INSIGHTS INTO THE RENAL PROTECTIVE MECHANISMS OF mRNA
BINDING PROTEIN HUR
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
The Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By Mamata Singh Ohio State Biochemistry Program
The Ohio State University 2011
Dissertation Committee:
Professor Beth S. Lee, Advisor
Professor Richard W. Burry
Professor Arthur R. Strauch
Professor Ma Jiyan
ABSTRACT
Acute kidney injury is a common and serious problem in clinical medicine that can be roughly defined as an abrupt and sustained decrease in renal function resulting in failure of the kidneys to clear metabolic waste products.
Transient loss of blood flow to kidneys, or ischemia-reperfusion injury, is the most common cause of acute kidney injury, and results in damage to both renal tubule epithelia and endothelial cells, as well as generation of an inflammatory response. Under this type of stress, renal cells initiate programs to promote cell survival, although the nature of these adaptations is not well understood. Here we examine the function and expression of cell survival protein, HuR, in renal proximal tubule epithelia cells using an in vitro of ischemia-reperfusion injury.
HuR is a ubiquitously expressed protein that resides primarily in nuclei under normal growth conditions but translocates to the cytosol during stress, where it binds and stabilizes a select subset of mRNAs bearing adenine and uridine-rich sequences in their 3’ untranslated regions. Although HuR is generally regarded as an anti-apoptotic protein, recent studies have indicated that caspase cleavage of HuR may contribute to an amplified apoptotic response. To clarify
HuR’s role in renal stress, we knocked down or overexpressed it in proximal tubule cell lines that were subjected to ATP depletion, an in vitro procedure that mimics effects of ischemic injury to native kidneys. HuR was found to be protective against apoptosis that occurs when proximal tubule cells are ATP
ii depleted. Further, HuR was found to be necessary for full expression of the cell survival proteins Bcl-2 and Hsp-70. These results strongly indicate an anti- apoptotic role for HuR in cultured proximal tubules cells, and suggest that its function in native proximal tubules may be protective against ischemic stress.
To understand what other cell survival signaling pathways might be induced by HuR, PCR array analysis was used to compare gene expression levels in
ATP-depleted cells with normal or suppressed levels of HuR. We found that loss of HuR caused decreased levels of mRNAs associated with the PI3-kinase/Akt signaling pathway that promotes cell survival. We found that HuR plays a central role in PI3K/Akt signaling, as not only did knockdown of HuR suppress many members of this pathway, but inhibition of PI3K also resulted in suppression of
HuR levels. Here, we show that in renal proximal tubule cells, HuR performs a central role in cell survival by amplifying Akt signaling in a positive feedback loop.
Key to this feedback loop is HuR-mediated stabilization of mRNA encoding
Grb10, an adaptor protein that enhances Akt activity by aiding in its transport to sites of activation. Under normal growth conditions, stimulation of Akt by HuR-
Grb10 interactions then activates NF-κB, which further enhances HuR expression at a transcriptional level. This study demonstrates a central role for
HuR in cell survival mechanisms and reveals how only modest changes in HuR levels below or above normal can be amplified, resulting in cell death, or alternately, cellular transformation.
The regulation of gene expression is a multi-level process that is essential to cellular processes. The data presented here show that the expression of HuR
iii during ATP depletion and recovery is upregulated by both transcriptional and
translational mechanisms. It was previously demonstrated that HuR mRNA is
expressed as two isoforms with vastly different 5’ untranslated regions and
different translatabilities. Here we have demonstrated that one of these mRNAs
is responsive to the BMP-7/Smad 1/5/8 signaling pathway, is more highly
expressed during recovery from ATP depletion, and is the only form capable of
being translated during ATP depletion. We also demonstrated that translation of the alternate form occurs during normal growth and recovery, and occurs through regulation by a post-transcriptional control element (PCE) present in its 5’ untranslated region. We further hypothesize that the differential translation of the
HuR isoforms may be due to their alternate targeting to polyribosomes or stress granules.
Stress granules are sites of aborted translation formed during various types of cell stress and recovery, including heat shock, arsenite poisoning, and UV- irradiation. Recruitment of specific mRNAs into stress granules regulates their stability and potential translation. We demonstrated that ATP-depleted epithelial cells form stress granules and activated their upstream kinases presumably for the purpose of regulating mRNA stability and translatability. During ATP depletion, some HuR mRNA is stored temporarily in stress granules. We hypothesize that this may be to avoid aberrant translation of HuR, which can lead
to tumorigenicity. The presence of a PCE imposes another level of regulation on
the translation of HuR and thus the translation of HuR is controlled during
normal, stressed, and recovered conditions.
iv
Dedicated to my Father, in his loving memory
v
ACKNOWLEDGEMENT
This dissertation would not have been possible without the guidance and
the help of several individuals who in one way or another contributed and
extended their valuable assistance in the preparation and completion of this
study.
First and the foremost, I am heartily thankful to my supervisor, Dr. Beth Lee,
whose encouragement, guidance and support from the initial to the final level
enabled me to develop a good understanding of the subject. I offer her my
gratitude, as she has supported me throughout my research with her patience
and knowledge along with providing excellent atmosphere for doing research.
Then, I would like to thank my committee members Dr. Richard Burry, Dr.
Arthur Strauch and Dr. Jiyan Ma for their precious suggestions and valuable comments that aided me in improving the quality of my research.
Additionally, I would like to thank my colleagues Dr. Selvi Jeyaraj, Dr. Dina
Ayupova, Dr. Brooke Mc Michael, and Suman Govindaraju for their constructive
criticism, help, and moral support.
I would also like to thank Alaina Martinez, a rotation student and a good
friend of mine, for all her help towards the completion of one of my projects.
vi Last but not the least, my parents, my husband, and my daughter for being patient with me, during all those years. In addition, I am thankful to the omnipresent divine spirit, for giving me the strength to finish this journey.
In the end, I offer my regards and gratitude to all of those who supported me in any respect during the completion of the project.
vii
VITA
July 20, 1974 …………………………………..Born-Banaras, India
June, 1997………………………………………B.S. Biochemistry
June, 1999………………………………………M tech in Biochemical Engineering IT BHU, India 2003-Present…………………………………...Graduate Teaching Assistant and Graduate Research Associate, The Ohio State University
PUBLICATIONS
1. Singh M, Sharma R, Banerjee UC. Biotechnological Applications of Cyclodextrins. J BiotechnologyAdvances 2002; 20:341-359.
2. Yi W Shao J, Zhu L, Li M, Singh M, Lu Y, Lin S, Li H, Ryu K, Shen J, Guo H, Yao Q, Bush CA, Wang PG. Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide. J Am Chem Soc. 2005 Feb 23; 127 (7):2040-1.
3. *Ayupova DA, *Singh M, Leonard EC, Basile DP, Lee BS. Expression of the RNA-stabilizing protein HuR in ischemia-reperfusion injury of rat kidney. Am J Physiol Renal Physiol. 2009 Jul; 97(1):F95-F105. (*denotes equal contribution)
4. Jeyaraj SC, Singh M, Ayupova DA, Govindaraju S, Lee BS. Transcriptional control of human antigen R (HuR) by bone morphogenetic protein (BMP). J Biol Chem. 2010; 2;285(7):4432-40.
FIELDS OF STUDY
MAJOR FIELD: BIOCHEMISTRY
viii
TABLE OF CONTENTS
ABSTRACT...... ii
ACKNOWLEDGMENTS...... vi
VITA...... viii
LIST OF FIGURES...... xiii
ABBREVIATIONS...... xv
CHAPTER 1
INTRODUCTION...... 1
1.1 Ischemia reperfusion Injury (IR)...... 1
1.2 Apoptosis: a mechanism of cell death...... 3
1.3 Ischemia Reperfusion Injury and Apoptosis….…...... 5
1.4 Cellular Models of Ischemic Injury………………………………………………..6
1.5 Differential Gene Expression as a Result of Ischemia Reperfusion Injury…..8
1.6 PI 3-Kinase Pathway, Apoptosis and Cell Survival……………………………..9
1.7 Mechanisms of Gene Regulation during Cell Stress……………………….…11
1.8 HuR and Gene Regulation…………………………………………….…….…...12
1.9 Role of mRNA Untranslated Regions in Gene Expression and mRNA Regulation……………………………………………………………………………...13
1.10 Stress Response-Stress Granules and Post-transcriptional Control Element (PCEs)…………………………………………………………………………………..14
1.10.1 Stress Granules ……………………………………………………….15
1.10.2 Post transcriptional elements (PCEs)…………………….…………17
ix
CHAPTER 2
MATERIALS AND METHODS……………………………………….……………....24
2.1 Cell Culture………………………………………………………………………..24
2.2 Apoptosis Assays…….. ……………………………………………………..…...24
2.3 Antibodies ………………………………………………………………………....25
2.4 Inhibitors…………………………………………………………………………...26
2.5 Western Analysis……………………………………………………………….…26
2.6 Transfection……………………………………………………………………..…27
2.7 RT-PCR and Immuno-RT-PCR. ………………………………………………..28
2.8 Immunocytochemistry……………………………………………………………29
2.9 PCR Arrays………………………………………………………………………...29
2.10 Plasmids………………………………………………………………………….30
2.11 In Situ Hybridization……………………………………………………………..30
2.12 Sucrose Density Gradients……………………………………………………..31
2.13 Gel Mobility Shift Assay………………………………………………………...32
2.14 Chromatin Immunoprecipitation (ChIP)………………………………………32
CHAPTER 3
HuR Promotes Cell Survival in ATP-depleted Renal Proximal Tubule Cells…..34
3.1 INTRODUCTION………………………………………………………………….34
3.2 RESULTS
3.2.1. Nature and Time Course of Cell Death in ATP-depleted LLC-PK1 Cells…………………………………………………………………………...... 37
x 3.2.2. Determination of Role of HuR in Cell Survival…………………… 38
3.2.3. Overexpression of HuR Promotes Cell Survival…………………..38
3.2.4. HuR Promotes Cell Survival by Inhibiting Both Intrinsic and Extrinsic Apoptosis……………………….………………………………………….…40
3.2.5. HuR Stabilizes Cell Survival mRNAs Bcl-2 and Hsp70 under Cell Stress………………………….………………………………………...... 41
3.3 CONCLUSION..………………………………………………………..………. .41
CHAPTER 4
HuR mediates anti-apoptotic effect by amplifying Akt signaling through a positive feedback loop, via Grb10……………………………………………………………50
4.1 INTRODUCTION………………………………………………………………...50
4.2 RESULTS
4.2.1 HuR Expression is Regulated by PI3K/Akt Activity in Proximal Tubule Cells…………………………………………………………………………...52
4.2.2. NF-κB is Critical for HuR Expression during Normal Growth…....54
4.2.3. Akt Activity but not Akt Expression is Regulated by HuR Levels in Proximal Tubule Cells……………………………………………………….55
4.2.4 Expression of Adaptor Protein Grb10 is Regulated by HuR Levels in Proximal Tubule Cells……………………………………………………….56
4.2.5. Grb10 Stimulates Akt Activation and HuR levels…………...... 58
4.3 CONCLUSION…………………………………………………………………...60
CHAPTER 5
Transcriptional and Translational control of HuR………………………………...68
5.1 INTRODUCTION…………………………..…………………………………….68
5.2 RESULTS xi 5.2.1 Gel Shift Assay and ChIP Confirms Binding of Smad 1/5/8 to HuR Promoter Elements……………………………………………...…………..69
5.2.2 Confirmation of the Presence of BMP-7 Receptors in LLC-
PK1cells………………………………………………………………………...71
5.2.3 Regulatory Mechanisms Mediating Translational Control of HuR...72
5.2.4 Translatability of Alternate Forms of HuR mRNA under Normal, Stressed, and recovered conditons…………………………………………72
5.2.5 Stress Granules………………………………………………………...73
5.2.6 Formation of Stress Granules during ATP Depletion and Recovery……………………………………………………………………….74
5.2.7 Effects of Cycloheximide and Puromycin on Stress Granule
Formation in LLC-PK1 Cells…………………………………………………75
5.2.8 Distribution of Stress Granule Markers during ATP Depletion and Recovery ………………………………………………………………………75
5.2.9 Increase in the Phosphorylation Level of eIF2-alpha during ATP Depletion……………………………………………………………………….77
5.2.10 PERK is Activated by ATP Depletion of Proximal Tubule Cells….78
5.2.11 Post-transcriptional Control Element (PCEs) and Translation of HuR……………………………………………………………………………..79
5.3 CONCLUSIONS…………………………………………………………………...81
CHAPTER 6
DISCUSSION………………………………………………………………………….92
6.1 Anti-apoptotic Role of HuR………………………………………………………92
6.2 HuR, PI3 Kinase/Akt Pathway, and Apoptosis………………………………...94
6.3 Transcriptional and Translational Control of HuR……………………………..97
BIBLIOGRAPHY……………………………………………………………………...100
xii
LIST OF FIGURES
Figure 1.1 Schematic of Intrinsic and Extrinsic Pathways of Apoptosis.………...20
Figure 1.2 Schematic of PI3K/Akt Signaling in Cell Survival and Growth……….21
Figure 1.3 The Role of eIF2α Phosphorylation in Global Translational Repression…………………………………………………………………...………...22
Figure 1.4 Formation of stress granules as a result of translational silencing….23
Figure 3.1 Time Course of Apoptosis in LLC-PK1 cells during ATP Depletion………………………………………………………………………………..44
Figure 3.2 Effects of HuR Knockdown on Apoptosis in ATP-depleted LLC-PK1 cells……...………………………………………………………………………………45
Figure 3.3 Distribution of Native and FLAG-tagged HuR in Stably Transfected
LLC-PK1 Cells……….………………………………………………………………...46
Figure 3.4 Effects of HuR Overexpression on Apoptosis in ATP-depleted LLC-
PK1 cells……………………..…………………………………………………………47
Figure 3.5 Effects of HuR Knockdown on Intrinsic and Extrinsic Pathways of Apoptosis……………………...... ……………………………………………………..48
Figure 3.6 Knockdown of HuR in ATP-depleted LLC-PK1 Cells Results in Suppression of Cell Survival Proteins…………………………..………………….49
Figure 4.1 HuR levels are regulated by PI3K/Akt signaling……………………...62
Figure 4.2 HuR levels are regulated by NF-κB signaling………………………..63
Figure 4.3 HuR regulates Akt activation and Grb10 expression…………………64
Figure 4.4 HuR regulates Akt activation and Grb10 expression…………………65
Figure 4.5 Knockdown of Grb10 inhibits Akt activation and HuR expression….66
xiii Figure 4.6 Schematic of HuR’s role in promoting a positive feedback loop of Akt signaling………………………………………………………………………………67
Figure 5.1 Smad 1/5/8 Binding Sites are Present in the HuR Promoter……….83
Figure 5.2 ATP Depletion and Recovery Induces Expression of ALK2………...84
Figure 5.3 Ribosomal Fractionation Indicates Differential Translation of HuR mRNAs during Normal Growth, ATP Depletion, and Recovery………………...85
Figure 5.4 ATP Depletion Induces Stress Granule Formation………………….86
Figure 5.5 Inhibitors of Translation Alter Stress Granule Formation in ATP- depleted LLCPK1 cells……………………………...………………………………..87
Figure 5.6 Neither eIF2-alpha nor a Large Ribosomal Subunit Co-Localize with
Stress Granules in LLC-PK1 Cells………………………………………………….88
Figure 5.7 Translation Initiation Factors, Small Ribosomal Proteins, and poly(A) mRNA Segregate to Stress Granules in LLC-PK1 Cells…………………….…..89
Figure 5.8 ATP Depletion Induces Phosphorylation of eIF2-alpha and PERK..90
Figure 5.9 HuR mRNA Contains a Post-transcriptional Control Element……....91
xiv
ABBREVIATIONS
AKI acute kidney injury
AMPK AMP-activated protein kinase
ARE AU rich element
ATN acute tubular necrosis
AUF1 AU rich binding factor-1
BMP bone morphogenic protein
DCP-1 decapping enzyme 1
ER endoplasmic reticulum
GAPDH glyceraldehyde-3-phosphate dehydrogenase
HuR human antigen R
HSP heat shock proteins
IRE iron response element
KIM-1 kidney injury molecule -1
MAP mitogen-activated protein
PABP poly A-binding protein
RHA RNA helicase A
TGF-β1 transforming growth factor β1
TIA-1 T-cell restricted intracellular antigen - 1
TIAR TIA-1 related
xv TTP tristetraprolin
SG stress granules
PCE post transcriptional control element
CX cycloheximide
PM puromycin
xvi
CHAPTER 1
Introduction
Excretion of waste and harmful chemicals and maintenance of fluid and solute balances are the major functions of the kidney. The kidney participates in whole-body homeostasis by regulating acid-base balance, electrolyte concentrations, extracellular fluid volume and regulation of blood pressure. The working unit of the kidney, the nephron, accomplishes this task through the process of plasma filtration along with reabsorption or secretion of solutes and water. Each kidney contains approximately 1,000,000 nephrons that contribute to regulation of blood and urine composition. Transient disruption of blood supply to the kidneys, or ischemia reperfusion injury, is the major cause of acute kidney injury (AKI) in both native and transplanted kidneys. The mortality of patients with
AKI remains high and unchanged in the modern era of critical care medicine.
