-independent pathway in the activation of KLF2 by shear stress and the involvement of nucleolin5,25. Parmar et al.

showed that the flow-dependent activation of KLF2 is mediated via a

MEK5/ERK5/MEF2 signaling pathway, suggesting the involvement of MAPK family

in the regulation of KLF23. Van Thienen et al. demonstrated that shear stress sustain

KLF2 expression through mRNA stabilization via PI3K dependent pathway30.

However, Lingrel’s group demonstrated that KLF2 promoter can be transcriptional activated by shear stress, and the shear responsive element is located at position of

-195 to -95 bp in front of the transcription start site5. These results indicate the both

mRNA stabilization and transcription are important for the shear-regulation of KLF2 expression. Here, we identify the AMPK as a key upstream regulator for ERK5 signaling that leads to KLF2 expression under shear. In RAW 264.7 cells, the PI3K inhibitor Wortmannin has been shown to abolish the nicotine-induced AMPK

phosphorylation 26, indicating the potential interaction between PI3K and AMPK.

However, in our hands, blocking PI3K with Worthmannin or LY2943002 had little

effect on shear-induced AMPK activation and KLF2 expression (data not shown).

Further investigation will be needed to understand the role of PI3K in shear-regulated

71 AMPK-KLF2 pathway. It has been shown in ECs that AMPK is activated by two

kinases, Peutz-Jeghers syndrome kinase LKB1 and Ca2+/calmodulin-dependent

protein kinase kinase (CaMKK)27,28. Our earlier work15 identified LKB1 as the

upstream kinase causing AMPK Thr172 phosphorylation under steady shear, and we postulate here that PS activates AMPK via the same pathway. The mechanisms by which shear stress modulates the KLF2 expression, however, are complex and involve multiple signaling pathways.

Since both AMPK and KLF2 are regulated by shear stress and able to augment

nitric oxide production, the present study focused on elucidating the role of the

AMPK pathway in regulating the shear-induction of KLF2 expression. In contrast to

the previous observation that steady laminar shear stress led to a transient activation

(5-30 min) of AMPK in cultured BAECs16, we found that PS (12 ± 4 dyn/cm2 laminar

shear stress with 1 Hz pulsatility) caused a 4-hr sustained activation of AMPK in

cultured HUVECs, and such a sustained AMPK activation can last up to 16hr (data

not shown). Our studies of steady laminar flow in HUVECs also demonstrated a

sustained AMPK phosphorylation up to 16 hr (data not shown). The different of

shear-induced AMPK activation is likely due to the variations of endothelial cell

sources, bovine vs. human. In the current study, we identified the necessity of AMPK

72 for the shear-induced phosphorylation of ERK5 and MEF2, and expression of KLF2 and its downstream target genes. We also demonstrated that activation of AMPK (by

AICAR) is sufficient for the induction of KLF2 expression, as well as the phosphorylation of its upstream signaling molecules ERK5 and MEF2. These results indicate an important role of AMPK in modulating the shear-activation of the

ERK2/MEF2/KLF2 pathway, and establish a novel link between the AMPK and the

ERK5 signaling pathways.

To further validate this newly identified relationship between AMPK and

ERK5/MEF2/KLF2 pathway, in vivo experiments were performed. We investigated the expressions of KLF2 and the activity levels of ERK5 and MEF2 in AMPK α2 knockout (AMPKα2-/-) mice. While AMPK α1 is more abundant in ECs, α2 also plays an important role in EC functions, e.g. migration and tube formation29. Although our in vitro studies mainly addressed AMPKα1, it appears that AMPKα2 plays a similar role (see Figs. 2C and 3E). Since comparison of AMPK α1-/- versus α2-/- mice demonstrated that α2-/- plays more important roles in fuel sensing and downstream signaling in vivo19,21 and due to the availability of the α2-/- mice, we studied the AMPK-KLF2 signaling in the vessels of α2-/- mice. Our results demonstrated that KLF2 expression was significantly reduced and the

73 phosphorylations of ERK5, MEF2, and eNOS were greatly attenuated in the aortas of

AMPKα2-/- mice. Due to the EC-specificity of KLF2, we are confident that the

reduction aortic KLF2 expression in α2-/- mice occurs in endothelium. To further

demonstrate vascular location of the reduction of MEF2 activation, we performed en

face staining of phospho-MEF2 to confirm the reduction in aortic endothelium.

In summary, this study suggested AMPK is an upstream signaling molecule of

the ERK5/MEF2/KLF2 pathway, and documented the requirement of AMPK for the

activation of ERK5/MEF2 signaling pathway and the expression of KLF2 in the mouse vasculature. Our findings established that the PS-activation of the

AMPK/ERK5/MEF2/KLF2 pathway plays an important role in the regulation of vascular homeostasis.

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80 Figure 2-1

Fig. 2-1. PS activates AMPK and induces KLF2 in cultured ECs. Confluent HUVECs were exposed to PS for the indicated times. Static controls are represented as “time 0”. The mean shear stress was 12 dyn/cm2 (represented by dash line) with ± 4 dyn/cm2 oscillation at 1 Hz. (A, C) ECs were lysed, the lysates were separated by SDS-PAGE, and the membranes were probed with antibodies as indicated. (B) RNA samples were isolated, and the levels of KLF2 mRNA were determined and quantified by real-time PCR, with the results normalized by GAPDH. The quantitative graphs below show the ratios of phosphorylated AMPK or ACC to total AMPK or ACC, respectively, in (A), KLF2 mRNA to GAPDH mRNA in (B), and KLF2 to α-tubulin in (C) at various time points. The shear results were normalized by that of the corresponding static control. The data represent mean ± SD from 3 independent experiments. * indicates p<0.05 between the two groups being compared.

81 Figure 2-2

Fig. 2-2. AMPK regulates the PS-induced KLF2 and its target genes. HUVECs were treated with Compound C (15 µM) for 30 min (A), transfected with AMPKα1 siRNA (B), AMPKα2 siRNA (C), or KLF2 siRNA (D, E) for 48 hr. Control siRNA were used in the parallel experiments. The ECs were then subjected to PS for 4 hr. RNA samples were isolated and the levels of KLF2 (A-C), eNOS (D), and ET-1 (E) mRNA were quantified by real-time qPCR. The results were normalized with GAPDH. The data represent mean ± SD from 3 separate experiments. * indicates p<0.05 between the two groups being compared.

82 Figure 2-3

Fig. 2-3. AMPK activates KLF2 via the ERK5-MEF2 pathway in cultured ECs. (A) KLF2 and GAPDH transcripts were analyzed by quantitative RT-PCR in HUVECs treated with 1 mM AICAR for 8 hr (left panel), and with Ad-null or Ad-AMPK-CA for 24 hr (right panel). (B,C) HUVECs were infected with Ad-GFP together with Ad-MEK5-DN or Ad-MEF2-DN followed by AICAR stimulation. Total RNA was extracted and the levels of KLF2 and GAPDH mRNA analyzed by quantitative RT-PCR. The data represent mean ± SD from 3 separate experiments.

83 Figure 2-4

Fig. 2-4. AMPK upregulates ERK5-MEF2 in ECs. AMPKα1 (A) or AMPKα2 (B) was knocked down by siRNA as described in Fig. 2-2. The cells were then treated with 1 mM AICAR or vehicle control for 1 hr before being lysed. Cell lysates were analyzed by Western blotting with anti-p-AMPK(Thr172), anti-p-ERK5(Thr218) and p-MEF2(Thr312). The bar graphs below are the statistic analysis of the ratio of p-ERK5 or p-MEF2 to that of total ERK5 and MEF2 respectively. The data represent mean ± SD from 3 separate experiments. * indicates p<0.05 between the two groups being compared. (C) HUVECs were treated with Compound C (15 µM) for 30 min and then subjected to PS flow for 0.5 and 1.5 hrs. Cell lysates were analyzed by Western blotting with anti-p-ACC(Ser79), anti-p-ERK5(Thr218) and p-MEF2(Thr312). The data represent mean ± SD from 3 separate experiments. * indicates p<0.05 between the two groups being compared.

