Antiviral and Antitumor Functions of RNase L
By Geqiang Li
Submitted in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy
Thesis Advisor: Dr. Robert H Silverman
Department of Genentics
Case Western Reserve University
January 2005
Case Western Reserve University
School of graduate studies
We hereby approve the thesis/dissertation of
______Geqiang Li______
candidate for the ______Doctorate______degree.
( Signed ) ______Robert H. Siverman______(Chair of the Committee)
______Bryan G Williams______
______David Samols______
______Ganes C. Sen______
______Alexandtru Almasan______
( Date )______10-11-2004____
1 TABLE OF CONTENTS
TABLE OF CONTENTS……………………………………………… ……………2 LIST OF FIGURES…………………………………………………………………..7 LIST OF ABBREVIATIONS……………………………………………..………….9 ABSTRACT………………………………………………………………………...11
CHAPTER 1. INTRODUCTION 1.1. Overview of the interferon (IFN) system………………………………….…...14 1.2. 2-5A / RNase L system………………………………….…….…………..…..15 1.2.1. Properties of RNase L…………………………………………………...17 1.2.2. Antiviral activity of RNase L…………………………………..………..20 1.2.3. Apoptotic activity of RNase L……………………………………..……22 1.2.4 Involvement of RNase L in the biology of prostate cancer………….…23
CHAPTER 2. AN APOPTOTIC SIGNALING PATHWAY IN THE INTERFERON
ANTIVIRAL RESPONSE MEDIATED BY RNASE L AND C-JUN NH2- TERMINAL KINASE
ABSTRACT…………………………………………….…………………….……29 2.1 INTRODUCTION…………………………………..………..……………...…31 2.2 MATERIALS AND METHODS………………….…..………….……...... …..35 2.2.1 Cell culture………………………………….…………………………….35 2.2.2 Transfections……………………………………………….……...……..36 2.2.3 Viral Infections.….………………...……………...……………………...36 2.2.4 Measuring Protein Synthesis in Intact Cells..…………..……...…………36 2.2.5 RNase L Activity in Intact Cells……………………….…………………36 2.2.6 RNase L Activity in a Cell-free System……………………………….….37 2.2.7 2-5A Binding Assay for RNase L…………....…………………………...37 2.2.8 Cell Viability Assay………………………………………………………38
2 2.2.9 Western Blots………………………..………...………………….……...38 2.2.10 TUNEL Assay………………………………………………………….38 2.2.11 JNK Kinase Assay…………………….………………………………..39 2.3 RESULTS………………….…………………………………………………....41 2.3.1 Viral Activation of JNK and Apoptosis Are Deficient in Cells Lacking RNaseL…………………………………………………...….41 2.3.2 2-5A Activation of RNase L Results in Stimulation of JNK and Apoptosis ……………………………………………………….…42 2.3.3 Inhibition of JNK Impairs RNase L-induced Apoptosis………………...45 2.3.4 Ablation of JNK Suppresses Apoptosis Induced by 2-5A Activation of RNase L…………………………………………...…….…...46 2.4 DISCUSSION………………..………………...……… …………………..….49 2.4.1 Essential Role of RNase L in Viral Activation of JNK…….…………...49 2.4.2 JNK Participation in RNase L-mediated Apoptosis………………….…51
2.4.3 The RNase L Apoptotic Signaling Pathway………………………….....52
CHAPTER 3. RNASE L IS A NEGATIVE REGULATOR OF CELL MIGRATION
ABSTRACT…………………………….…………………………………….…..73 3.1 INTRODUCTION………………………………...………………….……….75 3.2 METERIALS AND METHODS…………………………..……………...... 80 3.2.1 Cell culture and treatment…………..…………………….…….……...…80 3.2.2 Cell Adhesion assay………………………………………….….………80 3.2.2 Migration assays……………………………………..……………...... 80 3.2.3 In vitro kinase assays…………………………………….…….…….....81 3.2.4 Detection of protein-protein interactions…………………….…………82 3.2.5 Immunoprecipitations and immunoblot analysis…………….…………83 3.2.6 Immunofluorescence Microscopy……..………………………..……....83 3.3 RESULTS…………………………………………………………….….….…85
3 3.3.1 RNase L inhibits cell migration but not adhesion……………………....85 3.3.2 Integrin β1 mediated cell migration is inhibited by RNase L…….……86 3.3.3 RNase L inhibited adhesion dependent FAK activation……….……….87 3.3.4 Adhesion dependent of JNK activation and C-Jun phopshorylation is increased in RNase L-/- MEF cells…………………………………...87 3.3.5 The JNK inhibitor SP600125 suppresses cell migration more significantly in RNase L-/- than in RNase L+/+ MEF cells…………....…88 3.3.6 RNase L is able to interacts with integrin β1, and FAK………...……..89 3.3.7 RNase L can be phosphorylated by FAK in vitro……………………..91 3.3.8Activation of RNase L by 2-5A inhibits cell migration……...………....91 3.3.9 2-5A promote integrin migrating to cell membrane………….………...92 3.4 DISCUSSION……………..…………………………………………………..93 3.4.1 RNase L is a negative regulator of cell migration………….....………..93 3.4.2 JNK is involved in RNase L mediated inhibition of cell migration…....94 3.4.3 RNase L interact with integrin β1 and FAK…………………...……....95 3.4.4 2-5A induce inhibition of cell movement……………………………..97
CHAPTER 4 PMA INDUCED RNASE L PHOSPHORYLATION AND PREVENT 2-5A INDUCED APOPTOSIS
4.1 INTRODUCTION……………………………………………………...…...112 4.2 MATERIALS AND METHODS………………………………………...….116 4.2.1 Protein interaction assays……………...……………….……………..116 4.2.2 RNase L immnopricipitation………………………….………………116 4.2.3 2D gel electrophoresis…………...…………………….………….…..116 4.2.4 Silver Staining…………………………………………………...……117 4.2.5 Western blot analysis……………………………………………...….118 4.3 RESULTS AND DISCUSSION……………………………………….……119 4.3.1 PMA stimulate RNase L phosphorylation……………………………119 4.3.2 PMA blocked 2-5A induced apoptosis………………………………..119
4 4.3.3 Interaction of RNase L with PKCα……………………………………….120
CHAPTER 5. SUMMARY AND FUTURE DIRECTIONS
5.1 SUMMARY………………………………………………..…………..……125 5.2 FUTURE DIRECTIONS…………………………………..……………..…125 5.2.1. Investigate the mechanism of 2-5A induced JNK activation……..…127 5.2.2. Investigate involvement of RNase L in PKC signaling pathways...... 128 5.2.2.1 To determine if PKCα is the only protein responsible for phosphorylating RNase L in response to PMA………….129 5.2.2.2 Examine the effect of PMA treatment on RNase L interaction with cytoskeleton……………………..……..129 5.2.2.3 To determine the PKC signaling pathway affected by RNase L……………………………………………………...130 5.2.2.4 PMA induced gene expression file in RNase L+/+ and RNase L-/- fibroblasts………………………………....130 5.2.3. Investigate the role of RNase L in cell migration……..……………...131 5.2.3.1 Expression of DN-JNK in RNase L-/- cells and examine its effect on migration………………………..….….131 -/- 5.2.3.2 Expression of RNase L and RNase L∆EN in RNase L fibroblasts, and then examine the effect on cell migration…...132 5.2.3.3 Investigate the effect of RNase L on FAK autophosphorylation...... ….132 5.2.3.4 Examine the effect of RNase L in inhibition of tumor metastasis in nude mice…………………………………….. ...132
REFERERENCE…………………………………………………………………134
5 LIST OF FIGURES
CHAPTER 1
Figure 1.1 Antiviral mechanism of interferon action………………………….....…..15 Figure 1.2 The IFN-regulated 2-5A system, a RNA decay pathway with antiviral and apoptotic activities…………………………………..……..16 Figure 1.3 The structure of RNase L…………………………...………………..…..18 Figure 1.4 Functional model for the activation of RNase L by 2-5A…………..…..19 Figure 1.5 Comparison of the domains and motifs in human RNase L and yeast Ire1p………………….……..……...... 20 Figure 1.6 RNase L mutations in different populations of prostate cancer cases aligned to the domain structure of RNase L………………....…….23 Figure 1.7 Progression of prostate cancer and steps where RNase L could interfere……………………………………………..…....28
CHAPTER 2
Figure 2.1 Apoptosis and JNK activation are deficient in virus-infected. RNase L-/- cells……………………………………………….…….55-56 Figure 2.2. Activation of RNase L with 2-5A induces phosphorylation of c-Jun and JNK but not p38 and inhibits protein synthesis……....…57-58 Figure 2. 3. Apoptosis requires functional RNase L……………………………..…59 Figure 2.4 Bcl-2 overexpression blocks apoptosis in response to 2-5A activation of RNase L…………………………………………….....…60 Figure 2.5 Kinetics of c-Jun phosphorylation and rRNA cleavage in Hey1b cells……………………………………………..………...61-62 Figure 2.6 2-5A induces c-Jun phosphorylation in Hey1b cells pretreated with cycloheximide……………………………………….63 Figure 2.7 RNase L is involved in dsRNA mediated JNK activation………….…..64 Figure 2.8. The JNK inhibitor, SP600125, suppresses c-Jun phosphorylation
6 and apoptosis in response to 2-5A activation of RNase L………..…65-68 Figure 2.9 Suppression of c-Jun phosphorylation and apoptosis by transfecting cells with siRNA against JNK1 and JNK2………….…….69 Figure 2.10.Apoptosis in response to IFN and 2-5A is inhibited in Jnk1-/- Jnk2-/- MEF cells………………………….………………...70-71 Figure 2.11. Apoptotic signaling pathway in virus-infected cells mediated by RNase L and JNK……………………………………….…………….72
CHAPTER 3
Figure 3.1 RNase L inhibits cell haptotactic migration……………………...….98-99 Figure 3.2. RNase L doesn’t affect cell adhesion on fibronectin and laminin……100 Figure 3.3 RNase L inhibit β1 integrin mediated haptotaxis………………….….101 Figure 3.4 RNase L inhibit adhesion dependent FAK tyrosine phosphorylation….102 Figure 3.5. RNase L inhibit adhesion dependent JNK activation and c-Jun phophorylation……………………………….………..…….103 Figure 3.6. The JNK inhibitor, SP600125 suppresses cell migration in RNase L deficient cells………………………………………….……104 Figure 3.7. RNase L interacts with integrin β1 and FAK……….…….……..105-106 Figure 3.8 RNase L could be tyrosine phosphorylated by FAK in vitro…………107 Figure 3. 9 Activation of RNase L by 2-5A abolish cell haptotaxis………….108-109 Figure 3.10 2-5A transfection promote integrins migrate to plasma membrane…..110 Figure 3.11 RNase L suppressed integrin induced FAN and JNK activation……..111
CHAPTER 4 Figure 4.1 PMA stimulated RNase L phosphorylation………………………..….122 Figure 4.2 PMA treatment suppressed 2-5A induced apoptosis……………..…..123 Figure 4.3 RNase L interacted with integrin pkcα………………………..….…..124
7
Acknowledgements
I would like to thank Dr Robert H. Silverman, my advisor, for his tremendous enthusiasm to my thesis and very helpful comments on the text. I appreciate his generosity and kindness during my Ph. D. study in his lab. I would like to thanks the other members of my thesis committee, Drs. David Samols, Ganes Sen, Bryan
Williams, Alexandru Almasan, for their time and input at every stage of my training.
Especially, I am very grateful to Dr. Yan Xu for many insightful conversations and help in developing the ideas of the thesis. I would also like to thank Dr. Yan Xu’s lab colleagues for their technical support. My thanks also shall go to the people in Dr.
Silverman’s Lab, Beihua Dong, Ying Xiang, Elena Bulanova, Jayashree Paranjape,
Ross Molinaro, Shaija Shelby, Malathi Krishnamurthy, Chandar Thakur, Zachary
Novice, and former members: Aimin Zhou, Zhengu Wang, Gregory Wroblewski, and
Junko Munakami. They make my life in research enjoyable. I also appreciate people in Department of Cancer Biology and Cleveland Clinic for their many invaluable services and trainings. Finally, I am forever indebted to my family for their understanding, endless patience, and encouragement when it was most required.
8 LIST OF ABBREVIATIONS
2–5A 2’-5’ oligoadenylates
2D-GEL Two dimensional gel elctrophoresis
Ab antibody cDNA: complementary deoxyribonucleic acid
dsRNA double-stranded RNA
ECM extracellular
EMCV encephalomyocarditis virus
ERK extracellular signal-regulated kinase
FACS fluorescence-activated cell sorter
FAK focal adhesion kinase
FBS fetal bovine serum
FRET fluorescence resonance energy transfer
HPC hereditary prostate cancer
IFN interferon
JNK c-Jun NH2-terminal kinase
Kb kilo-base pair
MAPK mitogen-activated protein kinase
MEF mouse embryonic fibroblast
MOI: multiplicity of infection
mRNA messenger ribonucleic acid
9 MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt.
PAGE polyacrylamide gel electrophoresis
PARP poly(ADP-ribose)polymerase
PBS phosphate buffer saline
PCa prostate cancer
PKC protein kinase C
PKR protein kinase R
PMA phorbol ester
RNA ribonucleic acid
RNase L ribonuclease L rRNA ribosomal RNA
RT-PCR cDNA synthesis by reverse transcription followed by PCR
amplification
SAPK stress-activated protein kinase
SDS sodium dodecyl sulphate
SiRNA small interfering RNA
Tdt terminal deoxynucleotidyltransferase
TUNEL TdT dUTP nick end labeling analysis
10
The antiviral and antitumor functions of RNase L
Abstract
By Geqiang Li
RNase L is an endoribonuclease that functions in the molecular pathways of interferon (IFN) action against viral infections. Although the pro-apoptotic activity of
RNase L is well known, how RNase L activation during viral infection leads to apoptosis is not clear. Recently, RNase L was considered to be a tumor suppressor based on its pro-apoptotic activity and mapping to hereditary prostate cancer allele 1
(HPC1). While involvement of a viral pathogen in prostate cancer was proposed
(Silverman 2003), the unknown novel mechanism of RNase L in the suppression of prostate cancer progression still remains possible. Several lines of evidence implied that RNase L may possess an unidentified fuction: first, RNase L+/+ fibroblasts are different from RNase L-/- in morphology. Second, RNase L-/- mice showed a 5-day delayed rejection of skin allograft and a dramatic reduction in inflamatory infiltrates
(Silverman RH et al 2002), indicating that RNase L play a role in immunosystem.