1.1 Ischemia Reperfusion Injury (IR)
Restricted or transient interruption of the blood supply to tissues causes ischemic stress, which is a leading cause of the abnormalities resulting from myocardial infarction, cerebral ischemia, organ transplantation, and other short- lived traumas. Ischemic injury to tissues triggers a complex series of metabolic disturbances such as formation of oxygen radicals, changes in intracellular ion
1 concentrations, damage to the cytoskeleton, and alterations in gene expression
[Weinberg, 1991], often culminating in cell death. Ischemia is a state of tissue
oxygen deprivation accompanied by a reduced availability of the resulting metabolites [Bastin et al., 1987]. The absence of oxygen and nutrients from blood
creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than
restoration of normal function. Restoration of blood flow to the ischemic tissue is
known as reperfusion. Despite the undeniable benefit of reperfusion of blood to
an ischemic tissue, reperfusion itself can educe a cascade of unfavorable
reactions that injure tissue [Becker, 2005]. Reperfusion injury has been known to
cause organ damage in the brain, heart, lungs, liver, kidneys and skeletal muscle
[Maxwell, 1997]. The major obstacle to reperfusion after an infarct or successful organ transplantation is the susceptibility of tissue to ischemia reperfusion (IR) injury. A key event in ischemia is the failure of cells to maintain adenosine triphosphate (ATP) levels, particularly proximal tubule cells, which are most susceptible to ischemic damage due to the high dependence on mitochondrial
ATP [Canfield et al., 1991]. Damage results from defective synthesis of ATP and degradation to adenosine. In addition, cellular ATP depletion results in mitochondrial dysfunction that initiates the translocation of Bax, a proapoptotic
Bcl-2 family member protein, from the cytosol to the outer mitochondrial membrane. This causes mitochondrial swelling and induces the efflux of cytochrome c into the cytosol where cytochrome c activates effector caspases and initiates apoptosis [Padnilam et al., 1999]. Although ischemic injury is
2 primarily due to oxygen-deprived cell death, reperfusion also produces a wide
array of inflammatory responses that causes local damage, which leads to
general insult [Gobe et al., 1999]. A growing body of evidence, primarily from
animal models of IR and preliminary human studies has revealed that
inflammatory mechanisms play a major role in the pathogenesis of IR [Maxwell et
al., 1997].
1.2 Apoptosis: a mechanism of cell death
The word apoptosis is derived from Greek origins and means falling off or
senescence. Unlike the traumatic destruction of necrosis, apoptosis refers to a
regulated or programmed cell death, which is also called death by suicide [Kerr et al., 1972]. Apoptosis may play a positive role in organismal biology. During development, excess cells undergo programmed cell death to form final tissue structures. Alternately, dysregulated apoptosis results in negative consequences.
Excessive apoptosis causes atrophy of tissues, such as in ischemic damage, whereas insufficient levels of apoptosis may result in uncontrolled cell proliferation, such as cancer.
Morphological changes in apoptotic cells include cell shrinkage, deformation
and loose contact to neighboring cells, chromatin condensation, plasma membrane blebbing, and finally fragmentation into compact membrane-enclosed
structures, called “apoptotic bodies”. The apoptotic bodies can be engulfed by
macrophages and thus may be removed from the tissue without causing an
inflammatory response. Apoptosis leads to activation of proteolytic enzymes,
3 which eventually mediate the cleavage of DNA into oligonucleosomal fragments as well as the cleavage of a multitude of specific protein substrates which usually determine the integrity and shape of the cytoplasm or organelles [Saraste and
Pulkki, 2000]. Apoptosis is opposite to the necrotic mode of cell death in which cells suffer a major insult, resulting in a loss of membrane integrity, swelling, and disruption. During necrosis, cellular contents are released uncontrolled into the immediate environment which results in damage of surrounding cells and a strong inflammatory response in the corresponding tissue [Leist, 2001].
Apoptosis occurs through two main signaling cascades, the extrinsic and intrinsic apoptotic pathways. Any stimulus that causes oxidative stress, mitochondrial disturbances and DNA damage induces the intrinsic pathway. For example, cancer therapeutic agents, hypoxia, and ionizing irradiation can trigger the intrinsic pathway of apoptosis. When the mitochondrion is damaged, the outer membrane becomes permeable and facilitates cytochrome c release into cytoplasm. Further, cytochrome c binds to the caspase adaptor, Apaf-1
(apoptotic protease-activating factor-1), to form the apoptosome complex that consequently triggers the apoptotic cascade by activating procaspase 9. The newly activated caspase 9 activates many downstream effector caspases including caspases 3, 6 and 7, leading to DNA fragmentation and cell death
[Earnshaw, 1999]. On the other hand, the extrinsic apoptotic pathway is induced by ligand binding to death receptors. The major ligand-death receptor systems include tumor necrosis factor (TNF) with tumor necrosis factor receptor 1
(TNFR1), Fas ligand with Fas, and TRAIL with TRAIL receptors. Binding of the
4 receptors with ligands induces receptor oligomerization and recruitment of death
signal adaptor proteins. These form a complex termed DISC (death-inducing
signaling complex), which binds to initiator caspases 8 and 10. Consequently, the
caspase cascade is triggered to activate the caspases 3, 7 and 9, leading to
apoptotic events [Ashkenazi, 2002]. There exists extensive crosstalk between the
intrinsic and extrinsic apoptotic pathways, as activation of the caspase cascade
is the crucial component in the death process in either pathway. An overview of
these cascades is represented diagrammatically in Figure 1.1.
1.3 Ischemia Reperfusion Injury and Apoptosis
Renal tubular epithelial cell injury from ischemia was traditionally regarded
as a result of necrotic form of cell death. However, one of the first studies to describe apoptosis in ischemic acute renal failure was by Schumer et al. in 1991, in which DNA fragmentation in the kidney cortex was detected 12 hours after reperfusion. Similarly, several studies using immunohistochemical techniques reported the typical ladder pattern of DNA after subjecting kidneys to IR.
Additional evidence for the importance of apoptosis in IR has been provided by recent studies by Kelly et al. in 2003, who demonstrated the importance of guanosine triphosphate (GTP) depletion in apoptosis (versus ATP in necrosis) and the ability of guanosine and p53 inhibitors to protect against apoptosis and renal dysfunction.
In kidneys, the extrinsic pathway of apoptosis is initiated by activation of cell
death receptors for Fas and tumor necrosis factor-α (TNF-α) leading to activation
5 of procaspase-8, which, in turn, cleaves and activates procaspases-3. The
mitochondrial-dependent pathway is triggered by cytochrome c release from the
mitochondria [Castenada et al., 2003]. Cytochrome c binds to Apaf-1, leads to
caspase-9 activation. Differential regulation of caspases in kidneys subjected to
I/R injury have been shown [Devarajan, 2006]. The cleavage of caspase-1 and
caspase-3 is documented during reperfusion indicating activation of these
enzymes. In addition, a rat model of IR indicated that prolonged ischemia
induced pro-apoptotic mechanisms, including increases in the Bax/Bcl-2 ratio,
caspase-3 expression, poly [adenosine triphosphate (ADP)-ribose] polymerase
(PARP) cleavage, DNA fragmentation, and apoptotic cell number in renal
proximal and distal tubules [Wei et al., 2004].
In living cells, mitochondrial changes are predominantly prevented by
antiapoptotic members of the Bcl-2 family of proteins. It has been shown that the
activation of caspase-3 during hypoxia or ATP depletion is accompanied by the
translocation of Bcl-2 family member Bax from the cytosol to the mitochondria
and the release of cytochrome c from the mitochondria to the cytosol [Schwarz et
al., 2002]. However, transfecting the cells with Bcl-2 provided protection against
hypoxia-induced injury. Further, recent studies have recognized
phosphatidylinositol 3 (PI3) kinase/Akt phosphorylation as one of the signaling
pathways that blocks apoptosis and promotes cell survival in response to diverse apoptotic stimuli in different cell types [Devarajan, 2006].
1.4 Cellular Models of Ischemic Injury
6 Renal epithelial cell lines provide a convenient system for studying many
processes that occur in the kidney. Cell lines from many species exist that
represent the distal and proximal tubular epithelia. Because proximal tubules are
most susceptible to injury during renal ischemia, proximal tubule cell lines have
been well established as cellular models of renal ischemia. In particular, the LLC-
PK1 porcine proximal tubule cell line has been well studied in this regard. LLC-
PK1 cells were originally isolated from the kidney cortex of Hampshire pigs.
These cells maintain many characteristics of proximal tubule epithelia. Once
confluent, these cells maintain polarized basal and apical membranes, form tight
junctions between cells, form a brush border and continue cell transport systems
specific to this cell type [Amsler and Cook, 1982; Mullin, 1980]. The LLC-PK1 cell
line has also been well established as a model for studying stress to proximal
tubule epithelia. Depleting these cells of ATP results in alterations similar to the
ischemic kidney including activation of heat shock proteins [Van SK et al., 2003],
loss of cellular polarity due to disruption of the actin cytoskeleton [Canfield,
1991], and activation of protein kinases [Park KM et al., 2002].
Similarly, HK-2 cells are proven to have a potential usefulness as a tool for
studying injury and repair. HK-2 is an immortalized proximal tubule epithelial cell
line from normal adult human kidney. Phenotypically, the cell line HK-2 has the
same characteristics of adult normal tubular cells [Ryan et al., 1994]. In
particular, HK-2 cells maintain the brush border typical enzymatic activities (acid and alkaline phosphatase, leucine aminopeptidase, gamma
glutamyltranspeptidase). Furthermore, HK-2 cells retain functional characteristics
7 of proximal tubular epithelium (Na+ dependent/phlorizin sensitive sugar transport; adenylate cyclase responsiveness to parathyroid, but not to antidiuretic, hormone). Therefore, this cell line is used as a valuable tool for the study of physiological and pathophysiological human renal tubule, as well as the mechanisms of damage and repair at the level of tubular cell [Nadia et al., 2008].
1.5 Differential Gene Expression as a Result of Ischemia Reperfusion Injury
Global gene expression is altered during ischemia reperfusion injury. cDNA microarray has been a powerful tool to detect molecular changes during renal IR.
Altered gene expression includes changes in the levels of transcription factors, growth factors, signal transduction molecules, and apoptotic factors [Yoshida et al., 2002]. Upregulation of several transcription factors, such as AP-1, and Zf9 kruppel-like transcription factor has been noted. IR-regulated growth factors include heparin-binding epidermal growth factor (HB-EGF), insulin-like growth factor 1 (IGF-1), transforming growth factor-β1 (TGF- β1), and cell cycle proteins like p21 [Devarajan et al., 2003]. Signal transduction proteins that are induced during ischemia reperfusion events include transforming growth factor-1, interleukin 2 receptor, gamma interleukin 4 receptor, growth factor receptor bound (Grb) 2, Ras-like guanosine triphosphate binding protein, CD151, and endothelial nitric oxide synthase 3. In addition, some cytoskeletal proteins like claudin 7, cadherin 5 alpha, smoothelin, palladin, talin, vinculin, cortactin, tropomyosin 4, filamin, and β-tubulin also demonstrate changes in their gene profile [Supavakin et al., 2000].
8 Apoptotic factors such as Fadd, Bad, Daxx, Bax and p53 were found to be upregulated as suggested by micro-array analysis in a rat kidney ischemia- reperfusion model [Harris, 1997]. Both the death receptor-dependent (Fadd-
Daxx) and mitochondrial (Bad-Bak) pathways were found to be activated. This indicates that apoptosis is an important mechanism for the early loss of tubule cells following ischemia/reperfusion injury. In addition, activation of heat shock proteins 27, 72 and 70 as well as protein kinases JNK, MAPK kinase, and p38 has been observed during stress [Devarajan et al., 2003].
Understanding these changes in gene expression not only provides insight
into the pathogenesis of ischemia/reperfusion injury, but also can potentially
provide novel biomarkers and potential therapeutic targets for the treatment of
ischemic injury.
1.6 PI 3-Kinase Pathway, Apoptosis and Cell Survival
Signaling by TNF and related polypeptides is an active process in which
stimulation of cell death receptors induces apoptosis. Other signaling pathways
act in the opposite direction to promote cell survival by inhibiting apoptosis.
These pro-survival pathways control the fate of a wide variety of cellular survival
processes. One of the major intracellular signaling pathways responsible for
promoting cell survival is initiated by the enzyme PI3-kinase, which is activated
by either receptor tyrosine kinases or G protein-coupled receptors [Song et al.,
2005]. PI3-kinase phosphorylates the membrane phospholipid PIP2 to form
PIP3, which activates the protein serine/threonine kinase Akt. PIP3 recruits the
9 protein kinase Akt to the plasma membrane where it is activated as a result of
phosphorylation by PDK. Akt then phosphorylates a number of proteins that
contribute to cell survival. Akt (also known as protein kinase B) was originally
identified as the cellular homologue of the transforming oncogene of the AKT8
retrovirus. Several pathways downstream of PI-3/Akt phosphorylation have been
proposed for cell survival [Datta et al. 1999]. One of the well-studied molecules
that mediate cell survival by Akt phosphorylation is the proapoptotic Bcl-2 family
member Bad. Bad has the ability to directly interact and bind to antiapoptotic Bcl-
2 and Bcl-XL and block their survival function. Phosphorylated Akt can directly
phosphorylate Bad both in vitro and in vivo, and makes Bad incapable of binding
to Bcl-XL, thus restoring its antiapoptotic function. Sequestering phosphorylated
Bad in the cytosol also makes it unavailable to bind to Bcl-2, thus preventing it from damaging the mitochondria [Orike et al., 2001]. Akt can also phosphorylate human caspase-9, resulting in reduction of caspase-9 activity. Further, Akt
activates transcription factor NF-κB via regulating IκB kinase (IKK), resulting in
transcription of pro-survival genes [Datta et al., 1999].
In addition to these direct effects on components of the cell death
machinery, Akt phosphorylates transcription factors that regulate cell survival and
another protein kinase (GSK-3) that affects apoptosis as well as regulating cell
metabolism and protein synthesis. The PI3-kinase/Akt pathway thus regulates
cell survival through phosphorylation of a variety of downstream targets, extracellular growth factors or cell-cell interactions [Dancey, 2004]. A schematic
10 depicting the central role of PI 3-kinase/Akt signaling in cell survival is shown in
Figure 1.2.
PI3-kinase and Akt are activated after renal ischemia/reperfusion, leading to phosphorylation of FKHR and FKHRL1, which may affect epithelial cell fate in acute renal failure [Andreucci et al., 2003]. Inhibiting PI3-kinase is known to impose deleterious effects on serum blood urea nitrogen level and renal functions in mice [Xie et al., 2003]. PI3-kinase is also reported to activate PKC, which is involved in preconditioning, suggestive of its role in cell survival. Akt has been suggested to promote cell survival by intervening in the apoptosis cascade even before cytochrome c release and caspase activation [Kennedy et al., 1999].
In addition, PI3-kinase/akt activation leads to the activation of MEK/ERK survival signaling pathways through the reactive oxygen species-dependent
EGFR/Ras/Raf cascade [Kwon et al., 2006]. Activation of these kinases may be involved in the repair process during ischemia/reperfusion. Thus, PI3-K/Akt signaling pathway plays an important role in regulating the repair following renal
IR.
1.7 Mechanisms of Gene Regulation during Cell Stress
Following stress injury, cells undergo an adaptive response to alter gene
expression. Gene expression is under the coordinated control of mRNA
transcription, mRNA translation, and degradation. It follows that, since turnover of
certain mRNAs is an important form of regulation in mammalian cells, then
stabilization of certain mRNAs is vital for cell survival. Stable mRNAs permit a
11 longer translational window for highly expressed genes or a delayed expression
window when they are stably stored and expressed in a temporal manner.
Whereas the roles of transcription and degradation rates have been well studied,
the role of mRNA stability is now emerging as an important step in regulation.
There are many factors involved in determining mRNA stability. These include primary and secondary structure, translation rate, and intracellular location.
1.8 HuR and Gene Regulation
HuR is a widely expressed nucleocytoplasmic shuttling protein possessing
three well-conserved RNA recognition motifs [Peng et al., 1998]. Under normal
growth conditions, the majority of HuR is maintained in the nucleus but moves to
a more cytoplasmic distribution under stressed conditions [Jeyaraj et al., 2006].