84 Figure 2-5

Fig. 2-5. AMPK mediates KLF2 expression via the ERK5-MEF2 pathway in mouse models. Expression of KLF2 and phosphorylations of ERK5 and MEF2 are higher in the aorta of wild-type mice than those in AMPKα2-/- mice. Two aortic extracts from the same line were pooled and subjected to immunoblotting to determine (A) the levels of KLF2, eNOS, and α-tubulin expression, and (B) the phosphorylation of AMPK, ERK5, and MEF2; (C) en face immunostaining of phospho-MEF2 in thoracic endothelium. Fig. 2-5C: The thoracic aortas from C57BL6 and AMPKα2-/- mice were immunostained with anti-phospho-MEF2 followed by Alexa 647-conjugated secondary antibody. Nuclei were counterstained with DAPI. The immunostained images of anti-phospho-MEF2 and DAPI were obtained by confocal microscopy. Shown are representative images from 4 animals in each group. * indicates p<0.05 between the wild-type and ΑΜPΚα2-/- samples.

85 Figure 2-S1

Fig. 2-S1. Inhibition of KLF expression by Compound C is concentration dependent. (A) Compound C at 20, 15, or 10 mM was added to cultured HUVECs for 30 min before cells were subjected to 1-hr PS flow. A corresponding concentration of Compound C was included in the circulating media during the flow. The inclusion of Compound C completely inhibited PS flow activation of p-ACC. The high concentration (20 and 15 mM) of Compound C also suppressed the basal level expression of KLF2 (B) both in static ECs and in those under 1-hr PS flow. However, 10 µM of Compound C kept KLF2 expression at basal level in static condition, but significantly reduced the level of KLF2 induction under 1-hr PS flow. * P < 0.05 (in comparison to MeOH/Static). # P < 0.05 (in comparison to MeOH/PS). § P < 0.05 (in comparison to CC/Static).

86 Figure 2-S2

Fig. 2-S2. Optimization for KLF2 knockdown. Different amounts of KLF2 siRNA at 100 to 800 pmoles/slide were used to transfect HUVEC for 48hr. The RNA samples were isolated and the expression of KLF2 was quantified by real-time qPCR, and normalization to that of GAPDH. The optimal knock-down efficiency (~80%) was found to be at 400 pmoles/slide, which is equivalent to 40 nM.

87 Figure 2-S3

Fig. 2-S3. AICAR-induced ERK5 phosphorylation is compound C sensitive and MEK5 dependent. (A) AMPK was blocked by Compound C (CompdC) to test the role of AMPK in the downstream signaling events. The cells were then treated with 1 mM AICAR or vehicle control for 1 hr before being lysed. (B) Cells were infected with Ad-GFP or Ad-MEK5-DN for 24 hr before treatment with 1 mM AICAR for 1 hr. Cell lysates were analyzed by Western blotting with anti-p-AMPK(Thr172), or anti-p-ERK5(Thr218). The same blots were stripped and re-blotted with anti-a-tubulin to demonstrate the equal loading of proteins for the lanes.

88 Chapter 3

Flow-regulation of Krüppel-like Factor 2 Is Mediated

by MicroRNA-92a

3.1 Abstract

Background---Upregulated by atheroprotective flow, the transcription factor Krüppel- like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs

(miRNAs) are non-coding small RNAs that regulate gene expression at the post- transcriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow-regulated KLF2.

Methods and Results---Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells (HUVECs), suggesting that KLF2 is regulated by miRNA.

In silico analysis predicted that miR-92a could bind to the 3’ untranslated region (3’UTR) of KLF2. Overexpression of miR-92a precursor (pre-92a) decreased the expression of

KLF2 and the KLF2-regulated endothelial nitric oxide synthase (eNOS) and thrombomodulin (TM) at mRNA and protein levels. A complementary finding is that a miR-92a inhibitor (anti-92a) increased the mRNA and protein expression of KLF2, eNOS, and TM. Subsequent studies revealed that, atheroprotective laminar flow downregulated the level of miR-92a to induce KLF2, and the level of this flow-induced KLF2 was reduced by pre-92a. Furthermore, miR-92a level was lower in HUVECs exposed to the atheroprotective pulsatile shear flow (PS) than under atheroprone oscillatory shear flow.

89 Anti-Ago1/2 immunopreciptation coupled with RT-PCR revealed that PS decreased the functional targeting of miR-92a/KLF2 mRNA in HUVECs. Consistent with these findings, miR-92a level was lower in the endothelium of atheroprotective than atheroprone areas of the mouse aorta. Furthermore, mouse carotid arteries receiving pre-

92a exhibited impaired vasodilatory response to flow.

Conclusions—Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.

3.2 Introduction

The vascular endothelium, located at the interface between the circulating blood and the vessel wall, is exposed to shear stress resulting from blood flow. The endothelium in straight parts of the artery tree is subjected to pulsatile shear stress with a significant forward direction, which is an important physiological stimulus enhancing vessel compliance and conferring anti-thrombotic, -adhesive, and -inflammatory effects. In contrast, disturbed flow patterns at the arterial bifurcations and curvatures can cause endothelial dysfunction, which initiates atherosclerosis1-4.

The transcription factor Krüppel-like factor 2 (KLF2) is a critical integrator for endothelial lineage development and vascular functions (reviewed in references 5-7).

KLF2 can be highly induced by shear stress with a significant forward direction (whether steady laminar flow or pulsatile flow), as well as HMG-CoA reductase inhibitors (i.e., statins). Previous studies have shown that KLF2 can be induced by shear stress at both transcriptional and post-transcriptional levels. The induction of KLF2 mRNA by laminar

90 shear stress has been suggested to be mediated through the MEK5/ERK5/MEF2 pathway, which is AMPK dependent8-13. Shear stress increases the stability of KLF2 mRNA, with attendant elevation of KLF2 protein14. The molecular basis of such an increased stability of KLF2 mRNA has not been established.

MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level15-17. Ranging from 18 to 24 nt (22 nt in general), miRNAs bind to the 3’ untranslated region (3’UTR) of their target mRNAs, with ensuing suppression of protein translation or enhancement of mRNA degradation. Analysis of miRNAs expressed in arterial walls and cultured vascular endothelial cells (ECs) has shown that approximately 40 miRNAs are highly expressed in ECs18-20. These miRNAs play important roles in regulating blood vessel development, wound healing, redox signaling, inflammatory responses, and angiogenesis (reviewed by reference 21). Using miRNA microarrays, we and others have shown that laminar shear stress upregulates a set of miRNAs in ECs in vitro and in vivo22-25. Functionally, miR-21 increases NO bioavailability and reduces EC apoptosis22, miR-19a and miR-23b regulate EC proliferation23, 24, and miR-10a is anti-proinflammatory25. Several recent reports demonstrated that the miR-17~92 cluster regulates cardiac development, EC proliferation, and angiogenesis (reviewed by reference 26). In this cluster, miR-92a was the first miRNA identified to regulate angiogenesis. Loss- and gain-of-function experiments showed that miR-92a inhibited angiogenesis in vitro and in vivo27. Importantly, miR-92a overexpression in human umbilical vein ECs (HUVECs) suppressed the expression of several KLF2-regulated genes such as endothelial nitric oxide synthase (eNOS) and

91 thrombomodulin (TM)27. The lack of miR-92a binding sites in the 3’UTR of these genes suggests that a mechanism other than direct targeting of miR-92a is involved.

Here, we report that atheroprotective flow causes downregulation of miR-92a in

ECs, which in turn elevates the KLF2 mRNA. The functional consequences of the flow- regulated miR-92a/KLF2, including the augmentation of eNOS and TM levels and increase of NO bioavailability, can be mimicked by inhibition of miR-92a. These findings have led to a new paradigm of mechanotransduction that involves the shear modulation of miRNAs, KLF2 expression, and vascular homeostasis.