Third, RNase L was found to interact with actin cytoskeleton (Tnani M et al 1998), and the interactions were abolished by PMA treatment of the cells. Finally, three inactivating mutations (M1I, E265X, and ∆157) and an additional nine missense variants (G59S, I97L, I220V, V247M, G296V, and S322F) in RNase L have been observed in prostate cancer cases. No mutation is mapped in the ribonuclease domain, indicating that ribonuclease domain might not be involved in RNtase L related
11 pathogenesis in prostate cancer. My thesis thus focused the possible mechanisms of
the antiviral and antitumor functions of RNase L.
In antiviral studies, my thesis showed that 2-5A, dsRNA and viral infection induced
activation of the c-Jun NH2-terminal kinases (JNK) family of MAP kinases and viral
induction of apoptosis are deficient in mouse cells lacking RNase L. In addition, 2-5A
resulted in specific ribosomal RNA cleavage products coinciding with JNK activation.
Moreover, 2-5A induced apoptosis was dramatically enhanced by IFN pretreatment in
JNK1 +/+ JNK2+/+ cells but not in Jnk1-/- Jnk2-/- cells. These findings suggest that JNK
and RNase L function in an integrated signaling pathway during the IFN response that leads to elimination of virus-infected cells through apoptosis.
In antitumor studies, my research established that haptotaxis of RNase L-/- MEF cells
increased about two fold, compared to their wildtype counterparts. Fibronectin
induced activation of FAK, JNK, and c-jun phosphorylation was increased in RNase
L-/- MEF cells. The JNK activity contributed to RNase L -/- enhanced cell migration.
Activation of RNase L by 2-5A also reduced cell motility and was independent of
protein synthesis inhibition. RNase L was associated with the cytoplasmic domain of
integrin β1 and FAK. Together, these results suggest that RNase L has a potential role
in the regulation of metastasis by inhibition of cell migration.
Overall, my thesis expanded knowledge and elucidated new mechanisms by which
RNase L mediated antitumor and antiviral signaling pathways. These findings have
12 important implications for understanding the role of RNase L in prostate cancer pathogenesis.
13 CHAPTER 1 INTRODUCTION
1.1. Overview of the interferon (IFN) system
Interferons are a family of multifunctional cytokines that are responsible for the
induction of cellular resistance to viral infection, modulation of innate and adaptive
immune responses, and inhibition of cell growth. Type I IFN (IFNα and IFNβ) are
produced by virus-infected host cells and constitute the primary response against viral
infection. Type II IFN (IFNγ) is produced by mitogen-activated T cells and natural
killer cells and is crucial for eliciting appropriate immune response and pathogen
clearance. The pleiotropic effects of IFNs are mediated by the JAK-STAT pathways
and interferon regulatory factors (IRFs) (Stark, Kerr et al. 1998), both leading to
transcriptional induction of the IFN-stimulated genes (ISG). The ability of IFNs to
confer the antiviral state on cells is due to several IFN-regulated pathways including:
the dsRNA-dependent protein kinase R (PKR) (Williams 1997), the 2-5A
system/RNase L, the p56 proteins (Guo, Peters et al. 2000; Hui, Bhasker et al. 2003),
and the Mx proteins (Horisberger 1995) (Figure 1.1). PKR can phosphorylate the
translation initiation factor, eIF2α, resulting in inhibition of protein synthesis and suppression of viral replication(McMillan, Carpick et al. 1995; McMillan, Chun et al.
1995; Williams 1997). In addition, PKR is required for activating transcription factors
NF-κB and IRF-1 by IFN to confer its transcriptional effects (Williams 1997). P56 can interact with translation initiation factor, eIF3 and inhibit protein synthesis (Guo,
14 Peters et al. 2000). Mx proteins are IFN-induced GTPases in the dynamin superfamily that interfere with the intracellular movement of some negative stranded RNA viruses(Horisberger 1995).
The dsRNA- The 2-5A system The Mix pathway The P56 protein Dependent Protein IFN Induces
PKR 2-5A synthetases Mx Proteins P56 ATP 3ATP Viral dsRNA GTP activates ADP 2P pppA2’p5’pA2’p5’A PKR- activates (“2-5A”) GDP+Pi eIF3 NF-κB RNase L P38 ssRNA -UpN3’P’ eIF2α eIF2α-Pi +ADP RNA Cleavage Transcritional Protein synthesis inhibition inhibition Inhibition of protein synthesis and transcritional control
Figure 1.1 Antiviral mechanism of interferon action.
1.2. 2-5A / RNase L system
The 2’-5’ oligoadenylate (2-5A) system is an interferon-inducible RNA degradation pathway which is responsible for many of the antiviral and antiproliferative effects of
IFN. The 2-5A pathway is composed of at least three types of enzymatic activities, 2-
5A synthetase, 2-5A-degrading enzymes, and RNase L ( Figure 1.2). The ds-RNA, produced when viruses infect cells, binds to and activates 2-5A synthetases. The activated 2-5A synthetases convert ATP to PPi and a series of short 2’, 5’-linked
15 oligoadenylates collectively referred to as 2-5A [px(A2’p)nA, 1≤x ≤3, 2≤n≤4]
(Cayley, Davies et al. 1984).In humans, there are four related genes (OAS1, OAS2,
OAS3, and OASL) encoding eight or more isoforms of 2-5A synthetase as a result of alternative splicing. To date, the only well established biochemical function of 2-5A is to activate RNase L . 2-5A binds with high affinity with RNase L, converting it from inactive, monomeric state to a potent, dimeric endonuclease (Dong and
Silverman 1995). The 2-5A must have at least one(in human) or two(in mice) 5’- phosphoryl groups and a minimum of three adenylyl residues in 2’, 5’ linkage
(Cayley, Davies et al. 1984). Upon activation, RNase L cleaves cellular rRNA
/mRNA 3’ of UpUp and UpAp sequences, leading to the inhibition of protein synthesis.
RNase L also can cleave viral mRNA and induce host cell apoptosis (Dong and
Silverman 1995; Dong and Silverman 1997).
3ATP virus
2-5A synthetases dsRNA activates Induces 2PPi Interferon α, β, and γ 2’PDE pppA2’p5’pA2’p5’A ATP+2AMP Induces (“2-5A”) 5’-P’tase A2’p5’pA2’p5’A+3Pi RNase L
Viral RNA degradation Cellular RNA degradation Inhibition of viral replication Apoptosis
Figure 1.2 The IFN-regulated 2-5A system, a RNA decay pathway with antiviral and
apoptotic activities
16
1.2.1. Properties of RNase L
RNase L has an interesting arrangement of structural and functional domains (Figure
1.3). The N-terminal half contains nine ankyrin repeats. Ankyrin (ANK) repeats are
relatively common protein sequence motifs which are present in many proteins whose functions are rather unrelated to each other, but typically mediate protein-protein
interactions. ANK proteins do not bind selectively to a single class of protein targets.
Rather, the diversity of the biological roles of ANK proteins is paralleled by the diversity of the unrelated proteins with which they interact. The unique feature of the
ANK repeats in RNase L is that they interact with a nucleic acid, 2-5A. ANK repeats
2 and 4 are involved in 2-5A binding (Figure 1.3) (Tanaka et al 2004). In its native
state, the N terminal half functions as a repressor of the ribonuclease domain in the C-
terminal half (Figure 1.4). The minimum repressor function can be mediated by just
three ankyrin repeats, 7, 8, and 9[(Dong and Silverman 1997; Dong and Silverman
1999). An isolated C-terminal half of RNase L can cleave RNA in the absence of 2-
5A (Dong, Xu et al. 1994; Dong and Silverman 1997). Therefore, the regulatory and
catalytic functions of RNase L are present in the N- and C-terminal halves,
respectively. 2-5A binding to the N-terminal half probably induces a conformational
change of the enzyme, causes the N-terminal repressor domain to release from the C-
terminal ribonuclease domain, and unmasks the dimerization domain. RNase L also
contains several protein-kinase-like domains in its C terminal half. However, several
highly conserved residues required for kinase activity are absent. Protein kinase
17 domain II contains a critical conserved residue, lysine392, that functions in binding to
α and β phosphoryl groups of ATP. RNase LK392R mutant, having a substitution
mutation of the conserved lysine to arginine, is defective for the ribonuclease and
dimerization activities(Dong and Silverman 1999), indicating a critical role of Lysine
392. However, there is no report that RNase L is a protein kinase.
2-5A Binding Domain Catalytic Domain
1 Ankyrin Repeats 335 364 587 720 741
N- 1 2 3 4 5 6 7 8 9 Kinase-Like RNase -C
2-5A
Figure 1.3 The structure of RNase L
RNase L has sequence homology with a yeast protein, Ire1p, which is an
transmembrane protein of the endoplasmic reticulum (ER)(Urano, Wang et al. 2000)
(Figure 1.5). Ire1p is an essential factor in mediating the unfolded protein responses
(UPR) in the lumen of the ER, leading to the splicing of the HAC1 mRNA, coding for a specific transcription factor (Urano, Wang et al. 2000). Interestingly, yeast Ire1p has both serine/threonine kinase and ribonuclease activities. Human Ire1α and Ire1β have also been shown to retain both the kinase and ribonuclease activity. Functional similarities between RNase L and Ire1p include sensor domains in the N-terminal regions, enzyme activation accompanied by oligomerization, and stimulation by
18 adenosine nucleotides such as ATP(Dong, Niwa et al. 2001). There are also some
important functional differences between RNase L and Ire1p. Ire1p can
transphosphorylate during the UPR, in contrast, RNase L lacks protein kinase activity. Another important difference is that Ire1p is a site-specific nuclease, whereas
RNase L is relatively nonspecific(Dong, Niwa et al. 2001).
RNA RNase L K392R R462Q 2-5A N N N
K K K K K A 2-5A
a) Inactive 2- A 2- A
c) Active b) Inactive
A = Ankyrin Repeats 2-5A=p A(2’p5’A) x=1 to 3 n>=2 K = Kinase-like Domain x n
N = Nuclease Domain Inactive dimer: pA2’p5’A
Figure 1.4 Functional model for the activation of RNase L by 2-5A(Dong, Niwa et al. 2001)
Although RNase L has general nuclease activity in cell-free systems, there is growing
evidence for more specificity in intact cells. Recently, RNase L was showed to
degrade mRNAs for two IFN stimulated genes, ISG43 and ISG 15 with some priority,
indicating that RNase L play a role in dampening the IFN response. Bisbal et al
19 (Bisbal, Silhol et al. 2000) showed that in C2 myoblast cells over-expressing RNase
L, the MyoD mRNA was much more quickly degraded compared to the control C2
cells transfected with an empty vector. The effect of RNase L activity on MyoD
mRNA was relatively specific because several other mRNAs were resistant to it. In
EMCV infected cells, viral RNA is preferentially degraded in comparison to cellular
RNA (Li, Blackford et al. 2000). Furthermore, PKR mRNA stability was enhanced and PKR level was increased by IFN in RNase L-null cells. Ectopic expression of
RNase L from a plasmid vector prevented the IFN induction of PKR. These results suggest a role for RNase L in the transient control of the IFN response and possibly of other cytokine and stress responses (Khabar, Siddiqui et al. 2003)
Figure 1.5 Comparison of the domains and motifs in human RNase L and yeast
Ire1p.(Dong, Niwa et al. 2001) SP: signal peptide; TM: transmembrane domain.