In the cytoplasm, HuR aids in regulating stability and translatability of ARE- containing mRNAs by binding and stabilizing its cognate mRNAs for future translation [Fan et al., 1998]. HuR binds to specific mRNAs that contain adenosine-uridine-rich elements (AREs) in their 3’ untranslated regions, and stabilizes them during stress [Lal et al., 2003].
HuR, as one of the regulators of mRNA stability in renal epithelial cells, is
found to be redistributed and up-regulated at multiple levels during energy
depletion and recovery. By binding to mRNAs containing specific AREs, it
contributes to the stability of hundreds to thousands of distinct mRNA transcripts.
These proteins belong to a several major categories, including modulators of cell
proliferation and differentiation, inflammation, cell survival and apoptosis. It is a
12 stress response protein and is known to play an important role in cell survival, as
its loss in vitro or in vivo results in heightened apoptosis [Wang et al., 2000]. The
expression of HuR as a result is tightly regulated, as its over expression is
associated with oncogenicity in multiple tissues including renal carcinoma cell
lines [Denkert et al., 2004].
HuR has been studied under several forms of stress in cell culture models;
however, its potential role in organ systems has not been well explored. Because
HuR appears to play a role in stabilizing numerous transcripts during cellular
stress and apoptosis [Wang et al., 2000], understanding the regulation of this
protein will give important insight into general mechanisms of survival during
environmental insults such as ischemic injury.
1.9 Role of mRNA Untranslated Regions in Gene Expression and mRNA
Regulation
As described above, HuR binds to cognate mRNAs through specific
sequences in their 3’ untranslated regions (UTRs). Gene expression is finely
regulated at the post-transcriptional level by untranslated regions both 5’ and 3’
to the mRNA coding region [Chen et al., 1995]. Features of UTRs that control
mRNA translation, degradation, and localization include stem-loop structures, upstream initiation codons and open reading frames, internal ribosome entry sites and various cis-acting elements that are bound by RNA-binding proteins.
UTRs are known to play crucial roles in transport of mRNAs into and out of the nucleus, translation efficiency [Velden and Thomas, 1999], subcellular
13 localization [Jansen, 2001] and stability [Bashirullah et al., 2001]. Regulation by
UTRs is mediated in several ways. Nucleotide patterns or motifs that are the
target of trans-acting RNA binding proteins located in 5' UTRs and 3' UTRs can interact with specific RNA-binding proteins. Secondary structures in 5' UTRs are also important in the regulation of translation. Structural features of the 5' UTR have a major role in the control of mRNA translation [Sweeny, 1996]. The importance of UTRs in regulating gene expression is underlined by the finding that mutations that alter the UTR can lead to serious pathology [Conne et al.,
2000].
1.10 Stress Response-Stress Granules and Post-transcriptional Control
Element (PCEs)
In response to stressful environmental conditions, cells educe a series of
adaptive changes known as the stress response. Central to the stress response
is translational repression of all but a minority of stress- induced proteins. In most
cases, this general translational repression is due to hyperphosphorylation of the
translation initiation factor elF2α, a subunit of eukaryotic initiation factor elF2.
eIF2 activity is regulated by a mechanism involving both guanine nucleotide
exchange and phosphorylation. Phosphorylation takes place at the α-subunit, which is a target for four different kinases that phosphorylate serine 51. Those
kinases act as a result of stress such as amino acid deprivation (GCN2), ER
stress (PERK), the presence of dsRNA (PKR) or hemoglobin deficiency (HRI).
During translation initiation, elF2α forms a complex with GTP and the initiator
14 methionyl-tRNA, and this complex binds to the small ribosomal subunit to initiate
protein synthesis. One subunit of elF2α, eIF2B, is a guanine nucleotide
exchange factor that regulates the activity of the elF2 complex. Exchange of
GDP for GTP is prevented by phosphorylation of elF2α, thereby reducing the
availability of the elF2-GTP-met-tRNA complex, as a result translation cannot
proceed [Kedersha et al., 2002]. The relationships among members of the eIF2
complex and their roles in translation initiation are indicated in Figure 1.3. Along
with this general translational repression under stress, other regulatory pathways exist that shuttle the mRNA templates of selected proteins into active polysomes.
1.10.1 Stress Granules
Under conditions of stress, cells form discrete cytoplasmic foci called stress
granules (SGs). These have been identified in plant, yeast, and animal cells and
are characterized as sites of stalled translation initiation [Kedersha and
Anderson, 2002]. The assembly of SGs is triggered by phosphorylation of elF2α, which effectively prevents formation of the eIF2-GTP-Met-tRNAi ternary complex
[Kimball et al, 1999]. The RNA binding proteins TIA-1 and TIAR act downstream of the phosphorylation of eIF2 and recruit these untranslated mRNAs to SGs
[Kedersha et al., 2002]. An overview of stress granule formation is depicted in
Figure 1.4. The purpose of stress granules may be to protect RNAs from harmful conditions, to promote cell survival, and to function as a decision point for untranslated mRNAs [Kimball et al., 2003]. These mRNAs subsequently can go
15 down one of three paths: further storage, degradation, or re-initiation of translation.
Stress granules are composed of silent pre-initiation complexes containing small ribosomal proteins, as well as numerous mRNA-binding proteins such as
TIA-1, TIAR, poly-A binding protein (PABP), HuR, tristetraprolin, and several translation initiation factors [Kedersha et al., 1999]. These granules appear to act as mRNA storage sites, regulated in part by mRNA binding proteins (such as
HuR and TIAR) that mediate the stability of specific transcripts. Nearly all of the mRNA in the cell is recruited to SGs during stress [Kimball et al., 2003], however, specific transcripts that are critical for cell survival during stress (e.g. Hsp70) are excluded from the stress granules and are shuttled into active polysomes.
Considering total cellular mRNA, these critical mRNAs are in minority. For example, viral infection causes cell stress, but only about 3 - 5% of cellular mRNAs become associated with polysomes during infection [Kedersha et al.,
2002].
Only certain types of environmental insults such as heat shock, osmotic shock, UV irradiation, oxidative stress, and energy depletion lead to the formation of stress granules, whereas other forms of insult, including serum starvation, inhibition of transcription, microtubule or microfilament depolymerization, and inflammatory cytokines, do not result in formation of SGs [Kedersha et al., 1999].
It has also been observed that energy-depleted cells are able to form stress granules in the absence of eIF2α phosphorylation and it has been suggested that the lack of GTP in the system inhibits formation of the eIF2-GTP-met-tRNA
16 complex, which is required for translation initiation [Kedersha et al., 2002].
However, this contradicts the notion that elF2α phosphorylation is necessary for
SG formation [Kimball et al., 2003].
1.10.2 Post-transcriptional Elements (PCEs)
Following viral infection or disease, efficient regulation of translation is important in response to adapt to environmental changes. Translational initiation starts with the formation of multi-protein complex eukaryotic initiation factor eIF-
4A, recruitment of the small ribosomal subunit, scanning for the initiation codon and subsequent recruitment of the large ribosomal subunit. Like all other steps of translation, this step is tightly regulated. Transcripts containing a short (<100 nt), relatively unstructured 5' UTR are generally good candidates for efficient ribosome scanning. Conversely, transcripts that contain a longer and highly structured (i.e. G+C-rich) 5' UTR are less efficiently scanned. The structural features of 5' UTR, and the type of ribonucleoprotein complex (RNP) binding, determines the ribosome scanning and the efficiency of translation initiation.
Retroviral proteins are known for their long transcripts, containing highly structured 5’ UTRs. Long 5’ UTRs having inhibitory characteristics prompt for alternative mechanisms for translation. Translation through and internal ribosome entry site (IRES) is an alternative to the traditional cap dependent translation.
IRESs recruit ribosomal initiation complexes directly to the translation start codon by avoiding the scanning procedure through a highly structured 5’ UTR.
Recently, a novel highly structured RNA element in the 5’ UTR of selected
17 retroviruses and a cellular mRNA has been revealed. This element, called the
posttranscriptional control element (PCE), facilitates, rather than inhibits, cap
dependent translation.
PCEs are redundant stem-loop RNA structures that were initially identified
in the 5' UTR of avian spleen necrosis virus (SNV) [Roberts et al., 2005] and the
later in retroviruses including Mason-Pfizer (MPMV) [Hull at al., 2002], human
foamy virus (HFV) [Russell et al., 2001], reticuloendotheliosis virus strain A
(REV-A), human T-cell leukemia virus type 1 (HTLV-1), feline leukemia virus
(FeLV), bovine leukemia virus (BLV) [Boris-Lawrie et al., 1995] and HIV-1 [Zapp
et al., 1989]. PCEs are highly structured 5’ UTR sequences that facilitate cap
dependent translation. A protein called RNA helicase A (RHA) is known to
associate with the PCE structures [Hartman et al., 2006]. RHA is a
multifunctional DEIH box helicase and RNA binding protein, and deregulation of
RHA has been associated with various cancers and autoimmune disease
[Tettweiler et al., 2006]. RHA operates as a selective translation control switch
and facilitates cap-dependent translation. RHA interacts with PCE-containing
mRNAs in the nucleus and cytoplasm, and contributes to RNA/RNP remodeling.
Through its helicase and/or RNPase activity, RHA assists the melting of the
complex structure of the PCE to enable its efficient association with polysomes.
Upon RHA knock down, PCE-containing mRNAs accumulate in the cytoplasm,
though they are translationally silent and are sequestered in RNA storage
granules. Similarly, non-functional PCE mutant RNAs accumulate in the
18 cytoplasm in stress granules as these transcripts lack efficient interaction with
RHA and are poorly translated [Ranji et al., 2010].
1.11. Overview
The following studies will be an exploration of the role of HuR in protecting renal tubule cells against apoptosis caused by ischemia-reperfusion injury and
ATP depletion, as well as the transcriptional and translational mechanisms by which HuR itself is regulated. These studies have been designed to illuminate key aspects of the mechanisms behind cell survival and repair during acute kidney injury, and to uncover the role of HuR in this process.
19
Figure 1.1. Schematic of Intrinsic and Extrinsic Pathways of Apoptosis. The figure is adapted from Cellsignal.com.
20
Figure 1.2. Schematic of PI3K/Akt Signaling in Cell Survival and Growth. Figure is adapted from sarcomahelp.org/research
21
Figure 1.3. The Role of eIF2α Phosphorylation in Global Translational Repression. During translation initiation, elF2α forms a complex with GTP and the initiator methionyl-tRNA, and this complex binds to the small ribosomal subunit to initiate protein synthesis. One subunit of elF2α, eIF2B, is a guanine nucleotide exchange factor that regulates the activity of the elF2 complex. Exchange of GDP for GTP is prevented by phosphorylation of elF2α (by various kinases like GCN2, PKR, HRI, PERK), thereby reducing the availability of the elF2-GTP-met-tRNA complex, as a result translation cannot proceed. Figure is adapted from deverlab.nichd.nih.gov.
22
Figure 1.4. Formation of stress granules as a result of translational silencing. The schematic in (A) illustrates normal translation initiation. In (B), phosphorylation of eIF2α by various protein kinases, including PKR, results in aborted translation initiation, recruitment of mRNA binding proteins such as TIA- 1, and failure of large (60S) ribosomal subunits to join the 40S complex. Figure adapted from Cell Stress Chaperones 2002; 7(2):213-221.
23
CHAPTER 2
Materials and Methods
2.1 Cell Culture
LLC-PK1 or HK-2 cells (American Type Culture Collection, Manassas, VA)
were cultured in Dulbecco’s modified Eagle’s medium containing
penicillin/streptomycin supplemented with fetal bovine serum (10%) at 37°C in
5% CO2. For depletion of ATP from these cells, cultures were first grown to
confluency, then medium was replenished and cultures were incubated
overnight. The cells were then rinsed twice with phosphate-buffered saline and
the culture medium was replaced with pre-warmed Dulbecco’s modified Eagle’s medium base supplemented with L-glucose, sodium bicarbonate, and 0.1 μM
antimycin A for 0–6 hours. In some experiments, ATP-depleted cells were
allowed to recover by returning the cells to normal growth medium.
2.2 Apoptosis Assays
For caspase assays of siRNA treated cells, LLC-PK1 or HK-2 cells were
seeded in 96-well plates, grown to confluence, and transfected with targeted or
control siRNAs. Cells were subjected to ATP depletion 48 hours post-transfection and assayed for caspase 3/7 activity using the Caspase Glo 3/7 Assay kit
24 (Promega, Madison, WI). HuR-overexpressing cells or controls were seeded at
12 x 103/well, grown to confluence, subjected to ATP depletion, and assayed for caspase 3/7 activity using the same kit. Each treatment was performed in triplicate, and three such independent experiments were performed. For enumeration of apoptotic nuclei, siRNA-treated, HuR-overexpressing, or control cells were cultured on glass coverslips in 24-well plates. Following ATP depletion, cells were permeabilized, fixed with 4% paraformaldehyde and stained with Hoechst stain (1 μg/μl). Samples were examined under a Nikon Eclipse 80i
epifluorescent microscope. Condensed or fragmented nuclei were scored as
apoptotic, and three similar fields of cells were enumerated for each time point.
To test the role of PI3 kinase signaling, HK-2 cells were treated with 50 µM LY-
294002 or vehicle for 16-18 hours prior to ATP depletion and subsequent
caspase assays.
2.3 Antibodies
A mouse monoclonal antibody (3A2) against HuR, a rabbit polyclonal against
Bcl-2, and rabbit polyclonal antibodies recognizing mouse Grb10 and Smad1/5/8
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse
monoclonal against Hsp70, TIAR and eIF3 was purchased from BD Biosciences
(San Jose, CA). Antibodies recognizing ALK2 and β-actin were purchased from
Abcam (Cambridge, MA). Rabbit antibodies against total Akt, phospho-Akt, eIF2
alpha, phospho-eIF2 alpha, PERK, phospho-PERK, and S6, were obtained from
Cell Signaling Technology (Danvers, MA). A rabbit antibody that detects human
25 Grb10 also was purchased from Cell Signaling Technology. Secondary
antibodies coupled to horseradish peroxidase were purchased from
Amersham/GE Healthcare (Piscataway, NJ). Secondary fluorescent antibodies like Alexa 488-or Alexa 568-conjugated goat anti-mouse antibodies were bought
from Molecular Probes (Eugene, OR).
2.4 Inhibitors
The PI3 kinase inhibitor LY-294002 (Cell Signaling Technology) was added to cultures, where appropriate, at a concentration of 50 μM for 16-18 hours prior
to ATP depletion experiments. The NF-κB inhibitor BAY11-7082 (Sigma-Aldrich,
St. Louis, MO) was used at a concentration of 2 µM. Translation inhibitors
cycloheximide and puromycin were obtained from Sigma-Aldrich and used at
concentrations of 100 µg/ml and 10 µg/ml, respectively.
2.5 Western Analysis
Cell lysates were prepared by incubating cells in MPER with protease and
phosphatase inhibitor cocktails (Pierce, Rockford, IL). Cell lysates were
subjected to centrifugation at 10,000 rpm for 5 minutes for the removal of cell
debris. The supernatant protein content was determined using a BCA protein
assay kit (Pierce). For Western blot analysis, proteins were separated by 12.5%
SDS-PAGE (Bio-Rad, Hercules, CA) and transferred onto Hybond-P membrane
(Amersham/GE Healthcare, Piscataway, NJ). The membranes were blocked with
3% milk for 1 hour and incubated with primary antibodies diluted in Blotto. Anti-
26 mouse horseradish peroxidase-conjugated antibody and SuperSignal West Pico
Chemiluminescent substrate (Pierce) were used for signal detection.
2.6 Transfection
For small interfering RNA (siRNA)-mediated knockdown of HuR, LLC-PK1 or
HK-2 cells were plated at 40–50% confluency on six-well plates and transfected transiently with a previously described siRNA [Jeyaraj et al., 2006] of the following sequence: 5’-GGAGGAGUUACGAAGUCUGtt-3’ (sense); 5’-
CAGACUUCGUAACUCCUCCtg-3’ (antisense). HuR siRNA or a non-targeting control (Ambion, Austin, TX) was transfected at 50 nM using Lipofectamine with
Plus reagent (Invitrogen, Carlsbad, CA). RNAi-mediated knockdown of Grb10 was performed by transfecting 100 nM commercially available siRNA (Santa
Cruz) into HK-2 cells using Lipofectamine and Plus reagent (Invitrogen).
For overexpression of a FLAG-tagged HuR cDNA, addition of FLAG to the
COOH-terminal end of the full-length murine HuR coding region was performed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s recommendations. This cDNA, subcloned in expression vector pcDNA3.1 (Invitrogen), or pcDNA3.1 alone as a control, was stably transfected into LLC-PK1 cells. Transfections were performed using
Lipofectamine and Plus reagent (Invitrogen). Forty-eight hours post-transfection,
cells were treated with 800 μg/ml geneticin (Invitrogen); following expansion of
cells, individual colonies were selected and FLAG-tagged HuR expression was
27 proved by Western blotting and immuno-cytochemical analysis, using an anti-
Flag M2 antibody (Sigma-Aldrich, St. Louis, MO).