3.3 Materials and Methods

3.3.1 Cell culture and reagents

HUVECs were cultured as described in chapter 2. BAECs and human embryonic kidney 293 (HEK293) cells were cultured in DMEM containing 10% FBS. All cell cultures were kept in a humidified 5% CO2-95% air incubator at 37˚C.

Western blot analysis was performed with the use of antibodies against eNOS

(Cell Signaling, Beverly, MA), TM (ABcam, Cambridge, MA), KLF2, histone H1 (Santa

Cruz Biotechnology, Santa Cruz, CA), and α-tubulin (Sigma, St Louis, MO). The anti-

KLF2 antibody was obtained by immunizing rabbits with 2 separate synthetic peptides

(CALSEPILPSFST-amide and Ac-ALSEPILPSFST-Ahx-C-amide) corresponding to human KLF2. The antiserum was purified by affinity column and tested by ELISA.

92 3.3.2 miRNA and mRNA Real-time quantitative reverse-transcription polymerase chain reaction analysis

Quantitative Real-time PCR (qRT-PCR) was performed to measure the level of miR-92a with the TaqMan miRNA reverse transcription kit and the TaqMan miRNA assay kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA) with IQ5 real-time PCR detection system (Bio-Rad, Hercules, CA). U6 small nucleolar

RNA was used as the housekeeping small RNA reference gene. For the quantification of

KLF2, eNOS, and TM mRNA, qRT-PCR was performed with the iQ SYBR Green supermix (Bio-Rad). The sequences of primer sets were: KLF2,

AGACCTACACCAAGAGTTCGCATC and CATGTGCCGTTTCATGTGCAGC; eNOS, TGGTACATGAGCACTGAGATCG and CCACGTTGATTTCCACTGCTG;

GAPDH, ATGACATCAAGAAGGTGGTG and CATACCAGGAAATGAGCTTG.

Expression levels of miRNA and mRNA were quantified employing the 2 (-ΔΔCT) relative quantification method.

3.3.3 Knockdown and overexpression of miR-92a and KLF2

miR-92a was knocked down by using anti-92a (Ambion Inc, Austin, TX). The overexpression of miR-92a was achieved by transfecting cells with miR-92a precursor

(pre-92a) (Ambion). Dicer1 was knocked down by using small interfering RNA (siRNA) obtained from Qiagen (Valencia, CA). For KLF2 overexpression in ECs, 0.4 g pcDNA-CMV-KLF2 was used to transfect 106 cells. The transfections were carried out by use of lipofectamine 2000 (Invitrogen, Carlsbad, CA).

93 3.3.4 Shear stress experiments

A parallel-plate flow system was used to impose shear stress on ECs cultured in flow channels as described in chapter 2. Laminar flow, pulsatile shear flow (PS), and oscillatory shear flow (OS) were applied to ECs with shear stresses of 12 dyn/cm2, 12 ± 4 dyn/cm2, and 0 ± 4 dyn/cm2(frequency = 1 Hz), respectively.

3.3.5 Plasmid construction and luciferase assay

The full length of human KLF2 3’-UTR sequence was amplified and inserted to the downstream of the luciferase reporter gene of the pMIR-REPORT vector (between

Hind III and Spe I site) (Ambion). The FLAG-KLF2 plasmid was constructed by fusing a CMV-driven FLAG tag with KLF2 cDNA (including 3’UTR). The primer set: Forward

(EcoRI): ccggaattcATGGCGCTGAGTGAACCC and Reverse (BamHI): cgcggatccAACCAACCCAGCAAAATC. Luc-KLF2 (Luc-mut) and FLAG-KLF2 (mut) with a mutated miR-92a binding site were created by using QuickChange site-directed mutagenesis (Stratagene, La Jolla, CA). The sequences of the mutagenic oligonucleotides were: GCCACTTAAATTATTGCGCCATTTTGAAAAACAAAACTCG (sense) and

CGAGTTTTGTTTTTCAAAATGGCGCAATAATTTAAGTGGC (anti-sense). The deletion of miR-92 binding site in pMIR-KLF2-3’UTR was constructed by two-step

PCR28. The sequences of the primer sets were:

GCCACTTAATGCCACCAAAACTCGTCAAGG,

CCTTGACGAGTTTTTGGTGGCATTAAGTGGC. The miR-92a reporter (Luc-92a) contained a luciferase reporter and 2 copies of sequences complementary to miR-92a

(Luc-2xmiR92a). The reporter constructs were co-transfected with pre-92a or anti-92a

94 (20 nM) into HEK293 cells or BAECs by use of lipofectamine 2000 (Invitrogen).

Luciferase expression was measured by luciferase reporter and β-galactosidase enzyme assays (Promega, Madison, WI).

3.3.6 NO bioavailability

HUVECs were transfected with anti-92a or BAECs were co-transfected with pre-

92a and pcDNA-CMV-KLF2 for 48 hr and then the medium was replaced by phenol-free and serum-free DMEM. After 8 hrs, the medium samples were removed for immediate determination of total nitrate/nitrite concentration with nitrate/nitrite florescence assay kit

(Cayman Chemicals).

3.3.7 In situ hybridization

To detect miR-92a expression, the aortic arch and thoracic aorta segment were processed and stained by modified protocol29. In brief, the mice were perfused with 10 ml of 4% paraformaldehyde (PFA) after the sacrifice. Aortas were collected and immersed in 4% PFA for 24 h at 4ºC, then in 0.5 M sucrose at 4ºC for 48 h. For inactivation of enzymes in tissues, samples were acetylated by incubating for 30 min in a 1 ml solution of freshly prepared acetylation solution, containing 0.06N HCl, 1.2% (v/v) triethanolamine and 0.6% (v/v) acetic anhydride. the samples were pre-hybridized with

500 μl of hybridization buffer containing 50% formamide, 5x SSC, 1x Denhardt’s solution, 0.3M NaCl, 20mM Tris-HCl, pH8.0, 5mM EDTA, 10mM NaPO4, pH8.0, 10%

Dextran Sulfate, 500 μg/ml yeast tRNA (Sigma), at 57ºC for 0.5 h. For hybridization, 5 pmol of FITC-labeled LNA probe diluted in 100 μl of hybridization buffer were applied.

95 The samples were incubated in a sealed humidified chamber for 4 h at a temperature 20ºC below the TM of the LNA oilgonucleotide probe. The samples were washed twice in 1 ml of a solution containing 50% formamide, 1x SSC, and 0.1% Tween-20 for 30 min at the same temperature as probe hybridization twice. Finally samples were washed in 1.5 ml

0.2x SSC for 15 min and once in 1 ml of 0.1% Tween in PBS at room temperature. Then the segments were incubated in anti-FITC-HRP (PerkinElmer Life Sciences) diluted in blocking solution overnight at 4ºC. The samples were incubated with 100 μl of TSA Plus

FITC System working solution for 10 min at 25ºC in the dark according to the manufacturer’s protocol (PerkinElmer Life Sciences). The samples were then washed three times in TNT buffer. Aortas were opened and mounted on slides with mounting medium containing DAPI (Invitrogen, CA) and samples processed for microscopy.

3.3.8 Local oligo delivery

Chemically modified oligonucleotides (antagomir) comprising a sequence complementary to the mature miR-92a (anti-miR92a) were used to inhibit miR-92a activity. All experiments were performed according to institutional protocols (University of California) using 8-week-old male mice. To deliver antagomir-92 and precursor miR-

92 into vascular tissue and to avoid any potential systemic side effects, an established local oligo delivery model via F-127 pluronic gel was used as previously described30.

Briefly, 15 μg of these oligonucleotides (Dharmacon, Lafayette, CO) were mixed with 50

μl 25% F-127 pluronic gel (Sigma) at 4°C. Immediately after anesthesia and incision, carotids were locally applied by the gel mixture. Pluronic poloxamer gel is liquid at 4°C

96 but rapidly solidifies at 37°C when in contact with mouse tissues. It is hydrophilic and degrades rapidly in an aqueous environment. After gelification, the skin was reapproximated by a reabsorbable suture.