1.2.2. Antiviral activity of RNase L
20 Induction of RNA decay by RNase L is one of the host cell responses to viral
infection. The most compelling lines of evidence that link the 2-5A system to specific antiviral effects were obtained by measuring: (1) 2-5A accumulation and RNase L
activation in virus-infected cells(Hearl and Johnston 1987); (2) antiviral effects in
cells expressing 2-5A synthetase cDNA (Schroder, Suhadolnik et al. 1992); and (3)
enhanced virus production and reduction of the antiviral effects of IFN, caused by
inhibiting RNase L activity in cells. For instance, it was shown that expression of the
40kDa form of human 2-5A synthetase from a cDNA in Chinese hamster ovary
(CHO) cells provided resistance to the picornavirus, mengo virus (Chebath, Benech et
al. 1987). Similarly, expression of the 40kDa human 2-5A synthetase cDNA in a
human glioblastoma cell line, T98G, and expression of murine 43kDa 2-5A
synthetase from a cDNA in mouse NIH 3T3 cells resulted in resistance to EMCV
replication (Rysiecki, Gewert et al. 1989). Another strategy to study the involvement
of 2-5A system/RNase L in the antiviral activity of IFN was to use the 2-5A analog,
CH3Sp(A2’p)2A2’pp3’OCH3, which binds to, but does not activate RNase L
(Defilippi, Huez et al. 1985; Defilippi, Huez et al. 1986). Transfection of the analog into IFN-treated, EMCV-infected murine L929 cells inhibited rRNA cleavage and increased virus production by up to 10 fold. Hassel et al(Hassel, Zhou et al. 1993) showed that expression of a dominant negative truncated RNase L in murine SVT2 cells blocked the rRNA cleavage and reduced about 250-fold the anti-EMCV effects of IFN compared with the IFN-treated, vector control cells. The RNase L knockout mice succumbed to EMCV and HSV-1(McKrae strain) infections more rapidly than infected wild type mice (Zhou, Paranjape et al. 1997). RNase L knockout mice
21 treated with IFN prior to EMCV infection also died several days earlier than wild
type mice with the same treatment. However, IFN treatment extended survival to
EMCV infection of both the RNase L wildtype and knockout mice, indicating multiple and overlapping antiviral pathways of IFN. So far, RNase L has been shown to have antiviral effects on many viruses such as EMCV, vaccinia virus, reovirus, herpes simplex virus(HSV) and SV40 (Baglioni, De Benedetti et al. 1984; Diaz-
Guerra, Rivas et al. 1997; Rivas, Gil et al. 1998). However, viral evasion of the 2-
5Asystem/ RNase L was also reported. Vaccinia virus E3L proteins sequestered the dsRNA from 2-5A synthetase (Rivas, Gil et al. 1998), whereas reovirus S4 gene encodes a dsRNA-binding protein σ3 with the same function (Beattie, Denzler et al.
1995)
1.2.3. Apoptotic activity of RNase L
At the cellular level, the antiviral effects of IFN may be partly due to apoptosis.
Indeed, activation of PKR and 2-5A synthetase by dsRNA has been shown to induce apoptosis. Several lines of studies link RNase L to apoptosis in cells in response to viral infection. RNase L-null mice showed enlarged thymuses and reduced levels of spontaneous apoptosis in both the thymus and spleen. In addition, thymocytes and lymphocytes isolated from spleen of RNase L-null mice were resistant to apoptosis induced by staurosporine and irradiation (Zhou, Paranjape et al. 1997; 1998; Rusch,
Zhou et al. 2000). Furthermore, overexpression of dominant negative RNase L in
cells reduced apoptosis whereas overexpression of wildtype RNase L enhanced the
22 apoptosis in response to viral infection(Hassel, Zhou et al. 1993). The apoptosis
induced by RNase L involves cytochrome c release, is caspase dependent, and is
inhibited by overexpression of Bcl-2(Castelli, Hassel et al. 1997; Rusch, Zhou et al.
2000; Silverman 2003). However, how RNase L induces apoptosis is still unknown
and is a major theme of my thesis.
2-5A Binding & Repressor Dimerization Catalytic Activity (Ankyrin GKT 1 10 200 300 400 500 60 700 N- 1 2 3 4 5 6 7 8 9 Kinase-Like RNase -C
I220V M1I R462Q G59S V247M Y529C I97L E265X
∆157 G296V D541E
S322F
Figure 1.6 RNase L mutations in different populations of prostate cancer cases aligned to the domain structure of RNase L.
1.2.4 Involvement of RNase L in the biology of prostate cancer
The prostate is a small male sex gland located below the bladder, that produces
seminal fluid (Figure 1.7). Carcinoma of the prostate is the second leading cause of cancer death in men and the most frequent visceral cancer in males (reviewed in silverman RH 2003). Prostate cancer occurs mostly in older men, it is rare before the
age of 50, and the risk increases with age. There has been an increase in the incidence
23 of prostate cancer since the early 1980's. World-wide about 395,000 men are
diagnosed with prostate cancer each year (www.cancer.org). There is strong evidence
for both genetic and environmental factors in prostate cancer development. About 9% of patients with prostate cancer belong to families with hereditary prostate cancer
(HPC). Linkage analyses in families with HPC have suggested that multiple genetic loci may harbor PC-susceptibility genes including HPC1(MIM 601518),at 1q24-q25;
ELCA2/HPC2(MIM 605367), at 17p11; PCAP(MIM 602759a), at 1q42.2-q43;
HPCX(MIM 300147), at Xq27-q28; CAPB (MIM603688), at 1p36; and HPC20(MIM
176807), at 20q13 (Ostrander and Stanford 2000; Stanford and Ostrander 2001). Only
three candidate susceptibility genes in these genomic regions have been identified.
HPC2/ELAC2 , was the first positionally cloned prostate cancer susceptibility gene,
encoding a novel protein with undefined function. However, several lines of
independent experiment shows elac2 has a minor or no role in HPC (Nupponen,
Wallen et al. 2004). The second identified prostate cancer susceptibility gene is the macrophage scavenger receptor 1 (MSR1) gene, located at 8p22-p23. Segregating germ-line mutations in MSR1 have been reported in 13 families affected with hereditary prostate cancer (Xu, Zheng et al. 2001; Xu, Zheng et al. 2003). In addition, these germ-line mutations have also been found in patients with non-HPC. One missense mutant Arg293X, was observed significantly more frequently in men with non-HPC than in unaffected individuals.
RNase L is the third prostate cancer susceptibility gene identified by a positional- cloning/candidate method. Germ-line mutations were reported to cosegregate within
24 families with hereditary prostate cancer (HPC) linked to HPC1 region at 1q24-25
(Carpten, Nupponen et al. 2002). Now, four genetic mutations from cases in US,
Finland, and Israel support the identification of RNase L as HPC1 (Figure 1.6). Two
HPC1 families contained germline inactivating mutation: a 795G→T substitution in
exon 2 of RNase L resulting in the conversion of a glutamic acid to stop codon
(E265X). This mutation terminated translation within the 2-5A binding domain of
RNase L, thus abolishing 2-5A binding ability (Rokman, Ikonen et al. 2002). The
second mutation occurred in the translational start codon, a methionine to isoleucine
missense mutation (3G→A) (M1L). The third mutations, a 1385G→A resulting in an arginine to glutamine substitution at amino acid 462 which causes a three-fold decrease in RNase L activity. The fourth mutations, 471∆AAAG, caused a frame shift at codon 157 and a translation stop after seven additional codons. This mutation appeared in Ashkenazi Jews at a relatively high frequency(4%) and in the prostate cancer cell line, LNCaP (Nupponen, Wallen et al. 2004). The frequency of the mutation was higher among prostate cancer patients (6.9%) than in elderly, unaffected men (2.4%) of the same population group. RNase enzymatic activities assay showed that M1L or E265X carriers had half the level of RNase L activity compared with homozygous wildtype RNase L family members (Xiang, Wang et al.
2003).
Recently the R462Q (Figure 1.6) mutation of RNase L was further implicated in
unselected prostate cancer cases in a study of US patients. A significant association of
the R462Q with HPC cases was observed, (p=0.011) . The odds ratios indicated that
25 carrying one copy of R462Q variant gene increased risk of prostate cancer by about
1.5 fold, while having two variant alleles doubled the risk(Casey, Neville et al. 2002).
On the other hand, a D541E variant, was not associated with increased risk of prostate cancer. Interestingly, R462Q has only a 3-fold decrease in enzymatic activity(Xiang,
Wang et al. 2003).
Although a relationship between the RNASEL gene and prostate cancer is established, much more work needs to be done to show how commonly it causes or modifies the clinical course of the disease. A larger number of men - both with and without strong family histories, and with and without prostate cancer - need to be studied to see how often mutations in the gene are associated with the disease and to find out how often mutations occur in men without the disease. Furthermore, how
RNase L functions as a tumor suppressor in prostate cancer tumorigenesis and/or metastasis is still unknown (Figure 1.7). According to our knowledge, there exist several possible mechanisms by which RNase L could function as an anti-tumor protein.
First, RNase L has anti-proliferative and pro-apoptotic activities, which suggested that RNase L may have a tumor suppressor function. The 2-5A /RNase L system is involved in cellular responses to several external stimuli that result in apoptosis, such as viral infections, and TRAIL and camptothecin treatments (Krishnamurthy M. et al
2004). Androgen and IFN can upregulate OAS (P42), thus potentially priming the pathway for activation (Xiang Y. and Silverman R.H. Unpublished data). The
26 prostate has been recently reported to be a host organ for multiple viral infections,
including the human polyoma JC virus and HPV (Zambrano, Kalantari et al. 2002).
RNase L is an established antiviral gene. Involvement of a viral pathogen in prostate
cancer development remains a possibility.
Second, recently, our lab has shown that after down-regulation of RNase L by siRNA
in the prostate cancer cell line DU 145, cells were resistant to TRAIL and
camptothecin induced apoptosis (Krishnamurthy M. et al 2004). These findings indicated that RNase L is involved in an apoptotic signaling pathway other than that
induced by 2-5A. More interestingly, it was reported that RNase L-/- mice showed a
delayed rejection of skin allograft, suggesting that RNase L may also contribute to
tumor rejection through the immune system (Silverman, Zhou et al. 2002).
Finally, it was also very interesting that RNase L interacts with cytoskeleton protein,
filamin A. Filamin A is associated with integrin and FAK, which is important for cell
cytoskeleton rearrangement and cell migration. It was further found that in the filamin
deficient melanoma cell line M2, enzymatic activity of RNase L was dramatically
reduced, while in the A7 cell line in which filamin A was reconstituted, RNase L
enzymatic activity was restored, indicating RNase L activities can be regulated by
protein-protein interaction. (Xiang Y, Yin Z and Silverman RH, unpublished data).
These data provide some new possibilities for RNase L function in prostate cancer.
27 Understanding the molecular events by which RNase L functions as a tumor suppressor is another theme of my thesis. RNase L
?? ?
Normal Prostatic Invasive Epithelium Intraepithelial Carcinoma Metastasis Neoplasia (PIN)
Figure 1.7 Progression of prostate cancer and steps where RNase L could interfere.
28 CHAPTER 2. AN APOPTOTIC SIGNALING
PATHWAY IN THE INTERFERON ANTIVIRAL
RESPONSE MEDIATED BY RNASE L AND C-JUN
NH2-TERMINAL KINASE*
ABSTRACT
Cellular stress responses induced during viral infections are critical to the health and
survival of organisms. In higher vertebrates, interferons (IFNs) mediate the innate antiviral response in part through the action of RNase L, a uniquely regulated enzyme.
RNase L is activated by 5'-phosphorylated, 2'-5' oligoadenylates (2-5A) produced
from IFN-inducible and double stranded RNA-dependent synthetases. We show that
viral activation of the c-Jun NH2-terminal kinases (JNK) family of MAP kinases and
viral induction of apoptosis are both deficient in mouse cells lacking RNase L. Also,
JNK phosphorylation in response to 2-5A was greatly reduced in RNase L-/- mouse
cells. In addition, 2-5A treatment of the human ovarian carcinoma cell line, Hey1b,
resulted in specific ribosomal RNA cleavage products coinciding with JNK activation.
Furthermore, suppression of JNK activity with the chemical inhibitor, SP600125,
prevented apoptosis induced by 2-5A. In contrast, inhibition of alternative MAP
kinases, p38 and ERK, failed to prevent 2-5A-mediated apoptosis. Short interfering
29 RNA to JNK1/JNK2 mRNAs resulted in JNK ablation while also suppressing 2-5A-
mediated apoptosis. Moreover, Jnk1-/- Jnk2-/- cells were highly resistant to the
apoptotic effects of IFN an 2-5A. These findings suggest that JNK and RNase L function in an integrated signaling pathway during the IFN response that leads to
elimination of virus-infected cells through apoptosis.
*(The content of chapter 2 has been published. Li G. et al J. Biol. Chem. 2003.)
Contributions by other people:
( Ying Xiang generated PIND RNase L and PIND RNase L∆EN cells; Sabapathy K.
generated the JNK1+/+ JNK2-/- fibroblasts.)
30 2.1 INTRODUCTION
1 The c-Jun NH2-terminal kinase (JNK) family of MAP kinases relays signals from a
wide range of extracellular stimuli including viruses, cytokines, and environmental
stress ((Chu, Ostertag et al. 1999; McLean and Bachenheimer 1999; Ludwig,
Ehrhardt et al. 2001; Weston and Davis 2002). The three JNK genes (Jnk1, Jnk2, and
Jnk3) encode ten different molecular mass species of JNK as a result of alternative
splicing, including 55- and 46-kDa isoforms from each gene (Davis 2000). Mice with
homologous disruptions of the individual Jnk genes are viable, as are Jnk1-/- Jnk3-/- and Jnk2-/- Jnk3-/- double gene knockout mice (Kuan, Yang et al. 1999). In contrast,
combined loss of ubiquitously expressed JNK1 and JNK2 results in embryonic
lethality associated with altered neuronal apoptosis and exencephaly (Sabapathy,
Jochum et al. 1999; Tournier, Hess et al. 2000). JNK3 is present mostly in brain, therefore Jnk1-/- Jnk2-/- mouse embryonic fibroblasts (MEFs) lack JNK and are an
important resource for determining the biologic functions of JNK (Sabapathy, Jochum
et al. 1999; Tournier, Hess et al. 2000). For example, Jnk1-/- Jnk2-/- MEFs are
deficient in both activator protein-1 transcriptional activity and stress-induced
apoptosis (Tournier, Hess et al. 2000; Ventura, Kennedy et al. 2003).