2.7 RT-PCR and Immuno-RT-PCR.
An internal standard for competitive RT-PCR of HuR was previously synthesized [Jeyaraj et al., 2006]. An internal standard for Grb10 was synthesized using the same methodology. Total RNA from HK-2 cells was isolated using Trizol (Invitrogen) following the manufacturer’s instructions. A mixture of internal standard RNA and cellular RNA was reverse transcribed using the Super Script cDNA synthesis system (Invitrogen). The resulting cDNA was subjected to PCR using Platinum Taq DNA polymerase (Invitrogen). For HuR,
PCR primers were of the sequences 5’-GGTTATGAAGACCACATGGCCG-3’
(sense) and 5’-AAGCCATAGCCCAAGCTGT-3’ (antisense). Human Grb10 primers were of the sequences 5’-
ACAAGGTGGAGCAGACAAGGTGGAGCAGACACCTC-3’ (sense) and 5’-
GTAAAGAACCCGGCGGTGAGC-3’ (antisense), while murine Grb10 primers were of the sequences 5'-GAATCCAGTGAACTTCTTCCC-3’ (sense) and 5'-
GCGTTGTACTGCTTCTTTCC-3' (antisense).
Immunoprecipitation of HuR followed by RT-PCR of associated mRNAs was performed as previously described [Jeyaraj et al., 2006]. Briefly, HK-2 cells were lysed in CEB buffer (10 mM HEPES, pH 7.5, 3 mM MgCl2, 14 mM KCl, 5%
Glycerol, 0.2% NP-40, 1 mM DTT) containing protease and RNAse inhibitors,
followed by immunoprecipitation of HuR using mouse monoclonal 3A2 (Santa
28 Cruz) and Protein A/G (Thermo Scientific). The resulting precipitate was washed
with CEB buffer, and RNA was extracted using Trizol. Following RNA isolation
and cDNA synthesis, the presence of Grb10 mRNA was assessed using the
PCR primers described above.
2.8 Immunocytochemistry
4 For immunolocalization studies, 4 x 10 LLC-PK1 or HK-2 cells were seeded on glass coverslips in 24-well plates and grown to confluence, at which time the cells were given fresh medium and cultured overnight prior to use. Following the necessary treatments, cells were fixed and permeabilized in 2% formaldehyde in stabilization buffer [Jeyaraj et al., 2006]. Cells were then probed with relevant primary antibodies. Alexa 488-or Alexa 568-conjugated goat anti-mouse secondary antibodies were obtained from Molecular Probes (Eugene, OR). Cells were visualized with a Nikon Eclipse 80i epifluorescent microscope with SPOT software (Diagnostic Instruments, Sterling Heights, MI), or with a Zeiss 510
META laser scanning confocal microscope at the Campus Microscopy and
Imaging Facility at The Ohio State University.
2.9 PCR Arrays
RT2 Profiler PCR arrays and associated reagents were purchased from
SABiosciences/Qiagen. To compare gene expression between control and HuR- suppressed HK-2 cells, total RNA was harvested from cells treated with control oligonucleotide or HuR siRNA 48 hours post-transfection, then subjected to ATP
29 depletion for 6 hours. cDNA synthesis was performed using an RT2 First Strand
kit. PCR arrays were analyzed using a Bio-Rad iCycler iQ Real-time Detection
System in the Plant-Microbe Genomics Facility at The Ohio State University.
Analysis of resulting PCR products was performed using RT2 Profiler PCR Array
Data Analysis software according to SABiosciences/Qiagen protocols.
2.10 Plasmids
Plasmids encoding wild-type and constitutively active Akt were a kind gift of
Dr. Michael Ostrowski (The Ohio State University). The cDNA encoding mouse
Grb10 eta was an I.M.A.G.E. clone obtained from the American Type Culture
Collection (accession number BC016111). Restriction endonuclease XbaI was
used to remove 2434 bases (82.4%) of the Grb10 3’ untranslated region for
experiments testing the role of the 3’ UTR in Grb10 expression. Other plasmids were previously derived in the Lee laboratory.
2.11 In Situ Hybridization
For in situ hybridization of total HuR mRNA, an antisense probe was transcribed in vitro using the MAXIscript system (Ambion). The XhoI-KpnI fragment was subcloned into pBluescript and an antisense probe was made by linearizing the plasmid with XhoI and performing transcription using T7 RNA
polymerase. Alexa 488-labeled UTP (Molecular Probes) replaced unlabeled UTP
in this reaction. The hybridization procedures were essentially as previously
described [Jeyaraj et al., 2006]. The probe was directed to 400 nucleotides
30 present in the coding region. Thus, both HuR mRNA isoforms can be detected
using this probe.
2.12 Sucrose Density Gradients
7 For ribosomal profile protein analysis, 1 x 10 LLC-PK1 cells were seeded in
a 100 mm dish. After 1 or 2 hours of ATP depletion and 4 hours of recovery in
normal growth medium, cells were incubated with 100 μg/ml cycloheximide for 20
minutes. Cells were harvested in PBS, washed twice in PBS and gently lysed on
ice with 500 μl chilled Mg buffer (10 mM HEPES, 10 mM NaCl, 3 mM CaCl2, 7 mM MgCl2, 0.5% NP40) with 1 mM DTT and 100 U/ml RNasin (Promega,
Madison, WI). Cell lysates were centrifuged for 2 minutes at 14,000 × g, then
layered onto a 10 ml linear gradient of 15% to 45% sucrose in 10 mM HEPES, 10
mM NaCl, 3 mM CaCl2, 7 mM MgCl2, 1 mM DTT. Lysates were centrifuged at
36,000 × g for 2.25 hours at 4°C in a Beckman SW41 rotor. Gradients were fractionated and monitored at A254 using an ISCO fractionation system (Lincoln,
NE). Proteins in each fraction were combined as required, precipitated with ice
cold 20% trichloroacetic acid (TCA) for 30 minutes on ice, and centrifuged for 20
minutes at 12,000 RPM at 4°C. Pellets were washed 5 times with acetone, and
then dried for 2 hours in a Speedvac. Pellets were resuspended in SDS loading
dye, boiled for 5 minutes, and loaded on a 12.5% SDS-polyacrylamide gel and
subjected to western blot. RNA was extracted with Trizol and RT-PCR was HuR
mRNA was detected using two different primer sets. Total HuR mRNA was
detected using primer pairs 5’-GGTTATGAAGACCACATGGCCG-3’ (sense) and
31 5’-AAGCCATAGCCCAAGCTGT-3’ (antisense) whereas long form HuR mRNA
was identified by using primer pairs 5’-CGCGCTGAGGAGGAGCC-3’ (sense)
and 5’-CCTGGGTCATGTTCTGAGGGAG-3’UTR (antisense).
2.13 Gel Mobility Shift Assay
Nuclear extracts were prepared from untreated or ATP-
depleted/recovered LLC-PK1 cells as previously described [Lee et al., 1988].
Double-stranded DNA oligonucleotide probes were synthesized that
corresponded to -148 to -109 or -77 to -37 of the porcine HuR 5’-UTR (numbered relative to the translational start). Probes were end-labeled with [γ-32P]-ATP and
T4 polynucleotide kinase using a gel shift assay system (Promega Corp.,
Madison, WI). Fourteen micrograms of nuclear extract were incubated with
labeled probe in the provided binding buffer at room temperature for 20 minutes
prior to separation in a nondenaturing 4% polyacrylamide gel. Supershift assays
were performed by pre-incubating 6 µg of anti-Smad 1/5/8 antibody (Santa Cruz
Biotechnology) with nuclear extracts for 1 hour at 4°C prior to the addition of
radiolabeled probe. Oligonucleotide competition reactions were performed by
pre-incubating the nuclear extracts with unlabeled oligonucleotide for 10 minutes
at room temperature prior to the addition of the labeled probe.
2.14 Chromatin Immunoprecipitation (ChIP)
ChIP assays were performed with the Magna ChIP A kit (Millipore,
Billerica, MA) according to the manufacturer’s protocol. Control and treated LLC-
32 6 PK1 cells (10 x 10 each) were fixed for 10 minutes at room temperature and sonicated according to the manufacturer’s protocol. DNA bound to Smad 1/5/8 was precipitated with anti-Smad 1/5/8 antibody N-18 (Santa Cruz Biotechnology).
To amplify the Smad-binding region in 5’-UTR of the HuR gene, the precipitated
DNA was subjected to PCR using Prime-STAR HS DNA polymerase with GC buffer (Takara Bio Inc.) with the following primers under the following conditions:
5’-GCTGAGGAGGAGCCGC-3’ (sense) and 5’-GGCTGCTCCGGGTCG-3’
(antisense); 3 minutes at 94 °C; and then 30 cycles of 98 °C for 10 seconds, 66
°C for 5 seconds, and 72 °C for 30 seconds; followed by a final extension at 72
°C for 2 minutes. The PCR product was electrophoresed in a 5% polyacrylamide gel and quantified using Image J software. The identities of the resulting bands were confirmed by DNA sequencing.
33
CHAPTER 3
HuR Promotes Cell Survival in ATP-depleted Renal Proximal Tubule Cells
Adapted from Ayupova & Singh et al., Am J Physiol Renal Physiol. 2009 July;
297(1): F95–F105.
3.1 INTRODUCTION
Renal ischemia-reperfusion (IR) injury is a major cause of acute kidney
failure, a clinical condition that despite decades of basic research and technical advances remains associated with high morbidity and mortality [Devarajan,
2006]. Thus understanding the molecular mechanisms of renal cell injury is of
particular importance to the prevention or treatment of acute renal failure.
Various cellular events contribute to IR injury, including reduced cellular
ATP levels, damage to the cytoskeleton, generation of reactive oxygen species,
accumulation of inflammatory cells, and other shifts in cellular activity [Weinberg,
1991]. A number of recent studies have examined whether activation or inhibition
of specific intracellular signaling pathways provides protection from IR injury. It
has been reported that preconditional activation of hypoxia-inducible factors
[Bernhardt et al., 2006], gene silencing of complement 3 and caspase 3 [Zheng
et al., 2006], and adenovirus mediated Bcl-2 and Bcl-XL gene transfer [Chiang et
al., 2005; Chein et al., 2007] ameliorate ischemic acute renal failure, whereas
34 pretreatment with cyclooxygenase (COX)-2 inhibitor augments the degree of
renal dysfunction and injury caused by IR [Patel et al., 2007].
Previous results from the Lee laboratory using the proximal tubule cell line
LLC-PK1 subjected to ATP depletion and recovery suggested a role for human
antigen R (HuR) in protecting kidney epithelia from injury during ischemic stress
[Jeyaraj et al., 2005, 2006]. HuR is a ubiquitously expressed member of the
ELAV family of proteins, participating in posttranscriptional regulation of mRNAs bearing uridine-rich or adenine- and uridine-rich elements (AREs) in their 3’ untranslated sequences [Lopez et al., 2004]. Through identification of its targets,
HuR has emerged as an important regulator in cell division, carcinogenesis, immune responsiveness, and the response to cellular stress [Gallouzi et al.,
2001]. The protective role of HuR during cellular stress is effected, at least partially, through its positive influence on the expression of antiapoptotic genes, as well as its negative effect on proapoptotic genes. HuR was shown to stabilize
SIRT, a major antiapoptotic protein, the human ortholog of Sir2 (the stress- response and chromatin-silencing factor in S. cerevisae) [Abdelmohsen et al.,
2007]. Prothymosin α (ProTα), an inhibitor of apoptosome formation, is regulated by HuR via elevation of its translation [Lal et al., 2005]. More recently, two other
HuR target mRNAs that encode antiapoptotic proteins, Bcl-2 and Mcl-1, have been identified [Abdelmohsen et al., 2007]. Available evidence indicates that
HuR contributes to maintaining their elevated levels, although the precise mode of its action remains to be elucidated. HuR also modulates the expression of numerous proteins that indirectly prevent apoptosis by involvement in regulation
35 of cell signaling [Levy et al., 1998], cell division [Wang et al., 2000], and other functions.
Under normal cellular growth conditions, HuR is localized predominantly in
the nucleus, while stabilization and regulation of ARE-containing mRNA is
associated with translocation of HuR to cytoplasm. In the nucleus, HuR has been
postulated to participate in the processing of pre-mRNA (likely splicing and
export), although these functions remain poorly understood. Recent data about
the function of HuR as an alternative Fas pre-mRNA splicing regulator provide
new insight about HuR as an antiapoptotic and survival factor [Izquierdo, 2008].
However, in spite of the abundance of studies demonstrating an antiapoptotic role for HuR, in some instances a proapoptotic role has been noted [Mazroui et
al., 2008]. It has been suggested that in response to a variety of stresses, HuR
may promote the expression of proapoptotic mRNAs. In one study, HuR cleavage was triggered in response to lethal stress, and the HuR cleavage products were capable of promoting apoptosis [Mazroui et al., 2008]. Therefore, it is important to establish the function of HuR in a given cell type and stress event. In work previously published by the Lee laboratory on the LLC-PK1
proximal tubule cell model, energy depletion at the cellular level caused
detectable net movement of HuR into the cytoplasm where it can stabilize target
mRNAs, followed by net movement of HuR back into the nucleus on reversion to
normal growth medium. Changes observed in HuR protein and mRNA levels
during ATP depletion and recovery also suggested a role for HuR in
preconditioning LLC-PK1 cells to multiple ischemic insults [Jeyaraj et al., 2006]. In
36 this study, the role of HuR expression in the protection of LLC-PK1 cells from
apoptosis was determined to further explore HuR’s function in protecting renal
epithelia from stress.
3.2 RESULTS
3.2.1. Nature and Time Course of Cell Death in ATP-depleted LLC-PK1 Cells.
To determine a role for HuR in renal cell survival, we first needed to establish the nature and time course of death in LLC-PK1 cells undergoing ATP
depletion. This was accomplished by incubating the cells in DMEM base medium
lacking serum and glucose and containing 0.1 μM antimycin, an inhibitor of
mitochondrial ATP synthesis, and measuring cell viability followed by assays for
apoptosis/necrosis. This methodology reduces ATP levels to ~1% normal within
1 hour [Jeyaraj et al., 2006]. Our initial experiments demonstrated that long-term
ATP depletion in our system (>4 hours) induces significant cell death. The
potential role of apoptosis in this death was analyzed using standard indicators
including Hoechst staining, which detects presence of chromatin condensation
and apoptotic bodies, or by measuring activities of caspase 3/7. In some cases,
apoptosis was assayed also by the use of a commercially available fluorescent
TUNEL assay (Roche Applied Science) that permits measurement of cleaved
DNA by fluorescent microscopy. An example of a TUNEL assay is shown in
Figure 3.1. Apoptosis was found to increase from 1 to 6 hours of ATP depletion
but was more pronounced between 4-6 hours. Necrosis was not prevalent until
after 6 hrs of ATP depletion (data not shown).
37
3.2.2. Determination of Role of HuR in Cell Survival
Once we determined the time course of LLC-PK1 cells undergoing apoptosis
during the early hours of ATP depletion, siRNA was used to knock down HuR
expression to determine a potential role of this protein in promoting cell survival.
As previously tested, 1-2 days of siRNA treatment was sufficient to suppress
HuR in LLC-PK1 cells [Jeyaraj et al., 2005]. Following this treatment, cells were
subjected to ATP depletion, and measurement of apoptosis was performed using
caspase 3/7 assays and scoring of apoptotic nuclei.
We have previously demonstrated the ability to use siRNAs to suppress
HuR in LLC-PK1 cells under normal growth conditions [Jeyaraj et al., 2006], and
Figure 3.2A demonstrates that knockdown was similarly efficient in cells
undergoing ATP depletion (knockdown averaged about 75%). Measurement of
caspase 3/7 activation (Figure 3.2B, left) or quantitation of condensed/fragmented nuclei (Figure 3.2B, right) demonstrated that ATP depletion of control cells resulted in increasing levels of apoptosis over a 6 hour
period. Suppression of HuR expression by siRNAs resulted in even greater apoptosis during ATP depletion. Similar results were obtained using the human proximal tubule cell line HK-2 (data not shown). These results indicate the protective role of HuR in proximal tubule cells during ATP depletion.
3.2.3. Overexpression of HuR Promotes Cell Survival
38 For overexpression of HuR in LLC-PK1 cells, a murine HuR cDNA was epitope tagged so it was distinguishable from the endogenous (porcine) LLC-PK1
HuR by Western analysis. For this purpose, a FLAG tag (8 amino acid residues) was appended to the C-terminus of HuR. The FLAG epitope tag was chosen because a previously made HuR-FLAG hybrid was shown to traffic appropriately in NIH 3T3 cells [Lal et al., 2005]. The same epitope tag was inserted at the C- terminal end of the coding region while still maintaining the integrity of the 3’
UTR, using the Quick-Change II site directed mutagenesis system (Stratagene).