3.3.9 Vasodilation

The carotid arteries were isolated immediately after sacrifice and were mounted on two glass cannulae in a perfusion myograph chamber which is connected to the SoftEdge

Acquisiton Subsystem. The vessel chamber was superfused with warmed PSS containing

130mM NaCl, 10mM HEPES, 6mM glucose, 4mM KCl, 4mM NaHCO3, 1.8 mM CaCl2,

1.18 mM KH2PO4, 1.2mM MgSO4, and 0.025mM EDTA, pH7.4. Carotid arteries were imaged by a video camera attached to a microscope. A video dimension analyzer (Living

System) measured external diameter and data was collected via BioPac MP100 hardware and Biopac AcqKnowledge software (BioPac, Goleta, CA). The arteries were maintained at a intraluminal pressure of 100mmHg for the duration of the experiment. The vessels were equilibrated for 30 min before extraluminal administration of 1 μM phenylephrine.

After the maximal constriction, flow rate was increased to 400 μl/min, which corresponded to a shear stress of 12dyn/cm2. The diameter changes induced by flow will be recorded.

3.3.10 Computational analysis for KLF2-regulated miRNAs

The transcriptional start sites (TSSs) of the selected miRNAs were obtained from miRStart database (http://mirstart.mbc.nctu.edu.tw/), which contains the predicted promoters of human miRNAs. The miRNA promoters were identified by the supports of

97 several experimental datasets derived from TSS-relevant experiments, including CAGE tags, TSS Seq tags and ChIP-seq of H3K4me3 enrichment.

JASPAR31 was utilized to identify the potential binding sites of KLF2 within the promoter regions (flanking -3000~+500 according to TSS) of the reported shear- regulated miRNAs. The position weighted matrix (PWM) of KLF4 was used to identify

KLF2-regulated miRNAs since the binding motifs of KLF2 and KLF4 are highly similar.

3.3.11 Statistical analysis

All data were analyzed by Student’s t test (for paired testing between two groups used only once). Results are expressed as mean±SD from at least three independent experiments. P values < 0.05 were considered to be statistically significant.

3.4 Results

3.4.1 miRNAs are involved in the regulation of KLF2 expression.

To investigate whether the shear stress-induction of KLF2 can be mediated at the post-transcriptional level, HUVECs were pre-sheared with laminar flow for 6 hr and then treated with 5,6-dichloro-1-β-D-ribobenzimidazole (DRB) to terminate transcription so that KLF2 mRNA stability could be monitored. As shown in Fig. 3-1A, the degradation rate of KLF2 mRNA in HUVECs exposed to laminar flow was much slower than that under static controls. To explore whether miRNAs are involved in the regulation of

KLF2, we knocked down Dicer to block the miRNA biogenesis and found that the levels of KLF2 mRNA and protein increased (Fig. 3-1B,C). The levels of eNOS and TM,

98 which are downstream targets of KLF2, also increased in ECs with Dicer knockdown

(Fig. 3-1C).

By using miRanda, microCosm, and TargetScan, we explored the putative miRNA binding sequences at the 3’UTR of KLF2 mRNA. Twenty miRNA binding sites with 45 miRNAs were predicted (Fig. 3-1D). miRanda, microCosm, and TargetScan predicted, in common, that the segment between 228 to 249 nt contains binding sequences for 8 miRNAs (i.e., miR-25, -32, -92a, -92b, -363, -367, -29a, and -29b).

Analysis of the secondary structure of this segment by RNAcofold

(http://www.tbi.univie.ac.at) revealed that the miRNA binding locus was located in

“unstable” regions with multi-branching loops, characteristic of a miRNA target site.

Among these putative 8 miRNAs, miR-29 and miR-92a are highly expressed in ECs18-20.

Interestingly, a nucleotide sequence, GGUGCAAUA, complementary to the seed region of miR-92a, is highly conserved among the human, chimpanzee, mouse, and rat KLF2

3’UTR (Fig. 3-1E).

3.4.2 miR-92a targets KLF2 mRNA

To explore whether miR-92a targets KLF2 mRNA, HUVECs were transfected with pre-92a. qRT-PCR was performed and confirmed the increased expression of miR-

92a and decreased level of KLF2 mRNA in pre-92a-transfected cells, as compared with the control RNA-transfected HUVECs (Fig. 3-2A). Furthermore, the mRNA level of eNOS and TM decreased by ~50%. In complementary experiments, HUVECs transfected with anti-92a exhibited higher levels of KLF2, eNOS, and TM mRNAs (Fig.

3-2B); and protein (Fig. 3-2C).

99 To demonstrate the direct targeting of KLF2 3’UTR by miR-92a, we created a

CMV-driven expression plasmid encoding the wild-type KLF2 3’UTR fused with a

FLAG tag [FLAG-KLF2(WT)]. We also fused FLAG to a mutated KLF2 3’UTR in which the miR-92a binding site was altered [FLAG-KLF2(mut)] or deleted [FLAG-

KLF2(Δ)]. Together with pre-92a or control RNA, these KLF2 expression plasmids were transfected into HEK293 cells, which have a high transfection efficiency and a low level of endogenous KLF2. As shown in Fig. 3-2D, pre-92a, but not control RNA, decreased the level of FLAG-KLF2(WT) fusion protein. In parallel experiments, the expression of

FLAG-KLF2(mut) or FLAG-KLF2(Δ) was unaffected by the co-transfected pre-92a.

We also created reporter constructs containing luciferase fused to the wild-type

KLF2 3’UTR [Luc-KLF2(WT)] or the mutated KLF2 3’UTR [Luc-KLF2(mut)].

HEK293 cells were co-transfected with Luc-KLF2(WT) or Luc-KLF2(mut) together with pre-92a or control RNA. As shown in Fig. 3-2E, the transfected pre-92a decreased the luciferase activity of Luc-KLF2(WT), as compared with cells co-transfected with control

RNA but was unable to decrease the luciferase activity of Luc-KLF2(mut). Together, the data from Fig. 3-2 suggest that the 3’UTR of KLF2 mRNA contains a functional miR-

92a target site. The interaction of miR-92a with KLF2 mRNA through this site causes the degradation of KLF2 mRNA and/or decreased translation of KLF2.

3.4.3 Shear stress-regulation of KLF2 is mediated by miR-92a

Because shear stress upregulates, while miR-92a downregulates, KLF2 mRNA in

ECs, we examined the effect of laminar flow on miR-92a. As shown in Fig. 3-3A, the level of miR-92a in HUVECs decreased after exposure to laminar flow for 8 hr, and this

100 decrease lasted for at least 16 hr. To explore whether miR-92a is involved in the shear stress-regulated KLF2, HUVECs were transfected with pre-92a and then exposed to laminar flow. As shown in Fig. 3-3B, C, pre-92a transfection attenuated shear stress induction of KLF2 at both mRNA and protein levels. Furthermore, the shear stress- induction of eNOS and TM was downregulated by pre-92a in a similar manner (Fig. 3-

3D-F).

3.4.4 Differential regulation of miR-92a by PS versus OS

Because of the atheroprotective versus atheroprone natures associated with PS and OS, we compared the miR-92a levels in HUVECs subjected to PS and OS. Both qRT-PCR and miRNA microarray showed that the expression of miR-92a was attenuated in ECs exposed to PS as compared with OS (Fig. 3-4A, B). To further test the flow regulation of miR-92a/KLF2, we created a reporter construct in which luciferase was fused to 2 copies of the miR-92a binding site found in the KLF2 3’UTR (Luc-2xmiR92a).