JNKs often play a critical role in controlling the balance between cell survival and
death. For instance, JNK1 signals cell survival in transformed B lymphocytes
mediated in part by increased Bcl2 expression (Hess, Pihan et al. 2002). In contrast,
sustained activation of JNK in the absence of cell survival signals results in apoptosis
(Lei, Nimnual et al. 2002). A major function of JNK is regulation of the activator
31 protein-1 transcription factor through phosphorylation of c-Jun and related proteins
(reviewed in Ref. (Davis 2000). However, in addition to controlling new transcription,
JNKs participate in apoptotic signaling by regulating the activities of pre-existing
Bcl2-related proteins that mediate mitochondrial release of cytochrome c and subsequent caspase activation (Tournier, Hess et al. 2000). For example, proteolytic activation of Bid, a pro-apoptotic BH3-only Bcl2-related protein, occurs in UV-
treated wild type cells but not in similarly treated Jnk1-/- Jnk2-/- MEFs. Furthermore,
JNK activation failed to induce death of cells deficient in the pro-apoptotic proteins,
Bax and Bak(Lei, Nimnual et al. 2002) . The possible role of JNK in regulating
apoptosis through Bcl2 and Bcl-xL phosphorylation is controversial, in part because
results vary as to whether apoptosis would be enhanced or suppressed(Ito, Deng et al.
1997; Maundrell, Antonsson et al. 1997; Yamamoto, Ichijo et al. 1999) . In neurons,
the BH3-only Bcl2 member, Bim, and JNK are both implicated in apoptosis caused by nerve growth factor deprivation(Putcha, Moulder et al. 2001; Whitfield, Neame et
al. 2001). Although these studies suggest that JNK can control certain Bcl2-related
molecules at different levels, the molecular mechanism(s) by which JNK directly or
indirectly controls activation of different Bcl2 members remain to be elucidated.
Among the types of cellular stress stimuli that activate JNKs are agents that damage
cellular RNA, in particular ribosomal RNA (rRNA). For example, ribotoxins ( -sarcin and ricin A chain) and UV that damage the 3' end of the large (28 S) rRNA activate
JNK (Iordanov, Pribnow et al. 1997; Iordanov, Pribnow et al. 1998; Iordanov,
Paranjape et al. 2000; Iordanov, Ryabinina et al. 2000; Iordanov, Wong et al. 2000;
Iordanov, Choi et al. 2002). JNK is also activated by double stranded RNA (dsRNA)
32 treatment of cells, which results in the cleavage of 28 S rRNA by RNase L (Iordanov,
Paranjape et al. 2000). dsRNA, often viral in origin, activates the IFN-inducible 2-5A
synthetases that convert ATP to PPi and a series of 2'-5' oligoadenylates (p3A(2'p5'A)n, n = 2 4) collectively referred to as 2-5A (see Ref.: (Kerr and Brown 1978); reviewed
in Ref.: (Silverman 2003). The only well established function of 2-5A is activation of
RNase L, an endoribonuclease with nine ankyrin repeats and two P-loop motifs in its
N-terminal half and protein kinase-like and a ribonuclease domain in the C-terminal
half (Zhou, Hassel et al. 1993). Binding of 2-5A in the ankyrin-region of RNase L
converts the enzyme from inactive monomers to active dimers (Dong and Silverman
1995). Sustained activation of RNase L by 2-5A leads to apoptosis, thus limiting the
spread of viral infections (Castelli, Hassel et al. 1997; Zhou, Paranjape et al. 1997;
Rusch, Zhou et al. 2000). RNase L-/- mice are viable but have enhanced susceptibility
to encephalomyocarditis virus (EMCV) and are deficient in apoptosis in several
different cell types, including thymocytes, splenocytes, and fibroblasts(Zhou,
Paranjape et al. 1997). In addition, RNase L-/- mice are deficient in cellular immunity
both after skin allografts and during alphaviral-based DNA tumor vaccinations
(Silverman, Zhou et al. 2002; Leitner, Hwang et al. 2003). The pro-apoptotic activity
of RNase L may restrict tumor growth as suggested by the recent mapping of the hereditary prostate cancer 1 (HPC1) susceptibility allele to the RNase L gene
(reviewed in Ref. (Silverman 2003); see Ref. (Carpten, Nupponen et al. 2002).
Activation of RNase L in cells leads to mitochondrial release of cytochrome c and
caspase-dependent apoptosis (Rusch, Zhou et al. 2000). The apoptotic activity of
RNase L is suppressed by Bcl2, further implicating the involvement of mitochondria
33 in the cell death pathway initiated by 2-5A (Diaz-Guerra, Rivas et al. 1997; Diaz-
Guerra, Rivas et al. 1997). Here we demonstrate involvement of RNase L in JNK activation during viral infections, and furthermore we show that JNK is essential for apoptosis in response to RNase L activation.
34
2.2 MATERIALS AND METHODS
2.2.1 Cell culture
Human multiple myeloma cells IM-9, and human ovarian carcinoma cell line Hey1B
were routinely maintained in RPMI 1640 medium supplemented with 10% fetal
bovine serum (Invitrogen) and penicillin/streptomycin at 37°C and 5% CO2. pSFFV-
neo PCDNA3/Bcl-2 was used for stable transfection of IM-9 cells. Stably
overexpressing Bcl-2in IM-9 were maintained in 1 mg/ml G418 as described
previously (Zheng, Shi et al. 2004) JNK 1-/- JNK2-/-cells and JNK1+/+JNK2+/+ mouse embryonic fibroblasts were cultured in Dulbecco's modifed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and streptomycin
2.2.2 Transfections
Transfections with 2-5A, siRNA, or plasmids were performed using LipofectAMINE
2000 according to the manufacturer's protocol (Invitrogen). In experiments requiring
extended incubations, 3.5 h after transfection an equal volume of medium containing
20% fetal bovine serum was added. In experiments involving IFN, cells were
pretreated with IFNα (1,000 units per ml) for 16 h and then transfected with 2-5A. In
experiments involving MAP kinase inhibitors, cells were preincubated with inhibitor
for 1 h prior to 2-5A transfection.
35 2.2.3 Viral Infections
Cells were grown overnight, and medium was removed and replaced with serum-free
medium containing EMCV (American Type Culture Collection) at a multiplicity of
infection (m.o.i.) of 1 to 2 for 0.5 h at which time cells were washed, and medium
with 10% fetal bovine serum was added. Induction of RNase L expression from
ponasterone-inducible vectors was performed by pre-incubating cells with 5 µM
ponasterone for 16 h prior to EMCV infections.
2.2.4 Measuring Protein Synthesis in Intact Cells
Rates of cellular protein synthesis were determined as described previously (Rusch,
Dong et al. 2001). Briefly, cells were transfected with ps5'A(2'ps)3A for 3 h at 37 °C.
After removing medium and washing cells with phosphate-buffered saline, RPMI
1640 medium lacking methionine (Specialty Media, Phillipsburg, NJ) and
supplemented with 0.3 µCi per ml of [35S]methionine (>1000 Ci/mmol; Amersham
Biosciences) was added, and cells were incubated a further 2 h. Medium was removed,
and protein was precipitated with 5% (w/v) trichloroacetic acid as described (Rusch,
Dong et al. 2001). Precipitated protein was solubilized with 0.25 M NaOH and
neutralized, and radioactivity was determined by liquid scintillation counting.
2.2.5 RNase L Activity in Intact Cells
Hey1b cells were transfected with 5 µM 2-5A using LipofectAMINE 2000. At the
indicated times, the total RNA was isolated from transfected cells using Trizol reagent
(Invitrogen) and quantitated by measuring absorbance at 260 nm. RNA (2 µg) was
36 separated on RNA chips and analyzed with Bioanalyzer 2100 (Agilent Technologies).
The peak areas of 28 S and 18 S rRNA and their main cleavage products were
determined by using the Bio Sizing (version A.10.30 S1220) program (Agilent
Biotechnologies).
2.2.6 RNase L Activity in a Cell-free System
RNase L activity was determined by the fluorescence resonance energy transfer
(FRET) method.3 The assay uses recombinant human RNase L produced in insect
cells from a baculovirus vector and purified to homogeneity with Blue Sepharose
columns (Dong and Silverman 1995). The cleavable substrate consists of a 36-
nucleotide synthetic oligoribonucleotide sequence derived from respiratory syncytial
virus with the fluorophore, FAM, at the 5' terminus and black hole quencher-1 (BHQ-
1), at the 3' terminus (synthesized at Integrated DNA Technologies). The RNA
sequence contains several cleavage sites for RNase L (UU or UA). The reaction
buffer contains 100 nM RNA probe, 25 nM RNase L, 25 mM Tris-HCl (pH 7.4), 100
mM KCl, 10 mM MgCl2, 50 µM ATP, and 7 mM 2-mercaptoethanol with and without 50
nM psA(2'ps5'A)3 and SP600125 (25 µM). RNase L was the last component added to
the reaction mixtures, which were incubated for 30 min at 20 °C. Fluorescence was
measured with a Wallac 1420 fluorometer (PerkinElmer Life Sciences) (absorption
485 nm/emission 535 nm).
2.2.7 2-5A Binding Assay for RNase L
37 32 8 32 A P-labeled and bromine-substituted 2-5A analog, p(A2'p)2(br A2'p)2A3'[ P]pCp,
was cross-linked to RNase L present in cytoplasmic extracts of cells under ultraviolet
light (Nolan-Sorden, Lesiak et al. 1990). The cell extracts (200 µg protein) were incubated with the probe (0.02 µCi; 750 Ci/mmol) on ice for 60 min and then under
308 nm of light on ice for 60 min. Protein separation was by electrophoresis on
SDS/8% polyacrylamide gels followed by autoradiography of the dried gels.
Quantitation was by phosphorimage analysis.
2.2.8 Cell Viability Assay
The viability of Hey1b cells were determined by using the colorimetric Cell titer 96®
AQueous Cell Proliferation (tetrazolium conversion) assay (Promega). Hey1b cells were
seeded (1.5 x 104 cells per well) into 96-well plates. At various times of treatment
with 2-5A and/or SP600125, 50 µl of Cell titer 96® AQueous reagents were added to
each well, incubated at 37 °C for 2 h, and absorbance was measured at 490 nm with a
96-well plate reader (model Spectra max 340; Molecular Devices).
2.2.9 Western Blots
Cells were lysed in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM
EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM
Na3VO4, and 1 µg/ml leupeptin). Insoluble material was removed by centrifugation at
10,000 x g (at 4 °C for 10 min). Protein concentrations in the supernatants were
determined (Bio-Rad protein assay kit). Protein (30 µg per lane) was separated in 8 to
12% polyacrylamide gels containing SDS and transferred to polyvinylidene difluoride
38 membranes (Millipore). Membranes were incubated with different primary antibodies
according to the manufacturer's protocols.
2.2.10 TUNEL Assay
Terminal deoxynucleotidyltransferase (TdT) dUTP nick end labeling analysis
(TUNEL) for DNA fragmentation was carried out using an apo-Brdu kit (BD
Biosciences). Briefly, after the indicated treatments, media containing floating cells
were collected. Adherent cells were trypsinized and combined with medium
containing floating cells. Cell were fixed in 1% paraformaldehyde for 15 min and
stored in 70% ethanol at -20 °C until staining and analysis. DNA breakage was
determined by incorporating 5-bromo-2'-deoxyuridine and staining with a labeled
anti-bromodeoxyuridine monoclonal antibody. The total DNA content was
determined with propidium iodide, and analysis was performed by flow cytometry in
a fluorescence-activated cell sorter (FACS).
2.2.11 JNK Kinase Assay
JNK kinase activity was determined using the SAPK/JNK Assay Kit (nonradioactive)
(Cell Signaling Technology, Inc.). Briefly, cells were treated as described and lysed in
cell lysis buffer (provided by the manufacturer). Supernatants of 12,000 x g/10-min
centrifugations were incubated with an N-terminal c-Jun fusion protein bound to
glutathione-Sepharose beads to pull down SAPK/JNK. The beads were washed twice
to remove nonspecific bound proteins. The kinase reaction was in the presence of
ATP in 25 mM Tris-HCl (pH 7.5), 5 mM β-glycerophosphate, 2 mM dithiothreitol,
39
0.1 mM Na3VO4, and 10 mM MgCl2. JNK-induced phosphorylation of c-Jun was measured by quantitative immunoblotting with phospho-c-Jun (Ser-63) antibody.
40
2.3 RESULTS
2.3.1 Viral Activation of JNK and Apoptosis Are Deficient in Cells Lacking
RNase L
To determine the impact of RNase L on viral-induced apoptosis, RNase L+/+ or RNase
L-/- MEFs were infected with EMCV at an m.o.i. of 2 and were then monitored for
DNA breakage by FACS TUNEL assays at 8 h post-infection. Results show about
34% of wild type cells became TUNEL-positive after infection, in contrast to only 8%
of cells lacking RNase L (Figure 2.1B). In addition, in RNase L-/- cells induced to
express full-length human RNase L (from plasmid pINDRNase L), the level of virus-
induced apoptosis increased to about 20% whereas induced expression of a truncated
form of RNase L lacking the endoribonuclease domain (from plasmid pINDRNase L EN) produced only about 11% TUNEL-positive cells (Figure 2.1, A and B). These findings
show that RNase L contributes to EMCV-induced apoptosis in MEFs cells. EMCV
infections (m.o.i. = 1) of wild type and the reconstituted (pINDRNase L) MEFs also
caused robust phosphorylation of JNK (46- and 54-kDa isoforms, JNK1 and JNK2 both have 46/54 isoforms) by 6 h post-infection correlating with cleavage of the death
substrate PARP (Figure 2.1C). In contrast, at the same time post-infection in the
RNase L-/- MEFs there was relatively weak JNK phosphorylation (about 4-fold lower
+/+ than in the RNase L and pINDRNase L cells as determined by densitometry) and little
or no PARP cleavage. A more modest deficiency in virus-induction of JNK
41 phosphorylation was observed in cells expressing the nuclease-deleted polypeptide,
RNase L∆EN, and although a low level of PARP cleavage was present even in the
absence of virus there was no increase until a late time (8 h), post-infection.