Clonal LLC-PK1 lines expressing the HuR-FLAG hybrid or empty pcDNA3.1
vector were then made by isolating stably transfected cells expressing a
geneticin resistance marker. Following expansion of cells, individual colonies
were selected and FLAG-tagged HuR expression was proved by Western
blotting and immunocytochemical analysis. With the use of commercially
available anti-FLAG antibody, products of exogenously expressed clones were
detected. Expression and correct localization of FLAG-tagged HuR was checked
by immunocytochemistry. As shown in Figure 3.3, LLC-PK1 cells stably
expressing empty vector or FLAG-tagged HuR were grown under both normal
and ATP-depleted conditions. The vector-only cells demonstrate the distribution
of HuR seen in untreated LLC-PK1 cells; namely, that in normal growth medium,
HuR is primarily nuclear, but upon ATP depletion, its distribution becomes more
cytoplasmic. Similarly, detection of the FLAG tag in the HuR-overexpressing lines
shows that the tagged version also is distributed primarily in the nucleus in
normal growth medium but becomes cytoplasmic under ATP depletion
39 conditions. These results demonstrate appropriate trafficking of the HuR-FLAG hybrid protein.
FLAG-tagged HuR overexpressing clones were used to study the effect
of heightened levels of HuR on apoptosis. Although only one clonal line is
discussed here, a second line produced very similar results (data not shown).
As shown in Figure 3.4A, expression of HuR in these lines was approximately
2-3-fold times normal. These cells were subjected to ATP depletion for
different times and relative caspase 3/7 activity was measured (Figure 3.4B,
left), as well as numbers of apoptotic nuclei (Figure 3.4B, right). A decrease in
apoptosis was noted in HuR overexpressing cell lines in comparison to a line
stably transfected with empty vector. This further establishes the protective
role of HuR under conditions of ATP depletion.
3.2.4. HuR Promotes Cell Survival by Inhibiting Both Intrinsic and Extrinsic
Apoptosis Pathways.
Understanding the molecular events that lead to apoptosis in response to
ATP depletion, and how HuR delays apoptotic death, provides an understanding
of HuR’s function in renal ischemia related apoptosis. In order to observe
whether HuR mediates its anti-apoptotic effects through extrinsic and/or intrinsic
apoptotic pathways during ATP depletion, HuR was suppressed in LLC-PK1 cells
using siRNA HuR. Following the suppression, cells were ATP depleted for
various time points, and relative caspase 9 (intrinsic pathway, Figure 3.5A) and
caspase 8 (extrinsic pathway, Figure 3.5B) activities were measured.
40 Suppression of HuR expression by siRNAs resulted in greater apoptosis of both
pathways during ATP depletion. These results indicate that both pathways are
activated during ATP depletion of renal proximal tubule cells, and that HuR play a
protective role in each of them.
3.2.5. HuR Stabilizes Cell Survival mRNAs Bcl-2 and Hsp70 under Cell
Stress.
We wished to determine whether HuR might play a role in modulating
expression levels of proteins known to play protective roles in in vivo and in vitro
models of renal ischemia. The expression of Bcl-2, which is upregulated in
regenerating proximal tubule cells and is protective against renal ischemic injury
[Basile et al., 1997; Suzuki et al., 2008], was previously shown to be modulated
by HuR [Abdelmehsen, 2007]. As demonstrated in Figure 3.6, siRNA-mediated
suppression of HuR resulted in diminished Bcl-2 expression during ATP depletion in LLC-PK1 cells. In addition, suppression of HuR mildly diminished expression of Hsp70, a heat shock protein known to be upregulated primarily in
proximal tubule cells by renal ischemia and involved in a cellular-protective
response [Smoyer, 2000]. This is consistent with other studies demonstrating the
Hsp70 mRNA to be a target of HuR binding [Lopez et al., 2007]. Thus, HuR
appears to play a protective role in energy-depleted renal epithelia in vitro.
3.3. CONCLUSION
41 IR-induced changes include damage of renal tubular epithelial cells and especially cells of proximal tubules, known to be more susceptible to injury in this setting. Using an ATP depletion model in the proximal tubule cell line LLC-PK1, we have suggested a role for HuR in protecting kidney epithelia from injury during ischemic stress. Based on pull-down of HuR-mRNA complexes followed by microarray analysis, HuR has been predicted to bind several thousand cellular mRNAs [Lopez et al., 2004]. Here, we have demonstrated that suppression of
HuR in LLC-PK1 cells diminishes expression of at least two molecules key to cell survival following renal ischemia and reperfusion, Bcl-2 and Hsp70. With maturation of the kidney, Bcl-2 expression is detected in the parietal epithelium of
Bowman’s capsule, in the distal convoluted tubule and loop of Henle, and in widely scattered cells within proximal convoluted tubules and papillary collecting ducts. Bcl-2 resides in the mitochondria and prevents activation of the effector caspases. In addition, it is known that over-expression of Bcl-2 can block both apoptosis and necrosis [Supavaken, 2003], and protect ischemic tissue against reperfusion induced stress [Gobe, 1999].
Hsp70 is a stress response protein that interacts with cytoskeletal elements during the restoration of proximal tubule cell structure and polarity after renal ischemia [Bettina et al., 2000]. Hsp70 has been demonstrated to be protective in renal tubular cell apoptosis that is induced by inflammatory cytokine or ATP depletion [Supavaken, 2003]. In our findings, the level of both Hsp70 and Bcl-2 anti-apoptotic proteins decreased upon suppression of HuR (Figure 3.6). Taken together, our results show that HuR plays an antiapoptotic role in a proximal
42 tubule cell line, which suggests protective and preconditioning roles of HuR in renal ischemic injury.
43
Figure 3.1. Time Course of Apoptosis in LLC-PK1 cells during ATP Depletion. TUNEL assays were used to determine levels of apoptosis in LLC-
PK1 cells undergoing 30 minutes to 6 hours of ATP depletion. Apoptosis, as indicated by the fluorescent green labeling, steadily increased during this time.
44
Figure 3.2. Effects of HuR Knockdown on Apoptosis in ATP-depleted LLC-
PK1 cells. (A) Knockdown of HuR in LLC-PK1 cells with a small interfering RNA against HuR prior to ATP depletion resulted in an average knockdown of ~75%, as indicated by Western blot. Levels of β -actin are presented as a control for loading. The increase in HuR protein seen during ATP depletion was previously described [Jeyaraj et al., 2006]. (B) Knockdown of HuR resulted in increased apoptosis of LLC-PK1 cells during ATP depletion, as assayed by caspase 3/7 activity (left) or numbers of apoptotic nuclei (right). Values are mean + standard deviation; n = 3. *P < 0.05; **P < 0.01.
45
Figure 3.3. Distribution of Native and FLAG-tagged HuR in Stably
Transfected LLC-PK1 Cells. LLC-PK1 cells stably expressing a FLAG-tagged HuR construct were immunolabeled with an anti-FLAG antibody (top row). When cells were cultured in normal growth medium, the FLAG-HuR was primarily nuclear in distribution. However, ATP depletion for 4 hours resulted in movement of FLAG-HuR into the cytoplasm. The distribution of FLAG-HuR in these cells is consistent with that of endogenous HuR in LLC-PK1 cells transfected with an empty vector (bottom row) or untransfected cells [Lal et al., 2005].
46
Figure 3.4. Effects of HuR Overexpression on Apoptosis in ATP-depleted
LLC-PK1 cells. (A) Stable transfection of FLAG-tagged HuR into LLC-PK1 clonal lines resulted in increased overall HuR levels, as indicated by Western blot. In two separate lines, HuR expression was increased 2-3 fold. The lower panel indicates β -actin levels as a loading control. (B) HuR overexpression resulted in decreased apoptosis of LLC-PK1 cells during ATP depletion, as assayed by caspase 3/7 activity (left) or numbers of apoptotic nuclei (right). Values are mean + standard deviation; n = 3. *P < 0.05; **P < 0.01.
47
Figure 3.5. Effects of HuR Knockdown on Intrinsic and Extrinsic Pathways of Apoptosis. LLC-PK1 cells were treated with an siRNA to HuR or control oligonucleotides prior to ATP depletion for up to 6 hours. Relative caspase 8 or 9 activity was measured using a Caspase Glo Assay Kit (Promega). Knockdown of HuR resulted in increased apoptosis through both the intrinsic (caspase 9) and extrinsic (caspase 8) pathways. Values are mean + standard deviation; n = 3. *P < 0.05.
48
Figure 3.6. Knockdown of HuR in ATP-depleted LLC-PK1 Cells Results in Suppression of Cell Survival Proteins. HuR expression was knocked down in LLC-
PK1 cells with siRNAs prior to ATP depletion for up to 6 hours. Bcl-2 and Hsp70 levels were assessed by Western analysis. Knockdown of HuR resulted in loss of expression of both survival proteins. β-actin levels were assessed as loading controls.
49
CHAPTER 4
HuR mediates anti-apoptotic effect by amplifying Akt signaling through a positive feedback loop, via Grb10
4.1 INTRODUCTION
HuR (human antigen R) is a ubiquitously expressed regulator of post- transcriptional processing, with effects on pre-mRNA splicing, mRNA stability, and translation. HuR has been estimated to regulate ~8% of the human transcriptome, with broad effects on cell cycle, apoptosis, and inflammatory responses [Khabar, 2005]. Its most profound effects are on mRNA stability, in which it binds to adenine- and uridine-rich elements (AREs) in the 3’ untranslated regions of mature mRNAs and slows their rates of degradation. Regulation of
HuR expression must be very tightly controlled, as loss of HuR expression results in rapid cell death [Ghosh et al., 2009; Katsanou et al., 2009], while overexpression by only a few-fold results in tumorigenicity [Danilin et al., 2010;
Denkert et al., 2004a; Denkert et al., 2004b; Lopez de Silanes et al., 2003; Lopez de Silanes et al., 2005; Mazan-Mamczarz et al., 2008; Nabors et al., 2001]. We have demonstrated that in cultured renal proximal tubule cells, HuR stabilizes mRNAs and prevents apoptosis resulting from ATP depletion [Ayupova and
Singh et al., 2009; Jeyaraj et al., 2005]. In rat kidneys, ischemia-reperfusion injury results in overall increases in HuR mRNA throughout the nephron, but HuR
50 protein levels are increased only in proximal tubules [Ayupova and Singh et al.,
2009]. This distinction is notable, as proximal tubule cells are highly sensitive to
ischemic injury.
HuR’s capacity to bind mRNAs is enhanced during cell stress, when it switches from a typical nuclear steady-state location to the cytoplasm [Atasoy et
al., 1998; Fan and Steitz, 1998]. Stresses that trigger HuR activation include
energy depletion, ultraviolet light, hypoxia, nutrient deprivation, and heat shock,
among others. HuR has been shown to promote the mRNA stability and/or
translation of several anti-apoptotic proteins including Bcl-2, Mcl-1, prothymosin-
α, and XIAP. In addition, it was demonstrated that HuR promotes alternative
splicing of the mRNA for the apoptosis receptor Fas such that the resulting
protein no longer retains its pro-death functions [Izquierdo, 2008].
The PI3K/Akt pathway has been demonstrated to be critical to cell survival
and repair in multiple tissue injury models, including the kidney. Renal ischemia/reperfusion (IR) was shown to transiently increase Akt activation
[Andreucci et al., 2003], which was further demonstrated to play a protective role in IR-injured kidney function [Satake et al., 2008]. The PI3K/Akt pathway also has been shown to protect against acute kidney injury induced by cisplatin toxicity [Kuwana et al., 2008]. Akt, a family of highly related serine/threonine kinases, protects against apoptosis through phosphorylation of multiple proteins involved in regulating cell survival, including Bcl-2 family members, caspases and caspase inhibitors, the mTOR signaling pathway, and FoxO transcription factors.
Previous studies have suggested interaction between Akt signaling and HuR
51 function. In gastric tumor cells, it was shown that Akt activation could promote
HuR expression through stimulation of an NF-κB element in the HuR promoter
[Kang et al., 2008]. Conversely, it was suggested that HuR was required for Akt activation in renal tubule cells [Danilin et al., 2010]. Therefore, the precise interaction of HuR with the PI3K/Akt pathway is unclear.
Grb10 is a member of a family of adaptor proteins (including Grb7 and
Grb14) that functions downstream of multiple receptor tyrosine kinases such as
the insulin and insulin-like growth factor-I receptor [Liu and Roth, 1995; Morrione
et al., 1996], growth hormone receptor [Moutoussamy et al., 1998], and the Ret
receptor tyrosine kinase [Pandey et al., 1995], among others. Grb10 may also
interact with a number of non-receptor kinases, including Akt. It has been
proposed that Grb10 stimulates Akt function by translocating Akt to the plasma
membrane where it is phosphorylated and activated by PI3 kinase [Jahn et al.,
2002]. Here we show that HuR binds Grb10 mRNA and promotes Grb10
expression, leading to their participation in a positive feedback loop that amplifies
the pro-survival functions of Akt signaling in renal proximal tubule cells.
4.2. RESULTS
4.2.1. HuR Expression is Regulated by PI3K/Akt Activity in Proximal Tubule
Cells.
Numerous previous reports have established the importance of PI3K/Akt
activity in cell survival in energy-depleted renal epithelia [Andreucci et al., 2003;
Chen et al., 2008; Joo et al., 2006; Loverre et al., 2004; Sharples et al., 2004; Xie
52 et al., 2006]. To confirm these findings in vitro, the human proximal tubule cell line HK-2 was subjected to ATP depletion in the presence or absence of PI3K inhibitor LY-294002, and apoptosis was measured through assessment of caspase 3/7 activity. Figure 4.1A demonstrates that over the course of 6 hours, activity of these caspases increased approximately 7-fold and that the magnitude of this response was almost doubled by inhibition of PI3K signaling. In addition,
Figure 4.1B demonstrates that ATP depletion resulted in changes in the distribution of Akt. Control HK-2 cells demonstrated a primarily nuclear distribution of Akt, as has been demonstrated for a number of cell types including
HEK293, HeLa, PC12, and cardiomyocytes [Martelli et al., 2006; Miyamoto et al.,
2009; Wang and Brattain, 2006]. In contrast, ATP-depleted cells demonstrate a clear accumulation of Akt at the cell periphery (arrows), indicative of increased activity. ATP depletion also induces an increased in pAkt levels, as assessed by
Western blots (below). These results confirm a role for PI3K/Akt signaling in cell survival in HK-2 cells undergoing energy depletion.
PI3K/Akt signaling was previously shown to increase HuR expression in gastric tumor cells [Kang et al., 2008]. Because HuR, like PI3K/Akt, promotes cell survival in energy-depleted renal epithelia [Ayupova and Singh et al., 2009], we performed experiments to determine whether PI3K/Akt signaling enhanced
HuR expression in proximal tubule cells. The HK-2 cell line was ATP depleted in the presence or absence of LY-294002 and HuR expression levels were determined. As was previously demonstrated in the LLC-PK1 cell line [Jeyaraj et al., 2006], ATP depletion of HK-2 cells results in increased HuR protein
53 expression without increased HuR mRNA levels. Here, competitive RT-PCR
revealed that HuR mRNA levels did not change over 6 hours of ATP depletion in
control cells, but inhibition of PI3K by LY-294002 resulted in loss of HuR mRNA
expression (Figure 4.1C, top row). Figure 4.1C, middle row, demonstrates that
HuR protein levels increased during ATP depletion in HK-2 control cells.
However, consistent with the RT-PCR results, LY-294002 treatment caused a
dramatic loss of HuR protein. These results demonstrate that PI3K activity
regulates HuR expression in proximal tubule cells under both normal (timepoint
0) and energy-depleted conditions. Finally, to determine whether PI3K regulates
HuR expression via Akt, HK-2 cells were transfected either with wild-type or
constitutively active (myristoylated) Akt1. As shown in 4.1D, total Akt levels were
increased when either wild-type or CA-Akt was overexpressed. However, only
the constitutively active form caused an increase in HuR protein levels, indicating that PI3K/Akt signaling plays an important role in regulating HuR expression in proximal tubule cells under normal and stressed conditions.
4.2.2. NF-κB is Critical for HuR Expression during Normal Growth.
To determine whether PI3K/Akt signaling regulates HuR expression through
the NF-κB signaling pathway, normally growing HK-2 cells were treated with
inhibitor NF-κB inhibitor BAY11-7082, and HuR levels were assessed. Both
Western blot (Figure 4.2A) and competitive RT-PCR (Figure 4.2B) demonstrate
that inhibition of NF-κB activity in normally growing cells results in dramatic
decreases in HuR expression at both the mRNA and protein levels. To
54 demonstrate the relationship between Akt and NF-κB relative to HuR expression,
HK-2 cells overexpressing either WT or CA Akt were treated with NF-κB inhibitor
BAY11-7082. As shown in Figure 4.2C, inhibition of NF-κB resulted in loss of
HuR, indicating that Akt is upstream of NF-κB in this signaling pathway.