As shown in Fig. 3-4C, PS caused a 2-fold increase in luciferase activity, as compared with static controls. In contrast, OS significantly reduced the luciferase activity, as compared with static controls (Fig. 3-4D). Furthermore, PS increased whereas OS slightly decreased the luciferase activity of Luc-KLF2(WT) (Fig. 3-4E, F).

Because miRNA targeting mRNAs depends on the association of the miRNA/mRNA complex with Ago proteins to form miRNA-induced silencing complex

(miRISC), we investigated the association of miR-92a and KLF2 mRNA with Ago1 and

Ago2 in HUVECs under PS. As shown in Fig. 3-5A and C, the levels of miR-92a and

KLF2 mRNA associated with Ago1 or Ago2 immunoprecipitated from HUVECs

101 subjected to PS were lower than that in static controls. Similar experiments were performed with HUVECs exposed to OS. Contrary to PS, OS increased the miRISC- associated miR-92a and KLF2 mRNA (Fig. 3-5B, D). The expression of neither Ago1 nor Ago2 was affected by the applied PS and OS (Fig. 3-5E).

To determine whether miR-92a was differentially regulated by different flow patterns in vivo, we performed FISH on the aortic arch and thoracic aorta of C57BL6 mice. The endothelium experiences atheroprotective flow in the thoracic part and atheroprone flow in the aortic arch. As shown in Fig. 3-5F, the level of miR-92a in endothelium of the inner curvature of the aortic arch, where flow patterns are disturbed, was significantly higher than that in the thoracic aorta, which experiences undisturbed PS.

3.4.5 miR-92a regulates NO bioavailability

A functional consequence of KLF2-regulated eNOS is the increase in NO bioavailability in ECs. Hence, we examined whether NO production is affected by miR-

92a. As shown in Fig. 3-6A, anti-92a overexpression in HUVECs enhanced NO production. To show that miR-92a targeting KLF2 mRNA is involved in the enhanced

NO bioavailability, we co-transfected BAECs with FLAG-KLF2(WT) and pre-92a.

KLF2 overexpression indeed increased NO production, which was largely abolished by the co-transfected pre-92a (Fig. 3-6B).

To determine the role of miR-92a in regulating vascular functions in vivo, we delivered pre-92a into the mouse carotid artery by a local oligo delivery system. Carotid arteries were used because the associated flow conditions have been defined32 and gene expression in these vessels can be manipulated by pluronic gel-based gene delivery. As

102 shown in Fig. 3-6C, the expressions of KLF2 and eNOS were significantly lower in vessels with pre-92a treatment. The role of miR-92a in the flow-mediated vasodilation was then investigated. As shown in Fig. 3-6D, the flow-mediated vasodilation was suppressed in vessels with overexpressed miR-92a. Importantly, L-NAME administration inhibited the flow-mediated vasodilation in vessels receiving control RNA, but had little effect on vessels overexpressing pre-92a. These results indicate that miR-

92a is a critical molecule that regulates the eNOS-derived NO bioavailability in vivo.

3.5 Discussion

Shear stress with a forward direction has been shown to upregulate KLF2 in ECs5-7, which in turn modulates the expression of ~70% of the genes that are responsive to shear stress33. Early studies demonstrated that shear stress enhances KLF2 transcription via the binding of several transcription factors (TFs) to the promoter of the KLF2 gene (see

Table 4-1). Post-translational modifications (e.g., phosphorylation and acetylation/deacetylation) of these TFs play important roles in changing the transcriptional activity of KLF213, 34. Results from the present study show that shear stress also regulates KLF2 expression at the post-transcriptional level, which is mediated by the decreased level of miR-92a. Thus, the expression of KLF2 can be modulated at multiple levels, including transcription, post-transcriptional via miR-92a, and post- translational modifications.

miR-92a belongs to the miR-17~92 cluster. Also known as oncomiR-1, the miR-

17~92 cluster is located in the third intron of the C13orf25 locus at 13q31-q32.

Overexpression of miR-92a in ECs blocks angiogenesis in vitro and in vivo, which

103 suggests that miR-92a is a negative regulator of some endothelial functions27. It has since been found that other miRNAs of the miR-17~92 cluster, such as miR-17, -18a, -

19a, and -20a, are also anti-angiogenic35. Sharing the same promoter, various miRNAs within the miR-17~92 cluster would be suppressed by PS and/or induced by OS in a similar manner. Indeed, the expression of miR-17, -18a, -19b, and -20a in HUVECs was downregulated by PS but upregulated by OS (Fig. 3-7). However, some miRNAs within the miR-17~92 cluster may not target KLF2 mRNA, since only the seed sequence of miR-92a is complementary to the KLF2 3'UTR.

The expression of the miR-17~92 cluster can be modulated by several transcription factors or signaling molecules. Overexpression of c-Myc, cyclin D1, and

E2F increased the expression of the miR-17~92 cluster in cancer cells36-38. ChIP assay has shown the binding of c-Myc, E2F, and cyclin D1 to the upstream promoter region of the miR-17~92 cluster36-38. In addition, a binding site is present in this promoter39.

Given the positive effect of c-Myc, cyclin D1, E2F, and stat3 on the induction of the miR-17~92 cluster, one would assume that OS upregulates and that PS downregulates these proteins. Indeed, prolonged laminar flow has been shown to suppress the expression of these proteins23, 40-42. Since miR-92a targets the KLF2 3’UTR, the PS- downregulation of miR-92a should lead to an elevation of KLF2 expression at the post- transcriptional level. Consequently, the “de-suppressed” KLF2 transactivates its target genes (e.g., eNOS, and TM), which are otherwise suppressed under static or OS conditions. In addition to the regulation of NO and its consequent vasodilation, this mechanism involving miR-92a targeting KLF2 may also regulate other KLF2-dependent

104 genes such as von Willebrand factor, FLK1, and Tie-2, which are critical for EC lineage development and vascular functions27, 43, 44.

The results presented in Figs. 2 and 3 show that miR-92a regulates KLF2 at both mRNA and protein levels. When assembled into miRISC, the mature miRNAs can cause the degradation of their target mRNAs or interfere with the translational process15-17.

Among the Ago family members, Ago1 mediates the miRNA-induced translational inhibition16, 45. Recent studies have shown that the Ago1-involved miRISC could also destabilize target mRNAs by deadenylation and 5'→3′ decay after decapping16, 45.

Neither of the mechanisms requires a perfect match between miRNAs and the 3’UTR of the targeted mRNAs. However, mRNA degradation modulated by Ago2 requires a near- perfect complementary sequence in the 3′UTR of the target46, 47. The miR-92a/KLF2 mRNA targeting lacks a perfect match, since only the seed region of miR-92a (8 nt) is complementary to the KLF2 3’UTR. The RNA chaperone model48 suggests that Ago1 may facilitate the association of the guide strand of miR-92a with the KLF2 mRNA, and then Ago2 is recruited to the miRISC complex. This model explains the increased association of miR-92a and KLF2 mRNA with both Ago1 and Ago2 in ECs under OS

(Fig. 3-5B,D).

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112 Figure 3-1.