Previously we showed using the same cell lines that the RNase L-/- cells contained the
same levels of JNK as wild type MEFs (Iordanov, Paranjape et al. 2000). Therefore,
these results demonstrate that RNase L contributes to JNK phosphorylation
correlating with cell death during EMCV infections.
2.3.2 2-5A Activation of RNase L Results in Stimulation of JNK and Apoptosis
To determine the specific contribution of RNase L to JNK activation and apoptosis,
we transfected cells with 2-5A. Transient transfection of tetrameric 2-5A, p3(A2'p)3A
+/+ -/- (10 µM for 4 h), caused JNK phosphorylation in RNase L cells but not RNase L cells (Figure 2.2A, compare lanes 2 and 4). Similar findings were seen in
ponasterone-induced pINDRNase L stable cell expressing full-length wild type RNase L
but not in the uninduced cells (Figure 2.2A, compare lanes 6 and 8) (Xiang, Condit et
al. 2002). The ribonuclease domain was necessary for 2-5A induction of JNK as there
was no JNK phosphorylation in ponasterone-treated RNase L-/- cells containing
pINDRNase L EN (Figure 2.2A, lane 12).
To extend these findings to human cells, studies were performed on the ovarian
carcinoma cell line, Hey1b, treated with biostable phosphorothioate 2-5A analogs
(Xiang Y. et al 2003). A control compound, the diadenylate psA2'sp5'A, which is
unable to activate RNase L, failed to induce PARP cleavage or to cause significant
JNK phosphorylation (Figure 2B, lanes 2-6). In contrast, transfection with the active
42
2-5A analog, ps(A2'ps5')3A, resulted in phosphorylation of JNK and c-Jun
phosphorylation in a time- and concentration-dependent manner (Figure 2.2, B and C).
Activation of JNK peaked by 3.5 h after 2-5A transfection, preceding the cleavage of
PARP by several hours (Figure 2.2B, lanes 7-11). MAP kinase p38, on the other hand,
was not phosphorylated in the Hey1b cells treated with 2-5A. In contrast exposing cells to UV light did activate p38 in these cells (Figure 2.2D, lane 14). The tetramer,
ps(A2'ps5')3A, caused a dose-dependent (up to 8 µM) inhibition of protein synthesis,
<30% of the control, whereas the same concentration of the diadenylate, psA2'sp5'A,
only reduced the protein synthesis rate to 88% of that observed in the untreated cells
(Figure 2.2E).
To confirm the requirement of ribonuclease activity for apoptosis, we transiently
transfected HeLa M cells, which contain low endogenous levels of RNase L, with
cDNAs for either wild type RNase L or missense mutant RNase LR667A lacking
ribonuclease activity (see Figure 2.1A and Figure 2.3) (Dong, Niwa et al. 2001).
PARP cleavage, 55%, was observed at 24 h post-treatment with 2-5A of the control
cells (Figure 2.3, lane 3). In contrast, in HeLa M cells transfected with wild type
RNase L there was 68% PARP cleavage at 9 h and nearly complete cleavage by 24 h
post-treatment with 2-5A. On the other hand, transfection with the RNase LR667A showed only slight levels of PARP cleavage, 8% at 24 h, possibly a nonspecific
response because of protein over-expression (Figure. 2.3, lanes 8 and 9). These findings suggest that the ribonuclease activity of RNase L contributes to apoptosis.
43 It was reported that RNase L induced apoptosis was mediated by the mitochondrial
pathway (Rusch L et al 2000). We transfected 2-5A into human multiple myeloma
cell line, IM9 and the same cells that stably expressed Bcl-2 (IM9-pcDNA3-Bcl-2, a
gift from Dr. A. Almasan). Overexpression of Bcl2 fully blocked 2-5A induced
PARP cleavage. (Figure 2.4 A). Inhibition of 2-5A induced apoptosis was further
confirmed by TUNEL assays. While 2-5A induced more than 25% TUNEL positive
cells, over-expression of Bcl2 significantly reduced the TUNEL positive cells down
to 8%. This result confirmed that RNase L mediated apoptosis was through the
mitochondrial pathway. Bcl2 can be phosphorylated by JNK (Yamamoto, Ichijo et al.
1999) and plays a role in JNK induced cytochrome C release from mitochondria. This suggested that JNK might also be involved in RNase L induced apoptosis.
JNK activation as measured by c-Jun phosphorylation was compared with rRNA
cleavage as a function of time (Figure 2.5). The inactive, dimeric form of 2-5A
(p3A2'p5'A) did not induce JNK activation or rRNA cleavages in the Hey1b cells
(Figure 2.5, lanes 1-5). In contrast, treatment with natural 2-5A resulted in appearance
of both specific rRNA cleavage products and c-Jun phosphorylation beginning by 1 to
2 h post-2-5A treatment (Figure 2.5 B, lanes 6-11, and C). To determine whether JNK
activation by RNase L required ongoing protein synthesis, cells were pretreated with
cycloheximide (10 µg per ml) for 30 min prior to transfection with 2-5A (5 µM) for 3
h (Figure 2.6). Cycloheximide treatment by itself caused only weak activation of JNK
as measured by c-Jun phosphorylation (Figure 2.6, lane 3). In contrast, strong
phosphorylation of c-Jun phosphorylation occurred in response to 2-5A treatment regardless of prior inhibition of protein synthesis by cycloheximide treatment (Figure
44 2.6, lanes 2 and 4). Indeed, c-Jun phosphorylation was potentiated by cycloheximide
treatment. These results indicate that JNK activation by RNase L does not require
ongoing protein synthesis and therefore differs from the ribotoxic stress response
(Iordanov, Pribnow et al. 1997; Iordanov, Pribnow et al. 1998; Iordanov, Paranjape et al. 2000; Iordanov, Ryabinina et al. 2000; Iordanov, Wong et al. 2000; Iordanov,
Choi et al. 2002).
It was reported that RNase L is essential for double stranded RNA induced JNK
activation(Iordanov, et al 2000). Here we examine the effect of dsRNA on JNK
activation in RNase L+/+ and RNase L-/- cells. DsRNA induced JNK activation almost
at the same level (Figure 2.7A). It is contradictory that virus induced higher levels of
JNK activation in RNase L wildtype embryonic fibroblasts. However, virus infection differs from dsRNA transfections in that virus can induce cell protein synthesis inhibition and apoptosis more effectively. In the protein synthesis inhibitor, 2-5A significantly induced more JNK activation. We then transfected dsRNA (10µg/ml) with cycloheximide (2µg/ml). As expected, dsRNA induced dramatically higher levels of JNK activation in RNase L wildtype cells, compare to RNase L deficient cells (Figure 2.7B). Overexpression of human RNase L in RNaseL-/- cells restored
the JNK activation to a level that was similar to wildtype cells.
2.3.3 Inhibition of JNK Impairs RNase L-induced Apoptosis.
To determine whether there was a causal relationship between JNK activation and
apoptosis, Hey1b cells were pre-treated with an anthrapyrazolone inhibitor. SP600125
45 is a reversible, ATP-competitive inhibitor for JNKs and other several other kinases
(Diaz-Guerra, Rivas et al. 1997; Bennett, Sasaki et al. 2001; Bain, McLauchlan et al.
2003). Hey1b cells were pre-incubated 1 h in the absence or presence of different
amounts of SP600125 prior to treatments with the active 2-5A analog, psA(2'ps5'A)3.
JNK activity was measured by the appearance of phosphorylated c-Jun after 3.5 h of
2-5A treatment. At 10 µM SP600125, activation of JNK was inhibited (Figure 2.8A, E
, lane 5, upper panel). In addition, cleavage of PARP measured after 12 h was also
inhibited by 10µM SP600125. Accordingly, cell viability as measured by MTS
(tetrazolium conversion) assays, was greatly reduced by 9 h of treatment with 2-5A
alone, but cell viability remained unaffected when cells were preincubated with
SP600125 prior to 2-5A treatments (Figure 2.8B). To determine whether the effect
SP600125 directly inhibited RNase L, cell-free system assays were performed.
Purified RNase L was incubated with 2-5A in the presence or absence of SP600125.
Cleavage of a dual labeled RNA substrate was then measured by the FRET method.
Results showed that SP600125 had no effect on RNase L activity (Figure 2.8C).
Although the JNK inhibitor effectively prevented apoptosis, inhibitors of p38
(SB202190) and SB203580) and ERK (PD98059) had no effect on apoptosis induced
by 2-5A as measured by PARP cleavage (Figure 2.8D). Therefore, 2-5A-mediated
apoptosis requires JNK, but not p38 or ERK.
2.3.4 Ablation of JNK Suppresses Apoptosis Induced by 2-5A Activation of
RNase L
46 Because SP600125 is not specific for JNKs(Bain, McLauchlan et al. 2003), it was necessary to confirm involvement of JNKs by other means. To verify the role of JNKs in RNase L-induced apoptosis, JNK1 and JNK2 were simultaneous ablated in Hey1b cells by transfecting with an siRNA oligonucleotide to a conserved sequence in the
mRNAs for these two kinases (see "Materials and Methods") (Fig. 2.9). Down-
regulation in the levels of the JNKs prevented c-Jun protein phosphorylation and also
prevented PARP cleavage 9 h after 2-5A treatment (Fig. 2.9). In contrast, as a
control for nonspecific effects, siRNA to luciferase mRNA had no effect on 2-5A
induction of JNK activation or PARP cleavage (data not shown).
To provide further evidence for involvement of JNKs in RNase L-mediated apoptosis,
Jnk1-/- Jnk2-/- double gene knockout MEFs were used. JNK activity measured by in
vitro phosphorylation of c-Jun fusion protein was induced by natural 2-5A (10 µM) in
wild type MEFs but was absent in the Jnk1-/- Jnk2-/- cells (Figure 2.10A). In addition,
8% PARP cleavage was apparent by 6 h of 2-5A treatment increasing to 27 and 82% cleavage by 9 and 24 h in the wild type cells. In contrast, there was no observable
PARP cleavage at 6 and 9 h and only 32% cleavage after 24 h in the Jnk1-/- Jnk2-/-
cells treated with 2-5A (Figure 2.10 B). Apoptosis in response to 10 µM natural 2-5A
for 24 h was 8% in wild type MEFs and only 3% in the Jnk1-/- Jnk2-/- cells as
determined by FACS TUNEL assays. IFN treatments prior to 2-5A transfections are
known to enhance apoptosis (Zhou, Paranjape et al. 1997). Although IFN- treatments (1,000 units per ml) alone did not induce apoptosis in either wild type or
Jnk1-/- Jnk2-/- cells, treatment with IFN- prior to 2-5A transfection caused a large
increase in apoptosis (to about 38%) in the wild type cells. In contrast, the
47 combination IFN and 2-5A treatments only marginally increased the level of apoptosis in the Jnk1-/- Jnk2-/- cells, to 4.5% (Figure 2.10 C). The IFN stimulation of
2-5A-mediated apoptosis was because of a large IFN-induction of RNase L levels (3-
to 6-fold) as determined by covalently cross-linking of a 32P-labeled 2-5A analog to
RNase L (Figure 2.10 D).
48 2.4 DISCUSSION
2.4.1 Essential Role of RNase L in Viral Activation of JNK.
Our findings demonstrate that RNase L enhances viral activation of JNK. During
EMCV infections there was a deficiency both in JNK activation and apoptosis in
RNase L-/- cells compared with wild type cells. The possibility that epigenetic
differences in the cell lines may have accounted for these findings was ruled out by
reversal of phenotype because of induced expression of full-length but not truncated
RNase L. Our results are also consistent with reports that RNase L is an important
contributor to apoptosis in response to infections with a wide-range of DNA and RNA
viruses, including vaccinia virus and poliovirus (Castelli, Hassel et al. 1997; Diaz-
Guerra, Rivas et al. 1997). Activation of JNK is a common cellular response to viral infections, including influenza virus, vesicular stomatitis virus, herpes simplex virus
type I, and EMCV (see Refs.(Castelli, Hassel et al. 1997; McLean and Bachenheimer
1999; Ludwig, Ehrhardt et al. 2001)and Fig. 2.1). JNK can potentially function in
innate immune responses to viral infections through two distinct pathways, one
transcriptional and the other through pre-existing apoptotic signaling factors. For instance, vesicular stomatitis virus induction of IFN- and IFN- promoters was
severely deficient in Jnk2-/- cells (Chu, Ostertag et al. 1999). In addition, influenza A
virus induction of the IFN-β promoter was dependent on JNK (Ludwig, Ehrhardt et
al. 2001). On the other hand, sustained JNK activation leads to the mitochondria-
mediated death pathway, involving Bcl2 family members Bak and Bax (Lei, Nimnual
49 et al. 2002). Interestingly, JNK inhibition is known to increase viral yields compared
with control-infected cells (Ludwig, Ehrhardt et al. 2001).
Viruses are believed to activate JNK, at least in part, though the viral pattern
recognition molecule, dsRNA (Chu, Ostertag et al. 1999; Ludwig, Ehrhardt et al.