We were unable to assess the effects of NF-κB inhibition in cells undergoing
ATP depletion, as the two treatments together resulted in rapid (within minutes)
cell death. However, as we previously published, HuR protein levels increase during ATP depletion without an associated change in HuR mRNA levels [Jeyaraj et al., 2006]. Therefore, it seems unlikely that NF-κB signaling plays a significant
role in modulating HuR levels during ATP depletion.
4.2.3. Akt Activity but not Akt Expression is Regulated by HuR Levels in
Proximal Tubule Cells.
The previous data demonstrate that HuR levels are increased by PI3K/Akt
signaling in proximal tubule cells. Interestingly, other studies have suggested
that this relationship may be reciprocal; that is, that HuR can also modulate Akt
activity [Danilin et al., 2010; Meng et al., 2002]. Indeed, our own preliminary
studies using PCR arrays to examine changes in gene expression with HuR
knockdown suggested that HuR levels do affect Akt signaling (not shown).
Therefore, we performed Western analysis in our cell model to determine
whether siRNA-mediated suppression of HuR affected Akt expression or activity.
Figure 4.3A, top row, illustrates that HuR protein expression, which increases
during ATP depletion, can be strongly inhibited by HuR siRNAs in HK-2 cells.
55 Figure 4.3A also demonstrates that Akt is activated during ATP depletion and
that HuR knockdown almost completely abolishes this activation. In contrast,
total Akt expression is unaffected by the knockdown, demonstrating that HuR
modulates activation, rather than the expression, of Akt.
4.2.4 Expression of Adaptor Protein Grb10 is Regulated by HuR Levels in
Proximal Tubule Cells.
Because HuR is a post-transcriptional regulator of gene expression and not
a kinase, it follows that HuR’s effects on Akt activity are likely to be produced by alteration in expression of an upstream activator of Akt. To determine what members of the PI3K/Akt signaling pathway might be regulated by HuR, PCR array analysis was performed on control HK-2 cells and those in which HuR expression was suppressed by RNAi. In two independent experiments, the mediator of Akt signaling most strongly affected by HuR suppression showed at least a 10-fold loss of expression in HuR-suppressed cells. This protein, Grb10, is a member of the Grb7 family of adaptor proteins that has been shown to be a key regulator of PI3 kinase activity downstream of the insulin and insulin-like growth factor-1 receptor tyrosine kinases [Lim et al., 2004; Riedel, 2004]. To confirm the PCR array analysis, Grb10 mRNA levels were compared in control cells and cells in which HuR was suppressed by RNAi. As shown in Figure 4.3B,
Grb10 mRNA levels increased throughout ATP depletion; however, suppression of HuR inhibited Grb10 expression.
56 This result suggests that Grb10 mRNA may be a target of HuR-mediated
post-transcriptional control. To determine whether HuR binds Grb10 mRNA,
HuR from control or ATP-depleted cells was immunoprecipitated, the co-
precipitating mRNAs were isolated, and the presence of Grb10 mRNA was
assessed by RT-PCR. As shown in Figure 4.3C, under both normal and
stressed conditions, HuR was capable of co-precipitating Grb10 mRNA. An
increase in HuR-associated Grb10 was noted in stressed cells, as is consistent
with HuR’s protective role (in 3 independent experiments, an average increase of
66 + 21% over control cells). A search of the ARED databases [Bakheet et al.,
2006; Halees et al., 2008], which identify ARE-containing mRNAs through in
silico analysis, indicated that Grb10 contains at least one of these sequences.
To determine whether the 3’ UTR does indeed contain a functional ARE, full- length mouse Grb10 or a mutant lacking most of the 3’ UTR was transfected into
HK-2 cells in the presence or absence of exogenous HuR. As shown in Figure
4.3D, the exogenous mouse Grb10 mRNA and protein were then detected by
RT-PCR (top row) or in Western blots using an antibody specific to the mouse protein (middle row). These experiments show that intense bands were detected in cells overexpressing wild-type Grb10 (lanes 3 and 5) but not truncated Grb10
(lanes 4 and 6). This result is consistent with a positive role for the Grb10 3’ UTR in its expression. Additionally, Grb10 levels were highest when co-transfected with HuR (lane 5). These results, coupled with the previous figure showing
HuR’s ability to bind Grb10 mRNA, demonstrate that HuR plays a positive role in
promoting Grb10 expression.
57
4.2.5. Grb10 Stimulates Akt Activation and HuR levels.
As described above, Grb10 has been implicated in some cell types as a positive regulator of cell survival [Lim et al., 2004; Riedel, 2004]. To determine whether Grb10 has any effects on cell survival and the PI3K/Akt signaling pathway in proximal tubule cells, RNAi was used to knock down its expression.
Four alternately spliced Grb10 isoforms with yet unknown specific functions are present in human cells. The siRNAs used in this study were designed to knock down all isoforms. Figure 4.4A demonstrates that Grb10 mRNA can be strongly suppressed in HK-2 cells using nanomolar levels of siRNA. This loss of Grb10 mRNA results in a corresponding loss of Grb10 protein (Figure 4.4B). To determine whether Grb10 has effects on cell survival, HK-2 cells treated with either Grb10 siRNA or a control oligonucleotide underwent ATP depletion, and apoptosis was measured by assays for caspase 3/7 activation. As shown in
Figure 4.4C, ATP depletion in control cells resulted in a ~10-fold increase in caspase 3/7 activity over 6 hours. However, suppression of Grb10 expression increased this response over time, and after 6 hours, caspase 3/7 activity was
~50% higher in the siRNA-treated cells than the control cells. Thus, Grb10 plays an important role in cell survival during energy depletion.
Grb10 was previously shown to be a positive regulator of Akt signaling downstream of PI3 kinase activity [Jahn et al., 2002]. To test whether Grb10 has a similar effect in proximal tubule cells, Grb10 was knocked down by RNAi in cells undergoing ATP depletion, and total and activated Akt levels were assessed
58 by Western blot. As shown in the top row of Figure 4.5A, ATP depletion of
control cells induced Akt phosphorylation, as previously demonstrated in Figure
4.3A. The same row shows that suppression of Grb10 resulted in a dramatic decrease in Akt activation in non-stressed cells and prevented any increase in activation during ATP depletion. This was not due to alterations in total Akt levels, however, as these were unchanged by loss of Grb10 (Figure 4.5A, middle row). Interestingly, siRNA-mediated knockdown of Grb10 also resulted in severe loss of HuR (Figure 4.5A, bottom row). This result suggests that HuR participates in Akt signaling in a positive feedback loop, promoting expression of
Grb10, which then amplifies Akt activation through NF-κB to further enhance
HuR expression. To determine whether knockdown of Grb10 diminishes HuR
expression at the level of mRNA, control cells or those treated with Grb10 siRNA were assessed for HuR mRNA levels by competitive RT-PCR. As shown in
Figure 4.5B, suppression of Grb10 did indeed result in the loss of HuR mRNA.
This result is consistent with Grb10 activating Akt signaling, which then
stimulates expression of HuR.
Finally, we examined the relative distributions of Grb10 and phosphorylated
Akt. Figure 4.5C demonstrates an almost complete overlap of pAkt and Grb10 in
the nucleus under normal growth conditions. In addition, ATP depletion results in
Grb10, along with pAkt, being re-distributed to the cell periphery. As expected
from the results in Figure 4.5A, knockdown of Grb10 resulted in an almost
complete loss of pAkt as assayed by immunocytochemistry (not shown). These
59 results are consistent with a role for Grb10 in assisting in translocation of pAkt to
the plasma membrane in renal epithelia.
4.3 CONCLUSION
The studies described here suggest a central role for HuR within a positive
feedback loop that amplifies Akt signaling in proximal tubule cells. A schematic
of this proposed feedback loop, which is based both on the data presented here
as well as work published in the literature, is shown in Figure 4.6. In brief, we
propose that HuR expression stabilizes Grb10 mRNA, resulting in cytoplasmic
relocalization of pAkt, where it can increasingly stimulate NF-κB activity. NF-κB
activity, then, promotes HuR transcription. We hypothesize that this feedback
loop plays a key role in maintaining pAkt activity under normal growth conditions.
However, we were unable to determine whether NF-κB signaling is important for
HuR levels during energy depletion, since inhibiting this pathway during ATP
depletion rapidly killed the cells. However, this seems unlikely, as we found that
during ATP depletion, HuR protein levels increased without an accompanying
increase in HuR mRNA (this study and [Jeyaraj et al., 2006]). It is possible that
during stress, NF-κB activity is required to just maintain HuR mRNA levels, but it
is clear that an additional level of translational control is responsible for increases
in HuR protein. However, we demonstrated that PI3K/Akt signaling is indeed
involved in increased HuR protein expression during stress, as the PI3K inhibitor
LY-294002 strongly diminished HuR protein levels. Further complicating this
regulation is our finding that HuR mRNA is expressed as two isoforms with
60 different 5’ untranslated regions and different translatabilities in rabbit reticulocyte lysates [Ayupova et al., 2009; Jeyaraj et al., 2010]. Our preliminary studies indicate that these are differentially translated during normal growth and cell
stress (not shown), so experiments are underway to define the timing of these events.
These studies demonstrate how HuR plays a central, positive role in
proximal tubule cell survival through the activities of Akt.
61
Figure 4.1. HuR Levels are Regulated by PI3K/Akt Signaling. (A) Apoptosis levels were assessed by measurements of caspase 3/7 activity in HK-2 cells treated either with vehicle or PI3K inhibitor LY-294002. *, P< 0.05; **, P<0.005. (B) Control or ATP-depleted HK-2 cells were immunolabeled with an antibody against Akt. Arrows indicate plasma membrane distribution of Akt. (C) The effects of PI3K inhibition on HuR levels were measured by competitive RT-PCR (top panel) or Western blot (2nd panel). β -actin was detected as a loading control. For competitive RT-PCR, relative HuR levels are indicated by comparing ratios of the top HuR band to the bottom internal standard band. (D) HK-2 cells were either left untransfected (unt), transfected with an empty expression vector (mock) or transfected with wild-type (WT) or constitutively active (CA) Akt1. Western analysis shows levels of total Akt, HuR, or β -actin as a loading control.
62
Figure 4.2. HuR Levels are Regulated by NF-κB Signaling. The effects of NF- κB inhibition on HuR expression were demonstrated by treatment of HK-2 cells with BAY11-7082. HuR levels were assessed by Western blot (A) and competitive RT-PCR (B). (C) HK-2 cells overexpressing wild-type (WT) or constitutively active (CA) Akt1 were treated with vehicle or BAY11-7082. HuR levels were assessed by Western analysis.
63
Figure 4.3. HuR Regulates Akt Activation and Grb10 Expression. (A) Western analysis demonstrates that ATP depletion increases HuR protein levels and Akt phosphorylation. siRNA-mediated knockdown of HuR strongly inhibits Akt activation without affecting total Akt levels. β-actin is shown as a loading control. (B) Competitive RT-PCR demonstrates that Grb10 mRNA levels increase during ATP depletion, but are diminished by HuR knockdown. (C) HuR was immunoprecipitated from ATP-depleted or control cells, and associated RNA was purified. The presence of Grb10 mRNA was assessed by RT-PCR. Controls demonstrate specificity by showing that a lack of HuR antibody or reverse transcriptase (RT) in the procedure fails to produce Grb10 mRNA. (D) The role of the Grb10 3’ UTR in controlling its expression was demonstrated by both RT- PCR (top row) and Western blot (middle row). Analysis of β-actin levels is included as a loading control for the Western blot (bottom row). Lane 1: untransfected cells; lane 2: cells transfected with empty expression vector; lane 3: transfection with full-length murine Grb10; lane 4: transfection with murine Grb10 lacking 3’ UTR sequences; lane 5: transfection with full-length murine Grb10 plus HuR; lane 6: transfection with murine Grb10 lacking 3’ UTR sequences plus HuR.
64
Figure 4.4. siRNA-mediated Knockdown of Grb10 Increases Apoptosis in ATP-depleted HK-2 Cells. (A) Competitive RT-PCR demonstrates efficient knockdown of Grb10 mRNA with nanomolar levels of siRNA. (B) Western analysis demonstrates efficient knockdown of Grb10 protein during ATP depletion. β-actin levels are assessed as a loading control. (C) Caspase 3/7 assays show that knockdown of Grb10 increases apoptosis during ATP depletion. *, P < 0.05; **, P< 0.01.
65
Figure 4.5. Knockdown of Grb10 inhibits Akt activation and HuR expression. (A) Western analysis of HK-2 cells treated with control oligonucleotides or Grb10 siRNAs show that suppression of Grb10 results in strongly diminished Akt activation and HuR protein expression. (B) Competitive RT-PCR demonstrates that knockdown of Grb10 decreases HuR expression at the mRNA level. (C) Immunocytochemical analysis demonstrates almost complete overlap between Grb10 and pAkt distribution in both control and ATP- depleted cells. ATP depletion results in movement of both proteins to the plasma membrane.
66
Figure 4.6. Schematic of HuR’s role in promoting a positive feedback loop of Akt signaling. Based on data presented here and in the literature, we propose the following positive feedback loop for HuR in Akt signaling. Green arrows represent the pathway found in normally growing cells, while red arrows represent the pathway found in ATP-depleted cells. In normally growing cells, (1) Akt activation stimulates NF-κB activity; (2) NF-κB promotes HuR transcription; (3) HuR binds to and stabilizes Grb10 mRNA, resulting in increased Grb10 expression; (4) Grb10 activates Akt by aiding in its transport to the plasma membrane where it is activated by PI3K. In ATP-depleted cells, the relationship between pAkt activation and HuR levels are not clear, but does not appear to involve NF-κB signaling.
67
CHAPTER 5
Transcriptional and Translational Control of HuR
Some portions adapted from Jeyaraj et al., J. Biol. Chem. 285(7): 4432, 2010
5.1 INTRODUCTION
Cellular levels of HuR must be tightly regulated. We and others showed that
decreased levels of HuR result in increased apoptosis, while levels only a few-
fold over normal can result in tumor formation. However, at the same time, HuR expression must be dynamic to counteract effects of various cellular stresses. An interest of the Lee laboratory has been the regulatory mechanisms that control
HuR expression [Jeyaraj et al., 2006]. We found that ATP depletion and recovery of LLC-PK1 cells results in an unusual decoupling of HuR protein and
mRNA levels. First, ATP depletion induces a slow translation-mediated increase
in HuR protein, without an accompanying increase in HuR mRNA. When cells
are allowed to recover from ATP depletion, HuR protein levels revert to normal,
but HuR transcription is mildly induced. A second insult after recovery results in
a rapid increase in HuR protein without increased mRNA production.
Significantly, this increase in protein is dependent on transcriptional activity
during the prior recovery period. These findings are reminiscent of ischemic
preconditioning; the phenomenon whereby brief ischemic episodes render cells
68 resistant to subsequent ischemic injury [Jeyaraj et al., 2006]. Therefore, the mechanisms by which HuR transcription and translation are regulated may have great impact on protection and recovery of cells from various stresses. The experiments described in this chapter elucidate many of the transcriptional and translational regulatory processes that affect levels of HuR expression during normal growth and stress conditions.
5.2 RESULTS
5.2.1 Gel Shift Assay and ChIP Confirms Binding of Smad 1/5/8 to HuR
Promoter Elements
Results from the Lee laboratory demonstrated that under basal conditions,
HuR mRNA is expressed in two forms: one that contains an approximately 20- base 5'-untranslated region (UTR) sequence with a very low G+C content and one that contains an approximately 150-base, G+C-rich 5'-UTR that is inhibitory to translation in rabbit reticulocyte lysates. Recovery from cellular stresses induced increased expression of the shorter, more translatable transcript and decreased expression of the longer form [Ayupova and Singh et al., 2009]. We predict that expression of the shorter mRNA is induced during recovery from stress to allow rapid translation of HuR upon potential subsequent insults.
Therefore, identification of the promoter elements that drive transcription of this mRNA may give clues to the regulatory mechanisms that protect cells from repetitive insults. Promoter analysis of HuR upstream regions revealed sequences necessary for regulation of the shorter mRNA. Through use of
69 promoter-luciferase reporter constructs, it was determined that sequences critical for transcription of the short mRNA resided within the exon encoding the 5’ UTR of the long mRNA. Within the G+C-rich 5'-UTR exist multiple overlapping copies of the alternate Smad 1/5/8-binding motif GCCGnCGC.