Fig. 3-1. miRNAs are involved in the regulation of KLF2 mRNA. (A) HUVECs were subjected to laminar flow (12 dyn/cm2) for 6 hr. After the addition of 5,6-dichloro-1-β- D-ribobenzimidazole (DRB; 2 μg/ml), then the cells were continuously exposed to laminar flow or static conditions for additional 2 and 4 hr. The levels of KLF2 and GAPDH mRNAs were measured by qRT-PCR, and the KLF2/GAPDH mRNA ratio is plotted as a percentage of that in the untreated static cells. (B,C) HUVECs were transfected with 20 nM Dicer siRNA or control RNA for 48 hr. RT-PCR (B) and Western blot analysis (C) were performed to detect the mRNA levels of KLF2 and eNOS and protein levels of KLF2, eNOS, and TM, respectively. Histone H1 served as the internal control of nuclear extracts. The bar graphs are mean ± SD from 3 independent experiments. * p<0.05 between Dicer siRNA and control RNA. (D) The miRNA binding sites in the KLF2 3’UTR are predicted by bioinformatics algorithms. (E) The seed region of miR-92a and its target sequences at the KLF2 3’UTR of several mammalian species: Homo sapiens, Pan troglodytes, Mus Musculus, and Rattus Norvegicus

113 Figure 3-2.

Fig. 3-2. miR-92a targets KLF2 mRNA and decreases KLF2 translation. HUVECs were transfected with 20 nM miR-92a precursor (pre-92a) or control RNA. At 48 hr, the mRNA levels of miR-92a and KLF2, eNOS, and TM as ratios to GAPDH were assessed by qRT-PCR. In (B) and (C), HUVECs were transfected with 20 nM miR-92a inhibitor (anti-92a) or control RNA. The mRNA levels of KLF2, eNOS, and TM as ratios to GAPDH were assessed by qRT-PCR. The protein levels of KLF2, Histone H1 and eNOS were determined by Western blotting. (D) HEK293 cells were transfected with the wild- type FLAG-KLF2 (WT), FLAG-KLF2 (mut) (miR-92a binding site mutation), or FLAG- KLF2 (Δ) (miR-92a binding site deletion) together with 20 nM pre-92a or control RNA for 48 hr. The cells were then lysed, and the level of exogenously expressed FLAG- KLF2 fusion proteins was detected by Western blot analysis with anti-FLAG. Shown in the bottom panel is the densitometry analysis of the protein amount normalized to that in the control RNA-transfected cells. (E) HEK293 cells were transfected with the wild-type Luc-KLF2-3’UTR(WT) or Luc-KLF2-3’UTR(mut) together with 20 nM pre-92a or control RNA and 0.1 μg CMV-β-gal. The luciferase activity was normalized to that of β- gal. The data represent mean ± SD from 3 independent experiments. * p<0.05 between the 2 groups being compared.

114 Figure 3-3.

Fig. 3-3. Shear stress-induction of KLF2 is mediated through miR-92a. (A) HUVECs were exposed to laminar flow for 4, 8 or 16 hr. RT-PCR was performed to detect the level of miR-92a, which was normalized to that of U6 RNA. * p<0.05 compared with static controls (time 0). (B-F) HUVECs were transfected with 20 nM control RNA or pre-92a for 48 hr and then exposed to laminar flow for 8 hr. (B) KLF2 mRNA level was detected by qRT-PCR and (C) protein level was assessed by Western blot analysis. (D) eNOS and (E) TM mRNA levels were detected by qRT-PCR and (F) protein level was assessed by Western blot analysis, and the results of statistical analyses are shown in the right. The data represent mean ± SD from 3 independent experiments. * p<0.05 between ECs transfected with pre-92a and control RNA.

115 Figure 3-4.

Fig. 3-4. PS downregulates, but OS upregulates, miR-92a expression in ECs. (A) HUVECs were exposed to PS (12±4 dyn/cm2) or OS (0±4 dyn/cm2) for 8 hr. qRT-PCR was performed to detect the level of miR-92a, which was normalized to that of U6 RNA. (B) The expression of miR-92a in ECs exposed to PS or OS flow assessed by miRNA microarray. (C,D) BAECs were transfected with Luc-2×miR92 reporter or control plasmid for 24 hr and then exposed to PS or OS flow for 12 hr. The luciferase activity was measured and normalized to β-gal activity. (E, F) BAECs were transfected with Luc-KLF2(WT) or Luc-KLF2(mut) for 24 hr and then exposed to PS or OS flow for 12 hr. The luciferase activity was measured and normalized to β-gal activity. The data represent mean ± SD from 3 independent experiments. * p<0.05 between the 2 groups being compared.

116 Figure 3-5.

Fig. 3-5. miRISC regulates miR-92a. HUVECs were exposed to PS (A,C) or OS (B,D) for 8 hr. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by IP with the use of anti-Ago1 (A,B) or anti-Ago2 (C,D). mRNA levels of miR-92a and KLF2 were detected by qRT-PCR and normalized to those of Ago1 or Ago2 protein. The data represent mean ± SD from 3 independent experiments. * p<0.05 for PS or OS vs. static controls. (E) Protein levels of Ago1 and Ago2 assessed by Western blot analysis with anti-Ago1 and anti-Ago2 and normalized to that of α-tubulin. (F) Levels of miR-92a in endothelium of the thoracic aorta and aortic arch of C57BL6 mice were assessed by in situ hybridization with LNA miR-92a probe (green). Nuclei were counterstained with DAPI (blue). Images are representative of 4 animals.

117 Figure 3-6.

Fig. 3-6. miR-92a regulates endothelial function in vitro and ex vivo. HUVECs were transfected with control or anti-92a RNA (A) or BAECs were co-transfected with pre-92a and CMV-KLF2 (B). After 48 hr, the NO bioavailability was detected by fluorometric assay and expressed as NOx. The data represent mean ± SD from 3 independent experiments. (C) pre-92a or control RNA was administered to the carotid arteries of C57BL6 mice by pluronic gel F-127. Five days later, the arteries were isolated. The expression levels of miR-92a, KLF2 and eNOS in the isolated vessels (n=6) were assessed by qRT-PCR. miR-92a level was normalized to that of U6 RNA, whereas KLF2 and eNOS levels were normalized to that of GAPDH. (D) The flow-induced vasodilation ex vivo was measured by use of the SoftEdge Acquisiton Subsystem in the presence or absence of L-NAME (1 μM). The flow-induced diameter changes in various experimental groups were compared with those of vessels receiving control RNA set as 1. The bars represent mean±SD from the indicated number of vessel specimens, with control vessels set as 1.

118 Figure 3-7.

Fig. 3-7. The expression of miR-17-92 cluster under the different flow pattern. HUVECs were exposed to a PS (12±4 dyn/cm2) or OS (0±4 dyn/cm2) for 24 hr and then lysed. The amount of miR-92a were assessed by miRNA microarray. The level in ECs exposed to OS, averaged from 3 experiments was normalized to that in cells exposed to PS.

119 Chapter 4

Conclusion and perspectives

1. Introduction

KLF2 is an important integrator of multiple endothelial functions. The study of the mechanism by which atheroprotective flow upregulates KLF2 expression and maintains ECs homeostasis can increase our understanding of atherosclerosis and help develop novel strategies for its prevention and treatment. In this study, I explored the regulation of KLF2 expression at both the transcriptional level and the post-transcriptional level in response to shear stress. The major findings are as follows:

(1) AMPK mediates shear stress-upregulated KLF2 transcription in ECs and in vivo by activation of the MEK5/ERK5/MEF2 pathway; and (2) Atheroprotective flow patterns (laminar flow and pulsatile flow) decrease the level of miR-92a, which in turn increases KLF2 expression at the post-transcription level. These two mechanisms synergistically function to to maintain endothelial homeostasis. Therefore, my study summarized in this dissertation provide a novel molecular mechanism by which different flow patterns can regulate KLF2 expression and modulate vascular function.

2. Shear stress regulates KLF2 expression at the transcriptional level

KLF2 is highly expressed in the endothelium of the atheroprotective regions of the vessel and reduced in that of the atheroprone region, such as the branch points or bifurcations1. Because both AMPK and KLF2 are regulated by shear stress and can

120 augment nitric oxide production, I describe my study of the role of the AMPK in regulating the shear stress-induction of KLF2 expression in chapter 2.

In my study, an AMPK inhibitor (Compound C) or siRNA to target AMPK were used to block AMPK function, and an AMPK activator (AICAR) and an adenovirus expressing a constitutively active form an AMPK were used to mimic a gain of function. Indeed, the inhibition of AMPK had a significant blocking effect on

the shear stress-induced KLF2 expression, and the activation of AMPK is sufficient for

the induction of KLF2 expression.