2001). dsRNA is necessary for production of 2-5A oligoadenylates from ATP by the
IFN-inducible 2-5A synthetases. 2-5A binds to RNase L inducing formation of active
dimers with potent endoribonuclease activity (Dong and Silverman 1995; Dong and
Silverman 1999). A previous report demonstrated the requirement of RNase L for
efficient JNK activation in response to dsRNA (Iordanov, Pribnow et al. 1997;
Iordanov, Wong et al. 2000). However, dsRNA has profound effects on signaling
pathways, other than the 2-5A/RNase L system, involving PKR, toll-like receptor 3,
ADAR, and transcription factor IRF3 (Liu, George et al. 1997; Alexopoulou, Holt et
al. 2001; Williams 2001; Peters, Smith et al. 2002). In addition, we have found that it
is possible to activate JNK with dsRNA even in cells lacking RNase L, although at
lower levels than in wild type cells (data not shown). Therefore, it was important to
show that RNase L could affect JNK activation by transfecting cells with 2-5A, the
highly specific activator of RNase L. 2-5A activation of RNase L did indeed cause
potent JNK activation in widely divergent cell types, mouse fibroblasts (MEF), and a
human ovarian carcinoma cell line (Hey 1B). JNK activation required the presence of
the full-length, functional RNase L. In addition, 2-5A-mediated apoptosis was
deficient in HeLa cells expressing an inactive missense mutant (R667A) form of
RNase L. Results indicate a dose-dependent inhibition of protein synthesis inhibition
up to the highest concentration of 2-5A tested (8 µM) (Figure 2.2E). The 2-5A
50 concentrations used in this study are within the physiological range, for instance
vaccinia virus-infected cells produce in the range of 5 µM 2-5A (Rice, Roberts et al.
1984).
2.4.2 JNK Participation in RNase L-mediated Apoptosis
Three separate methods were used to demonstrate an essential role for JNK in the
apoptotic signaling pathway induced by RNase L activation. Studies using a chemical
inhibitor of JNK, siRNA ablation of JNK, and JNK-null cells all demonstrated that
loss of JNK function causes resistance to RNase L-mediated apoptosis. SP600125, a
competitive inhibitor of ATP binding to JNK and some other kinases (Bennett, Sasaki
et al. 2001; Bain, McLauchlan et al. 2003), was able to greatly reduce apoptosis
mediated by 2-5A activation of RNase L. In addition, siRNA ablation of JNK1 and
JNK2 reduced apoptosis to undetectable levels at 9 h post-2-5A treatment. Apoptosis
did occur after 24 h even in the presence of JNK1/JNK2 siRNA, possibly because of
nonspecific induction of IFN (Sledz, Holko et al. 2003). The most dramatic results were obtained by comparing wild type and Jnk1-/- Jnk2-/- MEFs. Apoptosis by the combination of IFN and 2-5A, to induce and activate RNase L, respectively, was >7-
fold higher in the wild type cells than in the JNK-null cells. On the other hand, inhibitors of alternative MAP kinases, p38 and ERK, did not affect apoptosis in response to RNase L activation. These findings show that RNase L activation results
in a cellular stress response requiring JNK (but not p38 or ERK) for efficient
induction of apoptosis.
51 2.4.3 The RNase L Apoptotic Signaling Pathway
The viral-induced pathway mediated through the 2-5A/RNase L system is beginning
to emerge from the present studies and prior published findings (Figure 2.11). dsRNA
produced during viral infections leads to 2-5A synthesis and RNase L activation.
Although the proximal RNA substrate(s) of RNase L that trigger JNK activation are
unknown, rRNAs in intact ribosomes are candidates. Several different stimuli that
target the peptidyl transferase ring or the adjacent S/R loop in 28 S rRNA require
actively translating ribosomes to activate JNK (Iordanov, Pribnow et al. 1997). UVB,
UVC, protein synthesis inhibitors blasticidin S and anisomycin, and enzymes that
cleave ( -sarcin) and depurinate (ricin A chain) 28 S rRNA require actively
translating ribosomes to activate JNK, termed the "ribotoxic stress response"(Iordanov, Pribnow et al. 1997) . In comparison, RNase L cleaves 28 S
rRNA in the L1 protuberance implicated in formation of the exit or E site of the
ribosome, 324-326 bases 3' to the -sarcin and ricin cleavage/modification sites.
Cleavage of 28 S rRNA at the RNase L susceptible site could possibly interfere with
release of deacylated tRNA (Iordanov, Paranjape et al. 2000). However, it is unlikely
that JNK activation occurs solely as a result of protein synthesis inhibition, because several translation inhibitors, including emetine and pactamycin, completely inhibit
protein synthesis without activating JNKs (Iordanov, Pribnow et al. 1997). In addition,
pre-treatments of cells with cycloheximide or emetine did not prevent JNK activation
by 2-5A suggesting that the RNase L pathway to apoptosis occurs by a distinct
mechanism compared with the ribotoxic stress response (Figure 2.6 and data not
shown). Although rRNA cleavage is a candidate for the proximal signal generated by
52 RNase L, it is possible that other RNA substrates are important in the apoptotic
signaling pathway. For instance, the amphibian cytotoxic ribonuclease, onconase,
which degrades primarily tRNA, also induces JNK-dependent apoptosis of primary
fibroblasts (Iordanov, Ryabinina et al. 2000). Therefore, different ribonucleases
activate JNK after degrading different RNA substrates. Cleavage of RNA by RNase L is followed by JNK activation by an as yet undetermined mechanism. However,
previously SEK1/MKK4 was shown to be upstream of the dsRNA-induced JNK
activation pathway that is deficient in cells lacking RNase L (Iordanov, Paranjape et
al. 2000). JNK phosphorylation gradually increased upon 2-5A treatment and was
sustained until apoptosis occurred. Therefore, RNA damage caused by RNase L could
lead to JNK activation and apoptosis. It remains possible, however, that other events,
in addition to RNA damage, participate in JNK activation and/or apoptosis. For
instance, 2-5A could possibly affect interactions between RNase L and other proteins.
It is likely that the RNase L apoptotic pathway involves mitochondria. Previously,
cytochrome c release from mitochondria was observed during RNase L activation
whereas a caspase 3 inhibitor or Bcl2 overexpression suppressed the RNase L
pathway to apoptosis (Diaz-Guerra, Rivas et al. 1997; Diaz-Guerra, Rivas et al. 1997;
Zhou, Paranjape et al. 1998) (Figure 2.11). RNase L has also been implicated in the
turnover of mitochondrial mRNAs during IFN- treatment (Le Roy, Bisbal et al.
2001). Furthermore, sustained activation of JNK leads to the mitochondrial pathway
of apoptosis in which Bid, Bax, and Bak (and other Bcl2 family members) have been
implicated (Ito, Deng et al. 1997; Maundrell, Antonsson et al. 1997; Yamamoto,
Ichijo et al. 1999; Tournier, Hess et al. 2000; Putcha, Moulder et al. 2001; Whitfield,
53 Neame et al. 2001; Lei, Nimnual et al. 2002) Our present findings suggest that JNK is
an essential factor in the apoptotic signaling pathway mediated by RNase L. These results could have implications for both the antiviral and antitumor properties of
RNase L.
54
Figure 2.1 Apoptosis and JNK activation are deficient in virus-infected RNase L-
/- cells. A, diagrams of wild type and mutant forms of human RNase L. B, EMCV
55 induction of apoptosis in MEFs determined by FACS TUNEL assays. Cells were
infected with EMCV at an m.o.i. of 2 and analyzed for apoptosis at 8 h post-infection.
The percent TUNEL-positive cells with standard deviations are shown. Black
columns, no virus; white columns, EMCV-infected cells. MEF cell lines are wild type
+/+ -/- -/- (RNase L ), RNase L , and RNase L stably transfected with pINDRNase L or pINDRNase L EN (as indicated). B, Western blots probed with antibodies to phosphorylated JNK, PARP, and -actin were performed at different times post-
infection with EMCV (m.o.i. = 1). pINDRNase L cells and pINDRNase L EN cells were
pre-treated for 16 h with 5 µM ponasterone prior to infection (Xiang et al J. virol.
2003).
56
57
Figure 2.2. Activation of RNase L with 2-5A induces phosphorylation of c-Jun and
JNK but not p38 and inhibits protein synthesis. A, cells were transfected with natural
2-5A (10 µM) for 4 h with or without prior (16 h) treatments with ponasterone (5 µM) as indicated. B-D, Hey1b cells were transfected with psA2'ps5'A, an inactive 2-5A analog or ps(A2'ps)3A, a potent activator of RNase L for the indicated times (B) or and 3.5 h (C and D). In B oligoadenylates were used at a concentration of 4 µM. D,
2 lane 14, UV254 nm treatment (40 J/m ) was for 1 min followed by a 15-min recovery.
Results of Western blots probed with antibodies are shown. E, effect of 2-5A transfections on protein synthesis rates (see "Materials and Methods").
58
A PCDNA3 RNase L RNase LR667A
Time, h 0 9 24 0 9 24 0 9 24 Lane 1 2 3 4 5 6 7 8 9 Intact PARP Cleaved PARP
B PCDNA3 RNase L RNase LR667A
RNase L
Figure 2. 3. Apoptosis requires functional RNase L. HeLa M cells were transfected with pcDNA3neo vector control (lanes 1-3), pcDNA3/RNase L (lanes 4-6), or
pcDNA3/RNase LR667A (lanes 7-9) for 24 h. Cells were then transfected for 16 h with
4 µM natural 2-5A. Western blots probed with antibody to PARP and RNase L are shown. PARP cleavage determined by densitometry is shown as a percentage of the total level of PARP.
59
A IM9 IM9/pcDNA3-Bcl-2
spA2’spA5’A + + + + + + spA(2’spA5’A)3 + + + + + + Time, h 0 12 24 0 12 24 0 12 24 0 12 24 Lane 1 2 3 4 5 6 7 8 9 10 11 12 Intact PARP Cleaved PARP
B
30
25 psA2'ps5'A
psA(2'ps5'A)3 20
15 Cell Death (%) 10
5
0 IM 9 IM9/Bcl-2 Figure 2.4 Bcl-2 overexpression blocks apoptosis in response to 2-5A activation of
RNase L . Multiple myeloma cell line IM9 or stably expressing Bcl-2(IM9-pcDNA3-
Bcl-2) were transfected with 4µM spA2’spA5’A or spA(2’spA5’A)3 at indicated time(A) or overnight (B). Apoptosis were determined by PARP cleavae(A) or FACS
TUNEL aasay.
60
A
Dimer 2-5A
Time, h : 0 1 2 4 6 0 0.5 1 2 4 6 lane: 1 2 3 4 5 6 7 8 9 10 11 p-c-Jun
β-actin
B Dimer 2-5A
Time, h : 0 1 2 4 6 0 0.5 1 2 4 6
Lane: 1 2 3 4 5 6 7 8 9 10 11
28S rRNA
rRNA 18S rRNA Cleavage Products
61 C
4 Fold induction of c-Jun phospharylation Percentage of Percentage 100
3 c-Jun Phosphorylation 80 rRNA cleavage rRNA cleavage 60 2
40
1 rRNA Cleavage,( % ) 20
0 0 1 2 3 4 5 6
Time, h
Figure 2.5 Kinetics of c-Jun phosphorylation and rRNA cleavage in Hey1b cells transfected with dimer 2-5A, pppA2'p5'A, or natural 2-5A, both at 10 µM for the time periods indicated. c-Jun phosphorylation was determined in Western blots (A), and rRNA cleavages in 1.0 µg of total RNA (B) were measured by RNA chips and analyzed with an Agilent Bioanalyzer 2100 (Agilent Technologies). C, quantitation of the results shown in A and B.
62
2-5A: - + - + Cycloheximide: - - + + Lane: 1 2 3 4 p-c-Jun
β-actin
Figure 2.6 2-5A induces c-Jun phosphorylation in Hey1b cells pretreated with cycloheximide. Hey1b cells were incubated with or without cycloheximide (10 µg per ml) for 30 min prior to mock transfection or transfection with 5 µM 2-5A for 3 h (as indicated). P-c-Jun and β-actin levels were determined in a Western blot.
63 A RNase L-/- RNase L +/+
dsRNA(10µg/ml), time,h 0 0.5 1 2 4 6 0 0.5 1 2 4 6 Lane 1 2 3 4 5 6 7 8 9 10 11 12 p-P55-JNK p-P46-JNK
QuickTime™ and a Planar RGB decompressor β-Actin are needed to see this picture.
B
RNase L +/+ RNase L-/- dsRNA(10µg/ml)+ CHX(2 µg/ml) time, h 0 0.5 1 2 4 6 8 0 0.5 1 2 4 6 8 Lane 1 2 3 4 5 6 7 8 9 1011 12 13 14 p-P55-JNK p-P46-JNK β-Actin
PIND RNase L dsRNA(10µg/ml)+ CHX(2 µg/ml) Time, h 0 0.5 1 2 4 6 8 Lane 1 2 3 4 5 6 7 p-P55-JNK p-P46-JNK RNase L β-Actin
Figure 2.7 RNase L is involved in dsRNA mediated JNK activation. ( A) RNase L+/+,
RNase L-/- cells were transfected with 10µg/ml dsRNA (B). RNase L+/+, RNase L-/-
-/- cells, and RNase L stably transfected with pINDRNase L were transfected with
10µg/ml dsRNA plus 2 µg/ml Cycloheximide . At indicated times, the cells were
harvested and cell lysates representing equal number of cells were subjected to
immunoblot analyses with antibodies specific for the phosphorylated forms of JNK
and antibody for β-actin.