To determine whether the putative Smad 1/5/8-binding sites in the HuR 5’-
UTR are indeed capable of binding Smads, gel mobility shift assays were performed on nuclear extracts derived from both untreated LLC-PK1 cells and those that were ATP-depleted and allowed to recover. Figure 5.1A shows an example of this method, using a porcine probe corresponding to the sequence -
148 to -109 (relative to the start of translation) that encompasses five perfect and two imperfect copies of the GCCGnCGC motif. As shown, one major band was shifted in the presence of nuclear extracts. This band was routinely more intense in extracts from ATP depleted/recovered cells than untreated cells (an increase of 87 + 18%; n=5). The addition of an excess of unlabeled probe caused loss of this band, whereas addition of an irrelevant unlabeled oligonucleotide did not.
Preincubation of extracts with antibody against Smads 1/5/8 resulted in the disappearance of the band, rather than a supershift, indicating potential interference of the antibody with the probe-binding site (data not shown).
Therefore, we performed chromatin immunoprecipitation (ChIP) assays to confirm the identities of the relevant bands. As shown in the bottom panel of
Figure 5.1B, immunoprecipitation of chromatin with an anti-Smad 1/5/8 antibody isolated a fragment containing the HuR 5’-UTR sequences. Consistent with the gel mobility shifts, Smads 1/5/8 roughly doubled their association with the HuR
70 5’-UTR following ATP depletion/recovery (an increase of 86 + 15%; n = 3). These
results demonstrate that the HuR 5’-UTR is indeed capable of binding R-Smads
and that binding activity is increased in cells subjected to ATP depletion and
recovery.
5.2.2 Confirmation of the Presence of BMP-7 Receptors in LLC-PK1 cells
It is of interest that the stress and recovery from ATP depletion induces
transcription of a readily translatable HuR mRNA driven by the family of Smad
1/5/8 transcription factors. Activation of Smads 1/5/8 occurs through signaling by
bone morphogenetic proteins (BMPs). One BMP isoform in particular, BMP-7,
plays a key role in kidney development, as BMP-7-null mice exhibit a lack of
renal mesenchymal differentiation and an absence of nephrons [Dudley et al.;
Luo et al., 1995]. Further, recent reports have demonstrated that BMP-7 plays
an important role in recovery from multiple types of kidney injury, including
damage from ischemia and reperfusion [Zeisberg et al., 2003; Hruska et al.,
2000].
In order to determine whether BMP receptors are present in LLC-PK1 cells,
RT-PCR was performed using primers corresponding to ALK2 (also known as
Acvr1 and ActRI), a type I receptor that transduces signals from BMP-7 [Macías-
Silva et al., 1998] and is expressed in native proximal tubule cells [Gould et al.,
2003]. Semi-quantitative RT-PCR (Figure 5.2) showed the existence of this receptor and suggested that ATP depletion and recovery induced higher expression of ALK2 mRNA (an increase of 85 + 18%; n=4). To confirm this
71 finding at the protein level, Western analysis was performed on LLC-PK1 cells subjected to various times of ATP depletion/recovery. Figure 5.2 demonstrates similarly increased levels of ALK2 protein in stressed and recovered cells compared with those under normal growth conditions. This finding provides a mechanism by which ATP-depleted/recovered cells can amplify their responsiveness to BMP-7, thus inducing signaling through the Smad 1/5/8 pathway.
5.2.3 Regulatory Mechanisms Mediating Translational Control of HuR
We have shown that HuR gene transcription results in two alternate isoforms of mRNA that are the outcome of precise regulation to avoid deleterious affects of aberrant HuR production [Ayupova and Singh et al., 2009]. These isoforms contain vastly different 5’ UTRs and demonstrate markedly different levels of translatability in in vitro assays. Therefore, it was important to examine the mechanisms of translational control of these forms under differing conditions.
In the following sections, the effects of normal growth, stress, and recovery on
HuR translation will be explored.
5.2.4 Translatability of Alternate Forms of HuR mRNA under Normal,
Stressed, and Recovered Conditions
To determine under which cellular conditions the alternate forms of HuR mRNA are translated, LLC-PK1 cells were cultured normally or subjected to ATP
depletion and recovery. Ribosome fractionation was performed after lysate
72 collection via sucrose density gradient. HuR mRNA and protein were isolated
from these fractions. Using RT-PCR, the presence of these two different forms of
HuR mRNA was detected using different primer sets. One set was specific for
the mRNA1 (long form); the other detected total HuR mRNA, as we cannot
detect mRNA2 (short form) on its own as it is essentially a truncated version of
mRNA1. RT-PCR on the ribosomal fractions showed that total HuR mRNA is
distributed evenly among lighter (cytosolic, monosome) and heavier (polysomes)
fractions (Fig 5.3A). In contrast, mRNA1 is associated with polysomes only
during normal growth and recovery (Fig 5.3B) but was absent from polysomes
during ATP depletion. This finding suggests that mRNA1, and possibly mRNA2,
is capable of being translated during normal growth and recovery, while only
mRNA2 is translated during ATP depletion. This result is consistent with the
hypothesis that mRNA2 is a readily translatable form, and therefore is upregulated following an initial ATP depletion event as a preconditioning
mechanism.
5.2.5 Stress Granules
The ribosome fractionation results above demonstrate that the short form of
HuR mRNA is translated during ATP depletion, whereas the long form is
translated under normal and recovered conditions. This finding suggests that the
transcripts are inactive under different cellular conditions. We hypothesize that
the active transcripts are synthesized by potential partitioning into privileged
translation sites (i.e. polyribosomes), whereas during ATP depletion, the inactive
73 form (i.e. mRNA1) may be sequestered into stress granules (SGs), which are
aggregates of stalled translation initiation complexes that form under various
types of cellular stress. However, nothing is known about the formation of SGs in
renal proximal tubule cells. Therefore, we explored their formation under conditions of ATP depletion and recovery using markers known to accumulate in
SGs. Initial identification of SGs in proximal tubule cells was accomplished
through immunocytochemically labeling the related RNA-binding proteins TIA-1
and TIAR (Kedersha et al., 1999). Under normal conditions TIA-1 and TIAR are
concentrated in the nuclei of cells, but in response to stress, both proteins rapidly
accumulate in the cytoplasm and become robust markers of SGs.
5.2.6 Formation of Stress Granules during ATP Depletion and Recovery
To determine whether ATP depletion and recovery induces SG formation in
kidney epithelial cells, LLC-PK1 cells were ATP depleted, recovered in normal
growth medium, and subjected to immunocytochemical analysis. Discrete foci
became apparent within 30 minutes of ATP depletion, as assayed by immuno-
staining for the SG markers TIAR. These granules become larger and more
apparent over 3-4 hours of recovery (Figure 5.4). Similar results were obtained
when cells were labeled for the related marker TIA-1 (data not shown). These
data are suggestive that ATP depletion of renal epithelia supports stress granule
formation, although further characterization of these foci is required.
74 5.2.7 Effects of Cycloheximide and Puromycin on Stress Granule Formation
in LLC-PK1 Cells
The paradoxically antagonistic effects of different pharmacological inhibitors
of protein translation on SG assembly reveal the dynamic equilibrium between
polysomes and SGs. Drugs that stabilize polysomes by freezing ribosomes on
translating mRNAs (e.g. cycloheximide and emetine) inhibit the assembly of SGs
and actively dissolve them in the continued presence of both stress and eIF2α
phosphorylation (Kedersha et al., 2002). In contrast, drugs that destabilize
polysomes by releasing ribosomes from mRNA transcripts (e.g. puromycin)
promote the assembly of SGs (Kedersha et al., 2002). There is a dynamic equilibrium between polysomes and SGs, which has led investigators to propose that SGs are the sites of mRNA triage, where untranslated mRNAs accumulate during stress prior to degradation, reinitiation, or repackaging as mRNPs
(Kedersha et al., 2002).
To determine whether discrete foci identified during stress and recovery of
LLC-PK1 cells were indeed SGs, cells were ATP depleted/recovered along with
the addition of cyclohexmide or puromycin. Upon puromycin treatment these foci
became larger whereas cycloheximide treatment made them disappear, as
assayed by immunolabeling for the SG marker TIAR (Figure 5.5). These results
are consistent with the identification of these foci as stress granules.
5.2.8 Distribution of Stress Granule Markers during ATP Depletion and
Recovery
75 Stress granules are primarily composed of stalled 48S complexes
containing bound mRNAs derived from disassembling polysomes. These contain
poly(A) mRNA bound to early initiation factors (such as eIF4E, eIF3, eIF4A,
eIFG) and small, but not large, ribosomal subunits. To further characterize the
putative stress granules seen in our cell model, large ribosomal proteins and
eIF2-alpha, which are known to be absent from SGs, were tested for their presence or absence in the stress granules formed in our ATP depletion model.
Using immunocytochemical methods, we tested for the presence of eIF2α and the large ribosomal protein, Ribo-P antigen. As shown in Figure 5.6, their distributions showed no overlap with that of the SG foci. In addition, we used antibodies against individual components of the 48S pre-initiation complex (e.g. eIF3, eIF4E, and PABP) to determine whether these proteins were present in the
SG foci. Most components of the 48S pre-initiation complex were found to colocalize with TIAR at the foci (Figure 5.7). These results indicate that in LLC-
PK1 cells, the discrete foci represent SGs that are eIF2-eIF5–deficient, pre-
initiation complexes that are assembled during stress.
RNA-binding proteins that either promote [Gallouzi et al., 2000] or inhibit
[Kedersha et al., 2000] mRNA stability are also recruited to SGs. This suggests
that the SG is a site where the fates of specific transcripts are determined by the
activity of different RNA-binding proteins. Consistent with other stress models,
poly(A) mRNA was found to be present in stress granules induced by ATP
depletion (Figure 5.7), as assessed by probing cells with a fluorescent oligo-dT
76 oligonucleotide. This finding is consistent with previous studies showing that poly(A) mRNA is a component of mammalian SGs [Kedersha et al., 1999].
5.2.9 Increase in the Phosphorylation Level of eIF2-alpha during ATP
Depletion
Eukaryotic cells express a family of translation initiation factor 2 alpha
(eIF2α) kinases (PKR, PERK, GCN2, HRI) that are individually activated in response to distinct types of environmental stress. Phosphorylation of eIF2α by one or more of these kinases reduces the concentration of eIF2–guanosine triphosphate (GTP)–transfer ribonucleic acid for methionine (tRNA-Met), the ternary complex that loads tRNA-Met onto the small ribosomal subunit to initiate protein translation. When ternary complex levels are reduced, the related RNA- binding proteins TIA-1 and TIAR promote the assembly of a noncanonical pre- initiation complex that lacks eIF2-GTP-tRNA-Met. The TIA proteins dynamically sort these translationally incompetent pre-initiation complexes into stress granules.
It has been reported in certain models that eIF2-alpha phosphorylation increases upon stress. This phosphorylation event is required to suppress general protein translation during stress. However, it has been suggested that
ATP depletion may inhibit translation by an alternate mechanism [Kedersha et al., 2002]. In order to determine the phosphorylation status of eIF2α in energy- depleted proximal tubules, LLC-PK1 cells were subjected to stress by ATP depletion and the phosphorylation state of eIF2α was determined by Western
77 blot. Phosphorylation of eIF2α increased during ATP depletion (Figure 5.8A) and
decreased during recovered conditions (Figure 5.8B), while total eIF2α remained unchanged. This finding suggests that general protein translation is diminished during ATP depletion. However, as we previously demonstrated, translation of
HuR (the short mRNA isoform) continues and indeed increases during ATP depletion. Thus, translation of this mRNA isoform appears to be able to circumvent the normal suppressive effects of eIF2-alpha phosphorylation.
5.2.10 PERK is Activated by ATP Depletion of Proximal Tubule Cells
The eIF2α subunit is the target of a family of serine/threonine kinases (PKR,
PERK, GCN2, HRI) that are activated by different forms of environmental stress.
For example, PKR senses heat, ultraviolet (UV) irradiation, viral infection, and
oxidative stress [Williams, 2001], whereas PERK (also known as PEK) detects
endoplasmic reticulum stress. GCN2 senses amino acid starvation [Kimball,
2001], and HRI monitors changes in the availability of heme during erythrocyte
differentiation [Han et al., 2001; Lu et al., 2001]. Each of these stress-activated kinases phosphorylates eIF2α on serine 51, a modification that increases the affinity of eIF2 for eIF2B, a GDP-GTP exchange factor that charges the eIF2-
GTP-tRNA-Met ternary complex [Kimball, 2001].
Endoplasmic reticulum (ER) stress, which can lead to activation of the eIF2
kinase PERK and eIF2α phosphorylation, has been shown to accompany
ischemic injury in several tissues, including kidney epithelia [Kumar et al., 2001].
Translational repression from ER stress has long been known to be a transient
78 phenomenon [Brostrom et al., 1998; Kaufman, 1999], and phosphorylation of
eIF2α by endoplasmic reticulum (ER) stress, which can lead to activation of the
eIF2 kinase PERK and eIF2α phosphorylation, has been shown to accompany ischemic injury in several tissues, including kidney epithelia [Bush et al., 2000].
More recently, renal ischemia-reperfusion was shown to induce phosphorylation of eIF2α via PERK directly [Montie et al., 2005].
Because PERK activity has been implicated in renal stress caused by ischemia-reperfusion, the phosphorylation status of PERK was determined by
Western analysis during ATP depletion of LLC-PK1 cells. It was observed that
phosphorylation of PERK increased within 15 minutes of incubation of cells in
ATP depletion buffer, while total PERK levels remained constant (Figure 5.8C).
Although this experiment does not rule out involvement of the other eIF2-alpha
kinases, it indicates a likelihood that PERK plays a key role in phosphorylation of eIF2-alpha and subsequent stress granule formation and translational repression in our model system.
5.2.11 Post-transcriptional Control Element (PCEs) and Translation of HuR
As described earlier in this chapter, one isoform of HuR mRNA (mRNA1)
contains a long, highly structured 5’ UTR. This mRNA was shown to be poorly
translated in rabbit reticulocyte lysates [Ayupova and Singh et al., 2009]. This
finding begs the question, what is the purpose of synthesizing a poorly translated
mRNA? One possibility is that this mRNA contains an internal ribosome entry
site (IRES). IRESs are elements found in highly structured 5’ UTRs that help
79 initiate ribosome binding near the initiation codon, rather than at the mRNA 5’ cap [Pestova et al., 2001]. This circumvents the need for ribosomal complexes to scan through a highly structured 5’ UTR. The possibility of HuR mRNA1
containing an IRES was highly attractive, as IRESs are often used during times
of cellular stress [Kullmann et al., 2003; Meng et al., 2005]. However, other work
in the Lee laboratory demonstrated an absence of IRES activity in the 5’ UTR of
HuR mRNA1 (data not shown). Therefore, it seemed likely that an alternate form
of initiation might be required to translate this mRNA.
Recently, another form of translation initiation was identified in retroviruses
and a few mammalian mRNAs, termed the post-transcriptional control element
(PCE). PCEs are extensively studied by the laboratory of Dr. Kathleen Boris-
Lawrie of the OSU College of Veterinary Medicine. PCEs are highly structured 5’
UTR sequences that facilitate, rather than inhibit, translation initiation. PCEs are
bound by RNA helicase A (RHA), which further recruits ribosomal complexes and
aids in unwinding the secondary structure of the 5’ UTR [Gritta et al., 2006],
allowing cap-dependent translation. PCEs form a tripartite stem-loop structure
composed of three required structural motifs. The HuR mRNA1 5’ UTR was
compared to the 5’ UTR of transcription factor JunD, which has been established
to contain a PCE [Hernandez et al., 2008]. Figure 5.9A shows high sequence homology between HuR and JunD within JunD’s established structural motifs.
Further, the laboratory of Boris-Lawrie, using a PCE reporter assay developed in
their group, found that HuR mRNA1 exhibits activity similar to that of a known
PCE-containing retrovirus (Figure 5.9B). Finally, in order to determine the
80 potential role of RNA helicase A (RHA) in regulating expression of HuR, we
knocked down expression of RHA using siRNAs developed by the Boris-Lawrie
lab. This knockdown in proximal tubule cells resulted in suppression of HuR
protein levels (Figure 5.9C). Taken together, these data strongly suggest the presence of a PCE in the 5’ UTR of HuR mRNA1.
5.3 CONCLUSIONS
Human antigen R (HuR) is an RNA-binding protein with protective activities
against cellular stress. Under basal conditions, HuR mRNA is expressed in two
forms: one that contains a 20-base 5’-untranslated region (UTR) sequence and
one that contains a 150-base, GC-rich 5’-UTR that is inhibitory to translation in
vitro. Previous results showed that recovery from cellular stresses such as
thapsigargin and ATP depletion induced increased expression of the shorter,
more translatable transcript (mRNA2) and decreased expression of the longer
form (mRNA1). Analysis of HuR upstream regions revealed sequences
necessary for regulation of the shorter mRNA that exist within the exon encoding
the GC-rich 5’-UTR of the longer mRNA. Gel shift and chromatin
immunoprecipitation analyses demonstrated the ability of Smad 1/5/8
transcription factors to bind to these sequences that contain overlapping copies
of the motif GCCGnCGC. Because Smad 1/5/8 are induced by bone
morphogenetic proteins (BMPs), these results suggest a role for HuR in
regulating the pleiotropic effects of BMP-7 in recovery from renal stress.