Furthermore, I explored the mechanism underlying AMPK regulation of shear stress-induced KLF2 expression. MEF2 is an important transcription factor for KLF2 in ECs; this finding was demonstrated by the identification of the MEF2 binding site in the shear stress response region of the KLF2 promoter. Post-translational modifications such as phosphorylation provide a mechanism to regulate the transactivation of MEF2. Previous studies have shown that ERK5 (BMK1) is capable of greatly enhancing the transactivation of MEF2C by phosphorylating

MEF2C at Thr 300 and MEF2A at Thr 3122. Parmar et al. showed that the flow-dependent activation of KLF2 is mediated via the MEK5/ERK5/MEF2 signaling pathway, which suggests the involvement of the MAPK family in the regulation of

KLF23. In chapter 2, I further identified that AMPK is necessary to activate

MEK5/ERK5/MEF2 signaling by use of AMPK siRNA to knockdown AMPK, which

in turn induced KLF2 expression in response to shear stress. I also demonstrated that

121 activation of AMPK by AICAR is also sufficient for the induction of the phosphorylation of ERK5 and MEF2, which was subsequently confirmed in vivo with

AMPKα2-/- mice. The phosphorylation of ERK5, MEF2, and eNOS was greatly attenuated and KLF2 expression was significantly reduced in the aortas of AMPKα2-/- mice. To further demonstrate the vascular location of the reduction of MEF2 activation, I performed en face staining of phospho-MEF2 to confirm the reduction in aortic endothelium of AMPK α2-/- mice. These results indicate that AMPK plays a critical role in modulating the shear stress activation of the ERK2/MEF2/KLF2 pathway and establishes a novel link between AMPK and ERK5/MEF2 signaling pathway.

In summary, my study suggested that AMPK is an upstream signaling molecule of the ERK5/MEF2/KLF2 pathway and is required for subsequent expression of KLF2 in ECs and in mouse vasculature. These findings established that the PS activation of the AMPK/ERK5/MEF2/KLF2 pathway plays an important role in the regulation of vascular homeostasis.

Remaining questions

1) Table 4-1 lists the TFs, including MEF2, that can bind to the KLF2 promoter region. Among these TFs, none are endothelial-specific, but the binding of these proteins to the KLF2 promoter is regulated by shear stress in ECs, which is probably due to the post-translational modification. For example, the binding of nucleolin to the KLF2 promoter is flow specific and PI3K dependent in ECs4. Nucleolin could be

122 highly phosphorylated at specific residues, which induces a conformational change leading to its active form5. Co-IP experiments showed that nucleolin can interact with

the p85 regulatory subunit of PI3K, but the phosphorylation site has not been

identified yet4. However, by use of our system, blocking PI3K with Wortmannin or

LY2943002 had little effect on shear-induced AMPK activation and KLF2 expression, which indicates that the AMPK-KLF2 pathway is independent to PI3K. Therefore, nucleolin may be phosphorylated by response of another kinase such as AMPK or

PKA to shear stress. Further investigation will be needed to understand how shear stress regulates the transactivation of TFs on KLF2 expresison.

2) In addition to phosphorylation, the transactivation of the MEF2 family has also been shown to be regulated by the recruitment of cofactors6. Wang et al. demonstrated

a novel role for histone deacetylase 5 (HDAC5) in flow-mediated KLF2 expression by

regulating MEF2A activity7. The authors showed that shear stress induced HDAC5

phosphorylation and nuclear export in endothelial cells via a

calcium/calmodulin-dependent pathway, thus leading to dissociation of HDAC5 and

MEF2 and enhancing MEF2 activity. The activated MEF2 subsequently increased the expression of KLF2 and eNOS. Moreover, the authors identified that S259 and S498 of HDAC5 are critical phosphorylation sites for this regulation, but they did not identify the upstream kinase of HDAC5. Previous studies, including ours, showed that CaMKK is a Ca-dependent kinase and is an upstream kinase of AMPK8. These

results suggest that AMPK may play a role in the phosphorylation of HDAC5 in

123 response to shear stress. In vitro kinase assays could be performed to examine

whether AMPK phosphorylates HDAC5 directly. The blockage of AMPK by siRNA

could be used to examine whether AMPK mediates shear stress-induced HDAC5

translocation to cytoplasm.

3. Shear Stress regulates KLF2 expression at the post-transcriptional level

Shear stress (improved by exercise training) is more potent than statins

(pharmaceutical) in increasing the levels of the KLF2 downstream targets (eNOS and

TM), despite being able to induce KLF2 transcription to a similar level. This result is due in part to the role of shear stress in promoting KLF2 stabilization. Results in chapter 3 show that shear stress regulates KLF2 expression at the post-transcriptional level, which is mediated by the decreased level of miR-92a.

In chapter 3, using RT-PCR, Luciferase reporter assays, and Fluorescence in situ Hybridization (FISH), I demonstrated that atheroprotective flow (LS and PS)

downregulates miR-92a expression and atheroprone flow (OS) upregulates miR-92a

expression in ECs (Fig.3-3,4). Consistent with my result, a recent report also

demonstrated by microarray analysis that miR-92a expression is increased in

HUVECs under PS compared to the static condition9. Moreover, miR-92a expression

was found elevated in the endothelium of the athero-susceptible aortic arch (AA) as

compared with that of the atheroprotective dorsal descending thoracic aorta (DT) in

swine10.

124 However, the mechanism by which the different flow patterns regulate the

expression of miR-17-92 cluster remains unknown. Oxidative stress, which is

relatively higher in the atheroprone region under disturbed flow, is a major

determinant of endothelial adhesiveness for monocytes and lesion formation11. A

recent study in our lab showed that both H2O2 and angiotensin II could increase the

expression of miR-92a. Similarly, IL-6, an oxidative stress inducing cytokine, is

related to pulmonary arterial hypertension, can upregulate the expression of the miR-17-92 cluster in HPAECs12. These results indicate that oxidative stress probably

plays an important role in the regulation of the miR-92a in response to different flow

patterns in ECs.

The previous studies revealed that c-Myc, E2F, STAT3 and cyclin D1, which

are related to the regulation of cell cycle, could bind to the miR-17-92 promoter and

upregulate its transcription13, 14. In contrast, p53 inhibits the transcription by

competing with the transcription machinery to bind to the same region of the

miR-17-92 promoter (TATA box)15. In addition to these TFs, 37 potential TFs binding

sites were revealed in the promoter region (-5000 to +500 bp) of the miR-17-92

cluster by in silico analysis. Several of these TFs may be regulated by laminar flow

(listed in Table 4-2), such as activating transcription factor 2 (ATF2) and PPARG.

The nuclear activation of ATF2, which can induce the expression of pro-inflammatory

genes, is inhibited by laminar flow16. Moreover, phosphorylated ATF2 protein level

is elevated in endothelial cells overlying early atherosclerotic plaques as compared

125 with healthy endothelium16. Considering the expression and function of ATF2, these

findings suggest that ATF2 probably mediates flow-regulated KLF2 expression.

The function of miR-92a in ECs

MiR-17-92 was considered an oncomiR because of its overexpression in several

types of lymphoma and solid tumors17, 18. In addition to the cluster’s role in

tumorigenesis, it is also a regulator of hematopoiesis and immune function19, 20. Even

though miR-92a is highly expressed in ECs, little is known about the function of

miR-92a in ECs. Gain-of-function experiments revealed that miR-92a does not influence apoptosis and proliferation of ECs. Overexpression of miR-92a in ECs

blocks angiogenesis in vitro and in vivo, which suggests that miR-92a is a negative

regulator of endothelial functions21. In chapter 3, I demonstrated that miR-92a can

directly bind to the KLF2 3’ UTR and decrease shear-induced KLF2 and its targets

(eNOS and TM) mRNA and protein levels. A functional consequence of this effect is

the increase in NO bioavailability in ECs. The enhanced overexpression of miR-92a

alleviated flow-induced vasodilation in mouse carotid artery. The result of L-NAME

administration, which blocked endothelial-dependent vasodilation, indicated that

miR-92a not only decreased endothelial-dependent but also endothelial-independent

vasodilation. The impaired vasodilatory ability is an indicator of EC dysfunction.