64
A
psA(2’sp5’A)3 (4 µM) - + - + + +
SP600125(µM) - - 25 1 10 25 P-c-Jun 3.5h actin
Intact PARP
Cleaved PARP 12h
actin
B
0.8
Cell Viability + 2-5A 0.6
0.4 - 2-5A
0.2
0 10 20 30 SP600125, µM
65 C
1800 Relative Fluoresence Relative RNase L Activity, 1600 1400 1200 1000 800 600
400 200 0 1 2 3
Sp600125 2-5A 2-5A (40nM)+ (25µM) (40nM) SP600125 (25µM) D
DMSO SB202190 PD98059 SB203580
2-5A - + + - + - + - + Lane 1 2 3 4 5 6 7 8 9 Intact PARP
Cleaved PARP
PD98059(25µM) - + + SB202190(2.5 µM) - - - + TNFα(10ng/ml) - + + SB203580(10 µM) - - + - p-ERK1/2 TNFα - + + + ERK1/2 p-P38 P38
66 E
+SP600125(25µM)
2-5A,Time, h 0 0.5 1 2 4 7 10 0.5 1 2 4 7 10 Lane 1 2 3 4 5 6 7 8 9 10 11 12 13 Intact PARP
Cleaved PARP
p-c-Jun
Figure 2.8. The JNK inhibitor, SP600125, suppresses c-Jun phosphorylation and apoptosis in response to 2-5A activation of RNase L. A, c-Jun phosphorylation,
PARP cleavage, and MTS assays (B) were performed on Hey1b cells treated the JNK inhibitor, SP600125, prior to 2-5A treatments. Pre-treatments with SP600125 were at the indicated concentrations (A) or at 25 µM (B,E) for 1 h prior to transfections with ps(A2'ps)3A. In A the times of treatment are shown to the right of the Figure. In B the
concentration of ps(A2'ps)3A was 4 µM. C, SP600125 does not inhibit RNase L.
ps5'A(2'psA)3 (50 nM) and SP600125 (25 µM) were incubated with purified RNase L.
Cleavage of a dual labeled RNA substrate by RNase L was determined by a FRET
assay (see "Materials and Methods"). D, inhibition of p38 and ERK did not affect 2-
5A-mediated apoptosis. Hey1b cells were treated with ERK inhibitor, 25 µM
PD98059, or p38 inhibitors (2.5 µM SB202190 or 10 µM SB203580) for 1 h prior to
mock transfections or transfections with 4 µM ps(A2'ps)3A for 1 h. E. Hey 1B cells
were incubated with JNK inhibitor 25µM for 1 h before transfection with 2-5A. At
indicated times, the cells were harvested and cell lysates representing equal number
67 of cells were subjected to immunoblot analyses with antibodies specific for PARP, cleaved PARP, and phospho-c-Jun.
68
-SiRNA +SiRNA
Time, h: 0 1 2 4 6 9 1 2 4 6 9
Lane: 1 2 3 4 5 6 7 8 9 10 11
Intact PARP
Cleaved PARP
P-c-Jun
JNK 55 kD JNK 46 kD
Figure 2.9 Suppression of c-Jun phosphorylation and apoptosis by transfecting cells with siRNA against JNK1 and JNK2. Hey1b cells were transfected with 100 nM siRNA to JNK1/JNK2 mRNA for 24 h, before transfection of ps(A2'ps)3A at 4 µM for 16 h.
69 A Jnk1+/+ Jnk2+/+ Jnk1-/- Jnk2-/-
Time, h: 0 1 2 4 6 0 1 2 4 6 Lane: 1 2 3 4 5 6 7 8 9 10 P-c-Jun c-Jun
B Jnk1+/+ Jnk2+/+ Jnk1-/- Jnk2-/-
Time, h: 0 1 2 4 6 9 24 0 1 2 4 6 9 24 Lane: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Intact PARP Cleaved PARP
C
45
40
35
30 )
25
20 Cell Death(% Cell 15
10
5
0 1 IFNα+ Ctr IFNα 2-5A 2-5A Ctr IFNα 2-5A IFNα+ 2-5A Jnk1+/+ Jnk2+/+ Jnk1-/- Jnk2-/-
70 D Jnk1+/+ Jnk2+/+ Jnk1-/- Jnk2-/-
IFNα: - + - + Lane: 1 2 3 4 RNase L
Figure 2.10. Apoptosis in response to IFN and 2-5A is inhibited in Jnk1-/- Jnk2-/-
MEF cells. A and B, Jnk1+/+ Jnk2+/+ and Jnk1-/- Jnk2-/- MEFs were transfected with 10
µM natural 2-5A for the indicated times. A, in vitro c-Jun phosphorylation assay was
performed in extracts of untreated and 2-5A transfected cells. B, PARP cleavage was
determined in Western blots assays. Levels of PARP cleavage are shown as a
percentage. C, cells were pre-treated with or without 1,000 units per ml IFN (BBDB) for 16 h prior to transfections with 10 µM natural 2-5A. Apoptosis was determined by
FACS TUNEL assay at 24 h post-treatment with 2-5A. Results shown are an average of three separate treatments of cells with standard deviations. D, IFN- treatment
(1,000 units per ml for 16 h) induced RNase L levels as determined by covalent cross- linking of a 32P-2-5A analog to RNase L (see "Materials and Methods") (Nolan-
Sorden, Lesiak et al. 1990). An autoradiogram of a dried gel is shown.
71
Figure 2.11. Apoptotic signaling pathway in virus-infected cells mediated by RNase L and JNK.
72 CHAPTER 3. RNASE L IS A NEGATIVE REGULATOR
OF CELL MIGRATION
ABSTRACT
RNase L, an endoribonuclease with nine ankyrin repeats at the N –terminal half and a kinase-like and ribonuclease domain at the C-terminal half, is activated by
5’phosphorylated, 2’5’-linked oligoadnelylates (2-5A) that functions in the antiviral
activities of IFN. Recent genetic studies have mapped the hereditary prostate cancer 1
(HPC1) allele to the RNase L gene, which indicates that RNase L may also functions
as a tumor suppressor gene(Carpten, Nupponen et al. 2002). Here we reported that in
RNase L-/- MEF cells, cell hapotactic migration toward fibronectin, laminin, and
vitronectin increased about two fold, compared with their wildtype counterpart cells.
Consistently, overexpression of RNase L in NIH3T3 cells reduced cell migration
about 2-3 fold. Fibronectin induced activation of FAK, JNK, and c-jun
phosphorylation was increased in RNase L-/- MEF cells. The JNK inhibitor,
SP600125, reduced migration of RNase L-/- MEF cells more dramatically than that of
wildtype counterparts, indicating that JNK activation is involved in RNase L induced
suppression of cell migration. Activation of RNase L by 2-5A also reduced motility
of the prostate cancer cell line, Du145. 2-5A -inhibited cell motility was independent
of protein synthesis inhibition. RNase L is associated with the cytoplasmic domain of
73 integrin β1 and with FAK. Together, these results suggest that RNase L has a potential role in the regulation of metastasis by inhibition of cell migration.
Contributions by other people:
Malathi Krishnamurthy constructed the DU 145-RNase Li and Du 145-RNase L –
MSM cell line.
74
3.1 Introduction
RNase L, is a regulated endoribonuclease, implicated in the IFN antiviral response. In
the absence of its activator, 2-5A (5’-phosphorylated, 2’5’-oliogoadenylate), the N-
terminal half of RNase L represses the ribonuclease domain of the c-terminal half
(Dong and Silverman 1995). The N terminal half of RNase L consists of nine ankyrin repeats, which is involved in mediating protein-protein interactions. There is also a protein kinase-like domain in the C-terminal of RNase L, although some conserved
kinase residues are absent. The kinase–like and ribonuclease regions of RNase L are
structurally homologous of the Ire1 kinase/ribonucleases that functions in the
unfolded protein response in organisms from yeast to human(Urano, Wang et al.
2000; Dong, Niwa et al. 2001).
Recent genetic studies have mapped the hereditary prostate cancer 1 (HPC1) allele to
the RNase L gene, which indicates that RNase L may also functions as a tumor
suppressor gene (Carpten, Nupponen et al. 2002; Silverman 2003). A number of
mutations of RNase L in different populations of prostate cancer cases were found.
Most of the mutations were aligned in the N-terminal ankyrin repeat domain and kinase-like domain. An interesting observation is that although ribonuclease
enzymatic activity is the only known function of RNase L, no mutations in the
ribonuclease domain of RNase L were found in prostate cancer patients (Silverman
2003).
75
Multiple lines of evidence indicate that anti-apoptotic or pro-apoptotic proteins always showed coordinate modulation of cell apoptosis and migration. For example, over- expression of Bcl-2 in glioma cells enhanced cell survival and promoted cell migration(Wick, Wagner et al. 1998). Furthermore, activation of pro-apoptotic protein kinase G induced cell apoptosis and inhibited cell migration (Deguchi,
Thompson et al. 2004). Overexpression of RNase L in NIH 3T3 cells induced changes in cell morphology; Morphology of RNase L-/- cell line is also different from wildtype (data not published), RNase L was found to interact with the actin cytoskeleton (Tnani, Aliau et al. 1998). All of these results indicated that RNase L might be involved in cytoskeleton rearrangements.
Cell movement is essential in many physiological and pathological processes including inflammation, wound healing, tumor growth, metastasis, and angiogenesis.
The integrin family of cell surface receptors are important mediators of cell migration.
Integrins are composed of non-covalently linked α and β subunits, each of which is a transmembrane glycoprotein with a single membrane-spanning segment and generally a short cytoplasmic domain(Damsky and Ilic 2002; DeMali, Wennerberg et al. 2003;
Humphries, McEwan et al. 2003). There are currently 15 known integrin α and 8 β subunits, assembling more than 20 different integrin receptors. In general, the combination of particular α and β subunits determines the ligand specificity of the integrin complex. In ligand binding, integrins aggregate into transmembrane complexes known as focal adhesion complexes, which are enriched in specific
76 cytoskeletal proteins including talin, vinculin, α-actinin,and actin(Burridge and Fath
1989). In addition to linking extracellular matrix (ECM) proteins with intracellular
structures, such as components of the cytoskeleton, integrins also transduce signals
into cells. One common cellular response to integrin clustering is activating a variety
of tyrosine kinases, including Ab1, Syk, FAK and Src-family kinases (Burridge, Fath
et al. 1988; Guan 1997; Guan 1997; Parsons and Parsons 1997; Schlaepfer and
Hunter 1998; Schlaepfer, Hauck et al. 1999; Sieg, Hauck et al. 1999). Integrin
activation results in FAK autophosphorylation at amino acid residue Y397, which
serves as a binding site for Src kinase. The Src-FAK complex leads to further
phosphorylation of FAK (Burridge, Fath et al. 1988; Guan 1997; Guan 1997; Hanks
and Polte 1997; Parsons and Parsons 1997; Schlaepfer and Hunter 1998; Schlaepfer,
Hauck et al. 1999; Sieg, Hauck et al. 1999). FAK is able to phosphorylate and
activate a number of cytoskeleton –linked proteins, which further transduce integrin
–generated signals such as the MAP kinases cascade(Schaller 2001; Schaller and
Schaefer 2001). However, the mechanism for regulation of FAK activation is still
unknown.
Among the many pathways activated by integrin and FAK, JNKs have recently
emerged as a crucial regulator of cell migration and the morphogenetic movement of
epithelial sheets(reviewed in(Xia and Karin 2004). Migration of JNK1-/-JNK2-/- embryonic fibroblasts is dramatically impaired(Javelaud, Laboureau et al. 2003). JNK has also been shown to be required for Drosophila dorsal closure (Riesgo-Escovar,
Jenni et al. 1996; Barr and Bogoyevitch 2001). Fibroblasts deficient of MEKK1, an upstream activator of JNK, are impaired in serum-factor stimulated JNK activation
77 and cell migration. However, MEKK1-/- mice are relatively normal, except a defect in eye closure (Yujiri, Ware et al. 2000). JNK was reported to exert its function in regulating migration by phosphorylation of paxillin, a focal adhesion adapter protein.
Rat bladder tumour epithelial cells, NB-II, expressing the Ser 178 Ala mutant of
paxillin (PaxS178A), which abolished the JNK phosphorylating site, showed
significantly reduced motility in single-cell-migration and wound healing assays
(Huang, Rajfur et al. 2003).
Although a number of genetic and epigenetic changes have been documented in the progression of prostate cancer(PCa), the cellular mechanism involved in the progression of localized PCa to metastasis remained largely unknown. One possible explanation is that a combination of complex genetic and epigenetic events changes cell adhesive properties allowing transformed cells to migrate, gaining access to the circulation and forming metastatic colonies. It is likely that some of these change occur at focal adhesion plaques. The linkage of RNase L to HPC1 and the interaction of RNase L with cytoskeleton (Tnani, Aliau et al. 1998) suggested that RNase L might directly, or indirectly suppresses one of many steps in prostate cancer metastasis. Therefore, we investigated whether RNase L was involved in integrin- induced signaling pathways. Here we found that in RNase L-/- MEF cells, cell
haptotactic migration increased by roughly two fold. Overexpression of RNase L in
NIH3T3 cells dramatically reduced cell migration by 3-4 fold. RNase L was able to
inhibit integrin-mediated cell migration and was physically associated with the
integrin β1 cytoplasmic domain and with FAK. RNase L negatively regulated FAK
78 activation. Activation of RNase L by 2-5A further reduced cell motility. 2-5A suppression of cell motility was independent of protein synthesis inhibition. 2-5A transfection weakened RNase L and integrin associations and promoted integrin movement to plasma membrane after fibronection or laminin exposure. These results suggested that RNase L has a potential role in the regulation of prostate cancer metastasis by regulating integrin-mediated cell migration.