81 Because HuR mRNA isoforms contain different 5’ UTRs and have shown
different levels of translatability in in vitro assays, the effects of normal growth, stress, and recovery on HuR translation were explored. HuR mRNA1 was shown to contain a PCE, which imposes a translational regulation and affects the levels of HuR expression during normal growth and recovered conditions. However, mRNA1 was not found to be translated during the stress of ATP depletion. Given these findings, as well as the presence of stress granules in ATP-depleted proximal tubule cells, we hypothesize that differential translation of the HuR mRNAs is likely to be a function of mRNA sequestration to stress granules or polyribosomes.
82
A
B
Figure 5.1. Smad 1/5/8 Binding Sites are Present in the HuR Promoter.
(A) LLC-PK1 cells were cultured in normal growth medium or ATP depletion medium for 2 hours and allowed to recover. Gel mobility shift assays were performed on nuclear extracts from untreated (unt) or ATP-depleted/ recovered
(rec) LLC-PK1 cells. One major band (designated by the arrow) was found to specifically bind Smad 1/5/8 consensus motifs in the porcine HuR 5’-UTR. The intensity of this band increased following ATP depletion/recovery. (B) In chromatin immunoprecipitation (ChIP) assays, antibody against Smad 1/5/8 precipitated DNA corresponding to the HuR 5’-UTR, as detected by PCR using specific primers. Although levels of input DNA were unchanged, Smad 1/5/8 antibodies precipitated more HuR DNA from ATP depleted/recovered cells than untreated cells, consistent with the gel mobility shift findings.
83
Figure 5.2. ATP Depletion and Recovery Induces Expression of ALK2. Two hours of ATP depletion followed by up to 4 hours of recovery caused increased expression of ALK2 mRNA (left panel) and protein (right panel). A beta-actin immunoblot is included as a loading control.
84
Figure 5.3. Ribosomal Fractionation Indicates Differential Translation of HuR mRNAs during Normal Growth, ATP Depletion, and Recovery. Ribosomal fractionation shows that total HuR mRNA is distributed across all the fractions (left panel), whereas mRNA1 (the long isoform) is heavily associated with polysomes during normal growth and recovery from ATP depletion (right panel). In contrast, mRNA2 (the short isoform) alone is translated during ATP depletion.
85
Figure 5.4. ATP Depletion Induces Stress Granule Formation. LLC-PK1 cells were plated on glass coverslips and were treated with D-glucose free media and 0.1 µM antimycin A for 1 to 4 hours followed by recovery in normal growth media for up to 4 hours. Cells were fixed, permeabilized, and immunolabeled with antibodies against TIAR.
86
Figure 5.5. Inhibitors of Translation Alter Stress Granule Formation in ATP- depleted LLC-PK1 cells. LLC-PK1 cells were plated on glass coverslips and were ATP depleted for 1 hour followed by 2 or 4 hours of recovery in media containing 10 µg/ml puromycin or 100 µg/ml cycloheximide. Cells were fixed, permeabilized, and immunolabeled with antibodies against TIAR.
87
Figure 5.6. Neither eIF2-alpha nor a Large Ribosomal Subunit Co-Localize
with Stress Granules in LLC-PK1 Cells. LLC-PK1cells were plated on glass coverslips and were ATP depleted for 1 hour followed by 2 or 4 hours of recovery in normal growth media. (top row) Cells were fixed, permeabilized, and immunolabeled with antibodies against TIAR in green and eIF2α in red. (bottom row) Cells were labeled with antibodies against large ribosomal subunit Ribo-P Antigen (green) and TIAR (red).
88
Figure 5.7. Translation Initiation Factors, Small Ribosomal Proteins, and poly(A) mRNA Segregate to Stress Granules in LLC-PK1 Cells. Initiation factors such as eIF2αP, eIF3, with TIAR show their presence in SGs along with small ribosomal protein S6 and poly(A) mRNA. HuR protein was also localized to SGs. Cells were fixed, permeabilized, immunostained with antibodies against phospho-eIF2-alpha, eIF3 and S6 in red, and HuR in green. Poly(A) mRNA was detected using a fluorescent labeled oligo-dT probe.
89
A
B
C
Figure 5.8. ATP Depletion Induces Phosphorylation of eIF2-alpha and
PERK. (A) LLC-PK1 cells were ATP depleted for up to 4 hours and lysates were assessed by Western blot for levels of total and phosphorylated eIF2-alpha. (B)
LLC-PK1 cells were ATP depleted 1 hour and recovered in normal medium to up to 4 hours. Lysates were assessed for levels of total and phosphorylated eIF2-
alpha as in (A). (C) LLC-PK1 cells were ATP depleted for up to 30 minutes and assessed by Western blot for levels of total and phosphorylated PERK.
90
Figure 5.9. HuR mRNA Contains a Post-transcriptional Control Element. (A) PCE-like motifs in HuR mRNA1 shows high homology with the PCE elements in JunD. (B) The HuR mRNA1 5’ UTR shows comparable PCE activity to that of SNV PCE, assayed via reporter constructs. (C) siRNA mediated knockdown of RHA results in suppression of HuR protein levels as assessed by Western blotting. β-actin is included as a loading control.
91
CHAPTER 6
Discussion
6.1 Anti-apoptotic Role of HuR
HuR, as one of the regulators of mRNA stability in renal epithelial cells, is redistributed and regulated at multiple levels during energy depletion and recovery. By binding to mRNAs containing specific AREs, HuR contributes to the stability of hundreds to thousands of distinct mRNA transcripts. It is a stress response protein that is known to play an important role in cell survival, as its loss in vitro or in vivo results in heightened apoptosis [Wang et al., 2000]. By stabilizing and promoting translation of specific mRNAs during stress events,
HuR is protective against cellular damage. It binds to mRNAs that encode several major categories of proteins including modulators of cell proliferation and differentiation, inflammation, cell survival and apoptosis. Because HuR appears to play a role in stabilizing numerous transcripts during cellular stress and apoptosis [Wang et al., 2000], understanding the regulation of this protein gives an important insight into general mechanisms of survival during environmental insults such as ischemic injury.
Although HuR has the capacity to bind numerous mRNA targets, it is known to promote mRNA stabilization or translation of a few specific gene products that
92 play important roles in promoting cell proliferation or protecting against apoptosis
and senescence. Target mRNA ligands for HuR include cell cycle-associated gene products such as c-Fos, p21, p53, and various cyclins, anti-apoptotic proteins including prothymosin α, and SIRT1, and other stress-related proteins such as HIF1α. HuR has emerged as an elicitor of a broad antiapoptotic function through its influence on the expression of proteins directly (e.g., ProTα, SIRT,
Bcl-2, Mcl-1) or indirectly (e.g., p21, p27, EGF, VEGF, IGF-IR), preventing apoptosis [Lopez et al., 2005].
HuR, together with polypyrimidine binding protein, binds hypoxia-inducible
factor-1 α (HIF-1α) mRNA and promotes HIF-1α translation under hypoxia-like
conditions [Galban et al., 2008]. Activation of HIF-1α has been identified as an important mechanism of cellular adaptation to low oxygen, one of the conditions arising from ischemia at the cellular level. Under a decreased oxygen level, HIF-
1α regulates a broad range of physiologically relevant genes involved in erythropoiesis, angiogenesis, apoptosis, and energy metabolism [Maxwell, 2003].
It was shown that preconditional activation of HIF-1α protected against ischemic injury in rats [Bernhardt, 2006]. It is striking that many of HIF-1 α target genes are also regulated posttranscriptionally by HuR, including VEGF, COX-2, IL-6, IL-8, and GLUT1 [Gantt et al., 2006; Mrena et al., 2005], and likely further contribute to the protective and adaptive role of HuR under ischemic conditions. By acting upon various mRNA pools, HuR participates in numerous key cellular processes; thus it was of interest to know the mechanism of how HuR expression is regulated.
93 Our own studies have demonstrated the anti-apoptotic nature of HuR in
cultured renal proximal tubule cells subjected to ATP depletion and indicate Bcl-2
and Hsp70 as targets [Ayupova and Singh et al., 2009]. Therefore, the
expression of HuR in kidney epithelia subjected to ischemia/reperfusion injury
may be protective against apoptosis caused by the ischemic insult and may also
promote proliferation of newly regenerating proximal tubule epithelia.
6.2 HuR, PI3 Kinase/Akt Pathway, and Apoptosis
We demonstrated that a major effect of HuR knockdown in cultured
proximal tubule cells was suppression of anti-apoptotic mRNAs including those
within the Akt signaling pathway. Other mRNAs that were downregulated in HuR-
suppressed cells (not discussed above) were transcripts corresponding to anti-
apoptotic proteins including the Bcl-2 family, Mcl-1, NAIP, NOD-1, and BFAR.
We found that HuR plays a central role in PI3K/Akt signaling, as it enhances
expression of Grb10, which promotes activation of Akt, that further goes on to
upregulated HuR expression. Thus, HuR is involved in a positive feedback loop
promoting Akt activity and cell survival. This study demonstrates how HuR plays
a central, positive role in proximal tubule cell survival through the activities of Akt.
As described above, HuR has been demonstrated to regulate the mRNA stability and/or translation of critical cell survival proteins in addition to Grb10.
HuR’s involvement in PI3K/Akt signaling adds another layer of complexity to its
effects on cell survival. For example, expression of the caspase inhibitor XIAP (X
chromosome-linked inhibitor of apoptosis) is increased by HuR both at the level
94 of mRNA stability (Zhang et al., 2009) and translation (Durie et al., 2010). Here we show that HuR can act on XIAP at another level to enhance cell survival; that is, Akt phosphorylates XIAP, inhibiting its ubiquitination and degradation. Thus,
HuR directly interacts with XIAP mRNA to increase its expression, while Akt, which is indirectly activated by HuR through Grb10, increases XIAP’s expression post-translationally. In another example, HuR also has been demonstrated to increase Bcl-2 expression at the levels of both mRNA stability and translation
(Ishimaru et al., 2009). Bcl-2’s ability to enhance cell survival lies in part in its abundance relative to that of its pro-apoptotic family member BAD, which when
dimerized to Bcl-2 (or Bcl-XL), promotes apoptosis. Akt phosphorylates BAD, resulting in its dissociation from Bcl-2/Bcl-XL and association with 14-3-3, thus inhibiting apoptosis. Therefore, while HuR increases Bcl-2 expression by
interaction with Bcl-2 mRNA, it also increases Bcl-2 activity through Akt-mediated
degradation of BAD.
The ability of HuR to affect anti-apoptotic pathways at multiple levels
accounts for its importance in promoting cell survival. Knockout of HuR pre-
natally results in embryonic lethality [Katsanou et al., 2009], while knockout post-
natally results in rapid death [Ghosh et al., 2009]. Conversely, overexpression of
HuR by as little as three to four-fold has been associated with increased
tumorigenicity in numerous tissue types, including renal cell carcinomas [Danilin
et al., 2010]. For this reason, HuR expression must be tightly controlled.
We have identified the adapter protein Grb10 as a new target of HuR
regulation and have shown its importance in mediating cell survival during stress.
95 As briefly described above, Grb10 has been demonstrated to bind Akt and
transport it to the plasma membrane where this protein complex may associate
with various activated receptor tyrosine kinases through Grb10’s SH2 domain
[Jahn et al., 2002]. This association brings Akt in proximity to plasma membrane
PI3 kinase, resulting in Akt’s phosphorylation and activation. Additionally, other
studies have indicated that Grb10 can promote cell survival through MAP kinase
pathways as well as Akt [Kebache et al., 2007]. Interestingly, HK-2 cells under
normal conditions demonstrate a primarily nuclear localization of pAkt. While in
the nucleus, pAkt has access to its nuclear targets including the forkhead family
of transcription factors. However, ATP depletion results in pAkt movement to the
plasma membrane, where it may have access to a new set of targets. Grb10 is
required both for pAkt activation and its movement to the plasma membrane.
Although our studies and other indicate that Grb10 promotes cell survival, it
is a negative regulator of growth, as its disruption in mice leads to embryonic and
placental overgrowth [Charalambous et al., 2003]. This is most likely due to
inhibition of insulin and insulin-like growth factor receptor signaling. Interestingly,
HuR also has been noted to inhibit insulin-like growth factor receptor signaling,
through inhibition of translation of the type I receptor [Meng et al., 2005].
However, it must be noted that the Grb10 literature contains multiple examples of
this adapter playing both negative and positive roles in studies of insulin signaling in vitro [Lim et al., 2004; Riedel, 2004]. These discrepancies may be attributable
in part to tissue- and cell-specific differences or to artifacts associated with
overexpression of Grb10 protein. Grb10 mRNA is expressed as multiple
96 alternately spliced transcripts (3 in mice, 4 in humans) whose functions may or
may not differ. In the present studies, we used Grb10 siRNAs that knocked down
expression of all transcripts so that any isoform-specific effects would not occur,
and our RT-PCR primers and antibodies also detected all forms. Further, in our studies to determine the role of the Grb10 3’ UTR in its expression, we transfected into HK-2 cells a mouse Grb10eta cDNA. We expect the findings
from this experiment to be relevant to all murine Grb10 isoforms, as they contain
identical 3’ untranslated regions.
In our study, Grb10 has emerged a novel binding target of HuR, which
plays a key role in regulating the PI3K/Akt pathway for cell survival. Therefore,
HuR demonstrates a central role in cell survival as both an activator of PI3K/Akt signaling and as a downstream target of PI3K/Akt-mediated gene regulation in renal epithelia.
6.3 Transcriptional and Translational Control of HuR
We previously demonstrated that HuR mRNA is synthesized in two forms
that differ in their 5’ untranslated regions and thus are transcribed through the
action of alternative promoters [Ayupova and Singh et al., 2009]. The data
presented here provide an initial characterization of essential promoter elements
required for transcriptional activation of HuR mRNA2 during recovery of renal
epithelia from cellular stress. Characterization of promoter elements driving
transcription of an mRNA1 is underway. Genome wide analysis has revealed the
prevalence of numerous alternate promoters, in fact suggesting that up to half of
97 human genes produce variant transcripts [Tsuritani et al., 2007]. Transcription from alternate promoters has been shown to be induced under multiple circumstances, including developmental activity [Saleh et al., 2002] tissue specificity [kamat et al., 2002], and stress [Meshorer et al., 2004]. Further, alternative promoter transcripts previously have been shown effective in the control of translational machinery, as suggested here for HuR [Landry et al.,
2003].
Because HuR mRNA is present as two isoforms contain different 5’ UTRs
and have shown different levels of translatability in in vitro assays, the effects of
normal growth, stress, and recovery on HuR translation was explored. Although
the HuR mRNA bearing the short 5’-UTR (mRNA2) is under control of
BMP/Smad signaling and is readily translatable, the mechanisms by which the
longer transcript (mRNA1) is translated were not known. Our results indicate that
both isoforms of HuR mRNA are present during stressed conditions; however
mRNA1 was found not to be translated during this time. Interestingly, the 5’ UTR
of mRNA1 was shown to contain a post-transcriptional control element (PCE).
The PCE imposes a translational regulation and affects the levels of HuR
expression during normal growth and recovered conditions. Although the role of
PCEs in mammalian cells is not understood, we speculate that the presence of a
PCE in mRNA1 aids to keep a check on the translation levels of HuR during
normal and recovered conditions. However, when a cell is exposed to and
recovers from stress, synthesize of mRNA2 predominates so that during
repeated stresses, this more translatable transcript may be readily translated.
98 We also observed that cultured proximal tubule cells subjected to ATP depletion form the foci of stalled translation initiation known as stress granules.
Although translation may be inhibited under numerous types of stresses, not all of these induce SG formation. Here we found that ATP depletion does indeed cause formation of these aggregates, and that phosphorylation of eIF2-alpha by the stress kinase PERK is likely to play a key role in this process. We also determined that some HuR mRNA is associated with SGs during ATP depletion.
However, we have been yet unable to determine which mRNA isoform or isoforms is present in these granules. Because mRNA1 is not translated during
ATP depletion, we speculate this is due to its sequestration in SGs. This is further bolstered by the findings that RNA helicase A, the PCE binding protein, is present in SGs during oxidative stress brought about by arsenite exposure (Dr.
K. Boris-Lawrie et al., unpublished data). This finding is consistent with the PCE- containing mRNA1 also being sequestered in SGs during ATP depletion.
However, more experiments are required to definitively prove the identity of the
SG-associated transcript(s). Nonetheless, observing these findings, it is hypothesized that differential translation of the HuR mRNAs is likely to be a function of alternative mRNA targeting to stress granules or polyribosomes. In summary, it is proposed that modulating the expression of HuR during ischemia/reperfusion injury provides a system in which there are multiple levels of regulation working in concert to protect and restore the cell.
99
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