126 miRNAs regulates KLF2

Within the KLF2 3’ UTR are 20 predicated putative miRNA binding sites that

can be regulated by 45 potential miRNAs. In addition to miR-92a, several other

miRNAs are downregulated by PS and upregulated by OS; examples are miR-25, 30,

99 and 106. Further experiments could validate the regulatory role of these miRNAs

on KLF2 expression and endothelial function. MiR-92a and these other miRNAs might act in synergy to regulate the KLF2 and other targets in response to differential

flow pattern. These findings would provide information on a novel and fine regulatory

layer of endothelial function.

miRNA subcellular localization

Much evidence indicates that the components of the miRISC may not localize in

the cytosol, but occurs in different cellular organelles or structures21. Both Dicer, the

key enzyme for miRNA biogenesis, and Ago, the key component of miRISC, have

been reported to be associated with the endoplasmic reticulum (ER) and Golgi membranes, which could facilitate the local biogenesis or action of miRNAs in their vicinity22-24. miRISC can also accumulate in discrete organelles known as P-bodies, which contain the enriched proteins involved in mRNA deadenylation, decapping, and degradation. In my study, FISH imaging showed that miR-92a mainly distributes in the cytosol close to the nucleus (Fig. 3-5). The possible explanation is that miR-92a mediates the binding of miRISC to ER, which directly connects to the nuclear envelope. Such a unique association may facilitate miR-92a targeting KLF2.

127 4. Shear stress regulates KLF2 expression at the post-translational level

To date, very little is known about the role of posttranslational modification of

KLF2 and the role in mediating its functions. The Lingrel group identified that WW domain–containing E3 ubiquitin protein ligase 1 (WWP1) can interact with the

inhibitory domain (110-267 aa) of KLF2, thus leading to the ubiquitination and

proteasomal degradation of KLF225. K121 is the critical site for ubiquitin

conjugation. In silico analysis revealed that this site and K148 are also the predicted sites (ψ-K-X-E) for SUMOylation, which probably modulates KLF2 function. KLF2

can interact with the transcriptional coactivator cyclic AMP response element–binding

protein (CBP/p300), inducing eNOS and inhibiting E-selectin and VCAM-126. p300

and CBP each contain a protein or histoneacetyltransferase (PAT/HAT) domain that can modulate the binding TFs and regulate their transactivation. Interestingly, K121 and K148 are also the potential acetylation sites for KLF2. In addition, the transactivation of KLF2 can be modulated by the recruitment of cofactors. For example, ERG, an endothelial-specific TF of ETS family, forms a physical complex with KLF2 and synergistically activates transcription of Flk1 during embryonic vascular development27. These experimental results and prediction indicate that

post-translational modification is important for the shear stress-regulated KLF2 function.

128 5. KLF2 regulates the expression of miRNAs

KLF2, functioning as a TF, regulates the expression of both genes and miRNAs.

The first evidence of regulating miRNA is that KLF2 induces the expression of

miR-126, an endothelial-specific miRNA, which results in activating Vegf signaling

and guiding angiogenesis during zebrafish development28. Recently, Harris et al. showed that KLF2 binds to the promoter region of miR-126 and directly regulates the transcription of miR-12629. In addition to miR-126, a set of shear stress-upregulated

miRNAs that have the KLF2 binding site in their promoter region were revealed by

bioinformatics analysis (Table 4-3). These miRNAs are probably induced by shear

stress through KLF2. The targets of KLF2 would be further identified by ChIP-Seq on a genome-wide scale.

6. Summary

From knowledge gained from the current study and that in the literature, Fig.

4-1 summarizes a network of molecular events leading to the induction of KLF2 in

ECs by atheroprotective flow. AMPK/MEF2 signaling and several TFs modulated

by the imposed flow are directly involved in the induction of gene (Table 4-1),

whereas other TFs may downregulate the miR-17~92 cluster (Table 4-2). Thus,

KLF2 is regulated at both transcriptional and post-transcriptional levels. The

upregulated KLF2, functioning as a TF, transactivates a panel of genes related to EC

lineage, as well as miRNAs28. Recently, Harris et al. showed that KLF2 regulates

the transcription of miR-126 directly29. In addition to miR-126, bioinformatics

129 analysis revealed a set of shear stress-upregulated miRNAs that have the

KLF2-binding site in their promoter region (Table 4-3). Moreover, the

downregulation of miR-92a leads to augmented IGTA5 and upregulation of miR-126

results in attenuated SpredI. Collectively, these miRNA-regulated events and KLF2

targets (e.g., eNOS, TM) maintain the EC lineage24, 26. These results provide an

integrated paradigm of how miRNAs and TFs enhance EC functions under

atheroprotective flow.

Because AMPK can regulate various signaling pathways, including KLF2,

activation of AMPK has become an emerging target for the prevention and treatment

of atherosclerosis. Because of its negative regulation of angiogenesis and vasodilation, miR-92a may also be an effective target for therapeutic agents for cardiovascular diseases. miRNA could be readily inhibited in vivo by use of the stable modified antagomiR, and this agent could be efficiently delivered into various tissues in animal models30-32. If miRNA-based therapeutics indeed becomes a reality, miR-92a will be

one of the miRNA candidates to be considered.

130

Fig. 4-1. Shear stress regulation of KLF2. The diagram shows the regulatory circuitry of the responses of transcription factors and miRNAs to atheroprotective shear flow. The circled Roman numerals refer to the Table numbers. Shear stress with a forward direction regulates the expression of KLF2 through AMPK/MEF2 signaling and other TFs (Tables I) and the expression of miR-92a through TFs (Table II). Serving as a transcription factor, KLF2 transactivates the expression of downstream genes such as eNOS and TM. In addition, KLF2 may bind to the promoter region of some miRNAs, including miR-126, to upregulate their transcription directly (Table III). In turn, the network of KLF2 and miRNAs regulates the expression of factors that control anti-inflammatory, -thrombotic, -proliferative, -angiogenic, -oxidant, and -fibrotic effects to enhance EC lineage development and maintain vascular functions. The methods for computational analysis are described in the supplements.

131 Table 4-1. Transcription factors regulating KLF2

Shear stress TF Regulation mode References regulated

MEF2A Activated yes 3, 33, 34 MEF2C Activated yes 3, 33, 34 BRG1 Unspecified unknown 35 p300 Activated yes 26, 36-38 PCAF Activated Yes 36, 38 hnRNP D Activated Yes 36, 38 hnRNP U Activated Yes 36, 38 Nucleolin Activated Yes 4 SP1 Activated yes 39 Oct-3/4 Activated unknown 40-43 Activated unknown 40, 41, 44

132 Table 4-2. Transcription factors regulating the miR-17~92 cluster

Shear stress TF Regulation mode References inducible

c-Myc Activated Inhibited 45, 46 E2F Activated Inhibited 47, 48 STAT3 Activated Inhibited 12, 49 cyclin D1 Activated Inhibited 50, 51 P53 Inhibited Activated 15, 52 RUNX1 Inhibited Inhibited 19 ATF2 predicted inhibited 16 CREB predicted Activated 53 PPARG predicted Inhibited 54 SP1 predicted Activated 55

133 Table 4-3. KLF2-targeted miRNAs

Gene Host gene Function Validated Reference hsa-miR-126 Egfl7 Angiogenesis Yes 9, 29 Anti-inflammation hsa-miR-30a C6orf155 angiogenesis predicted 9, 56 hsa-miR-483-5p IGF2 unknown predicted 51 hsa-miR-101-1 Intergenic Cell growth predicted 57, 58 hsa-miR-181d Intergenic differentiation predicted 57, 59 hsa-miR-15a DLEU2 apoptosis predicted 57, 60 hsa-miR-148a Intergenic Cell survival predicted 57, 61 hsa-miR-365-1 Intergenic unknown predicted 57

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