79
3.2 Materials and methods
3.2.1 Cell culture and treatment
NIH 3T3 and Hela M cells were cultured in Dulbecco's modifed Eagle's medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100
U/ml penicillin and streptomycin. NIH3T3-neo and NIH 3T3 stably expressing
human RNase L cDNA (NIH3T3-RNase L) were cultured in regular DMEM plus
400ng/ml G418. Human prostate carcinoma cell line, DU145 containing the vector
pSuper (Krishnamurthy M et al, 2004 ) encoding small interfering RNA (sequence:
5’-CGA AGA TGT TGA CCT GGT C-3', 109-nucleotide from the mRNA start site)
for knockdown of RNase L (Du145-RNase Li) or, a 3-bp mismatch control (5'-CGA
AcA TGa TGA CgT GGT C-3'), where the lower case letters indicate mismatch at
position 5, 9 and 14) (Du145-RNase L-MSM), were maintained in regular RPMI
medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100
U/ml penicillin, streptomycin and 2µg/ml puromysin.
3.2.2 Cell Adhesion assay
96 well plates were coated with fibronectin (FN), laminin (LN), vitronectin (VN), or
Collagen-1 (CN-1) (Chemicon) (10 µg/µl) at 37 °C for 2 h. The wells were then
blocked by 1% BSA. Serum starved cells (1 × 104) were added into each well in 100
µl serum-free medium, and incubated at 37 °C for 2 h. The unbounded cells were
80 washed off with PBS, and the bound cells were detached by trypsin and counted in a hemocytometer (Sengupta, Xiao et al. 2003).
3.2.3 Migration assays
Cells were starved in 0.1% FBS medium overnight. Migration assays were performed using 24-well Transwell chambers (Costar). The underside of the membrane was coated with 10 µl of 10 µg/ml FN, LN or VN in PBS. Starved cells were detached with cell dissociation buffer (Invitrogen), and washed twice with 1X PBS medium.
Cells (1 × 105) were added to the upper chamber and allowed to migrate for 4 h at
37°C, the non-migratory cells on the upper membrane surface were removed with a cotton tip, and the migratory cells attached to the lower membrane surface were fixed by methanol, and stained with hematoxylin. Migrated cell number were determined by counting stained cells by microscopy (Sengupta, Xiao et al. 2003).
The JNK inhibitor Sp600125, function-activating integrin, functional blocking integrin β1 or integrin β3 antibody(Chemicon), were added into cells after detached from plates and incubate for 1h before added into the chamber.
Each determination represents the average of three individual wells, and error bars represent the standard deviation.
81 3.2.4 In vitro kinase assays
FAK was immunoprecipitated from cytoplasmic extracts of prostate cancer epithelial
cells, Du145 and the immunocomplexes were washed extensively in kinase assay
buffer (20 mM HEPES pH 7.4, 10 mM MnCl2, 5mM glycerophosphate, and 0.05%
Triton X-100). Purified RNase L (Dong, Xu et al. 1994) was incubated with FAK immunocomplexes in 20 µl kinase reaction buffer on ice for 30 min, and then incubated in kinase assay buffer for 10 min at 30°C with the addition of 5 mM ATP.
The reactions were stopped by addition of SDS-PAGE sample buffer, then subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore).
Protein phosphorylation analysis was determined by the anti-tyrosine
phosphorylation antibody P-tyr-100 (Cell Signaling Co.).
3.2.5 Detection of protein-protein interactions
Expression of GST fusion proteins encoding full length human RNase L and a
ribonuclease-dead point mutant, R667A, C-terminal truncated mutant RNase L1-335 , and N-terminal truncated mutant RNase L∆N385 were already described (Dong, Xu et
al. 1994). Briefly, after inducing the protein expression (1mM IPTG for 3 h, 30 °C) cells were pelleted, washed, and resuspended in PBS-C buffer (PBS with 10% glycerol, 1 mM EDTA, 0.1 mM ATP, 5 mM MgCl2, 14 mM 2-mercaptoethanol, 1
µg/ml leupeptin, and 1 mM PMSF) (Dong, Xu et al. 1994), and lysed with a French
Press. The supernatants were collected after centrifugation at 16000×g for 20 min at 4
°C. The cleared supernatants were incubated with glutathione–Sepharose 4B beads
82 (1-2 h at 4 °C). Beads were washed 4 times with PBSC and incubated with cleared
Du145 cell lysate overnight at 4 °C. Beads were then washed with cell lysis buffer
[20 mM tris pH 7.35, 1% triton x-100, 150 mM NaCl,1 mM EDTA, 1 mM EGTA 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate , 1 mM Na3VO4, 1 mM
PMSF and 1 µM leupeptin ] three times, once with PBS-C buffer, and then proteins was eluted with 10 mM glutathione for 20min at room temperature. The eluted proteins were subjected to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane. Membranes were incubated with different primary antibodies according to the manufacturer’s protocol.
3.2.6 Immunoprecipitations and immunoblot analysis
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in a cell lysis buffer. Clarified cell extracts were immunoprecipitated with either mouse monoclonal anti-FAK (Chemicon), or mouse monoclonal anti-integrin β1
(Chemicon), or anti-RNase L (Dong, et al 1994) antibodies overnight at 4°C. The immunoprecipitates were washed 4 times with cell lysis buffer and subjected to SDS-
PAGE and immunoblotting. Membranes were incubated with either anti-FAK, anti- integrin β1 (Santa Cruz, polyclonal), or anti-RNase L antibody.
3.2.7 Immunofluorescence Microscopy
83 Du145 cells were transfected with or without 10 µM 2-5A mix for 2 h, and then trypsinized and plated into 10 µg/ml fibronectin or collagen-1-coated chamber slide
(Falcon). After incubation for 1 h at 37°C, cells were washed twice with PBS and fixed for 30 min at room temperature in 4% paraformaldehyde solution. The cells
were then washed with PBS. Cells were permeabilized with 0.2% Triton X-100 and
washed again. Cells were then blocked with PBS containing 0.02% Tween 20, 3%
BSA (Bovine serum album) and 3% goat serum for 1 h at room temperature. Cells were then incubated for 60 min with anti-RNase L antibody or anti-integrin β1
antibody, washed three times for 10 min, and incubated for 60 min with Alexa Fluor®
488 goat anti-mouse IgG1, or Alexa Fluor® 568 donkey anti-rabbit IgG
(H+L)(Molecule Probes). Cells were mounted with Vectorshield with DAPI.
Immunofluorescence was analyzed using a Leica DMRBE microscope equipped with
a ×63 objective lens and filters for epifluorescence. Images were captured using a
CoolSNAP digital camera (RP Photometrics, Tucson, AZ) and analyzed by Meta
Imaging SeriesTM software (version 5.0) from Universal Imaging Corp.
84 3.3 Results.
3.3.1. RNase L inhibits cell migration but not adhesion
Overexpression of RNase L in NIH 3T3 cells induces cell morphology changes. In addition, morphology of RNase L-/- MEF cell line is also different from wildtype
MEF cells, indicating that RNase L might be involved in cytoskeleton remodeling
(unpublished data). The above observation suggested that there was a possibility that
RNase L had an unidentified function involved in cell adhesion or migration. We
tested the haptotaxis of RNase L+/+ and RNase L-/- MEFs on different extracellular matrix (ECM) proteins including Laminin (LN), Vitronectin (VN), Fibronectin (FN), and Collagen-1 (CN-1)(Figure 3.1a). In the absence of ECM, both RNase L+/+ and
RNase L-/- MEFs are unable to migrate. In contrast, in the presence of the ECMs,
migration of RNase L-/- MEFs was increased roughly about 2-fold, compared to
RNase L+/+ MEFs. The effect of RNase L on migration was not specific, since RNase
L-/- motility toward each of the different ECMs increased to a similar extent. To
confirm the findings, motility of NIH 3T3 cell line stably over-expressing RNase L
(NIH3T3-RNase L) and the control cell line, expressing a parental empty
vector(NIH3T3-neo), were studied. FN and LN –induced cell migration was 2-3 fold
less in NIH 3T3-RNase L cells, compared to NIH3T3-neo (Figure 3.1b). The
expression level of human RNase L in NIH3T3-RNase L cells and NIH3T3-neo were
determined by immunoblotting (Figure 3.1c). Similar results were observed in the
human prostate cancer cell line, Du145. Du145 cells that contain a pSuper vector
85 encoding siRNA against RNase L mRNA (Du145-RNase Li) or, a 3-bp mismatch
control (Du145-RNase L-MSM) (Krishnamurthy M et al 2004). The level of RNase L
in Du145-RNase Li cells was roughly half of that in the parental DU145 cells or
mismatch control cell line Du145-RNase L-MSM (Figure 3.1e). The corresponding
haptotaxis increased by 2-fold in Du145- RNase Li cells, compared to parental Du145
cells or to the mismatch control cell line Du145-RNase L-MSM. (Figure 3.1d).
The effect of RNase L on cell adhesion was also examined. Overexpression of RNase
L in NIH3T3 cells, or down-regulation of RNase L in prostate cancer cell line DU145
did not change cell adhesion (Figure 3.2).
3.3.2 Integrin β1 mediated cell migration is inhibited by RNase L
To further characterize the RNase L inhibitory effect on cell migration, we preincubated Du145-RNase Li, its parental cell line, Du145, and mismatch control,
Du145-RNase L-MSM with either a function blocking β1 integrin or β3 integrin antibody. The results showed that β1 integrin antibody, but not the β3 integrin
antibody effectively blocked the cell migration in both three cell lines (Figure 3.3a).
Increased cell migration due to down-regulation of RNase L was also abolished by the function blocking β1 integrin antibody.
By contrast, a function stimulatory β1 integrin antibody partially reversed the inhibitory effect of RNase L on cell migration in NIH3T3 cells (Figure 3.3b). These
86 results showed that the inhibitory effect of RNase L was mediated by preventing, at least partially, integrin β1 mediated cell migration.
3.3.3 RNase L inhibited adhesion-dependent FAK activation.
FAK is an important component of the integrin-mediated signal transduction pathway and plays a key role in modulating cell morphology and migration. FAK is activated through tyrosine phosphorylation, which occurs after cells adhere to ECM proteins.
To determine whether FAK is involved in RNase L-induced inhibition of cell migration, NIH3T3 –neo or NIH3T3-RNase L cells were plated on plates precoated with 10 µg/ml FN for 1 h. Phosphorylation of FAK was detected by phosphotyrosine- specific antibody, P-Y-100 (Cell Signaling Co.). As shown in Figure 3.4a, over- expression of RNase L in NIH3T3 cells reduced FAK phosphorylation about 3 fold.
In contrast, FN induced similar ERK1/2 activation in both NIH3T3-RNase L and
NIH3T3-neo cells. Similar results were obtained in RNase L+/+ and RNase L-/- MEF cells (Figure 3.4b). FAK tyrosine phosphorylation was reduced about 3-fold in
RNase L +/+ MEFs, compared to that in RNase L-/- MEF cells. These data suggested
that RNase L induced inhibition of cell migration via FAK signal cascade.
3.3.4 Adhesion dependence of JNK activation and C-Jun phosphorylation is increased in RNase L-/- MEF cells
87 The C-Jun amino-terminal kinase (JNK) is generally thought to be involved in
inflammation, proliferation and apoptosis(Ip and Davis 1998; Rivas, Gil et al. 1998).
However, JNK activation is also involved in regulation of dorsal closure(Glise,
Bourbon et al. 1995; Sluss, Han et al. 1996). JNK1-/- JNK2-/- fibroblasts exhibit a
significantly reduced ability to close mechanically-induced cell layer wounds than
their wildtype counterparts. Wound healing in human dermal fibroblasts is
dramatically impaired by the specific JNK inhibitor, SP600125 (Javelaud, Laboureau
et al. 2003). Activation of JNK was reported to phosphorylate paxillin, a focal
adhesion adaptor. A ser178→Ala mutation in paxillin, which abolished the JNK
phosphorylation, impaired rapid cell migration (Huang, Rajfur et al. 2003). To
investigate whether JNK is involved in RNase L-induced inhibition of cell migration,
RNase L +/+ and RNase L-/- MEF cells were plated onto 10 µg/ml fibronectin
precoated plates at the indicated times (Figure 3.5a). JNK activation and c-Jun
phosphorylation were then examined. In RNase L knockout MEFs, JNK activation
and c-Jun phosphorylation were significantly enhanced, compared to their wildtype counterparts. The difference was not due to a defect in the JNK activation pathway, since LPS, interleukin 1 (IL1), and TNFα induced similar levels of JNK activation in both wildtype and knockout cells.
3.3.5 The JNK inhibitor SP600125 suppresses cell migration more significantly in RNase L-/- than in RNase L+/+ MEF cells.
88 Signal transduction pathway inhibitors were tested for activity in blocking enhanced
haptotactic activity by down-regulation of RNase L including the ERK inhibitor,
PD98059, PKC inhibitor, GF109203X, AKT kinase inhibitor, AKTi, P38 kinase
inhibitor SB203580, and JNK inhibitor SP600125. All of the inhibitors except the
JNK inhibitor had no effect on increased haptotactic migration of RNase L-/- cells.
(data not shown). However, treatment of RNase L-/- cells with the JNK inhibitor,
SP600125 for 30 min before the transwell migration assay, reduced cell haptotactic
ability in a dose-dependent manner. At a concentration of 25µM, SP600125 reduced
RNase L-/- cells migration to about 36% of the control level (Figure 3.6). SP600125 also showed inhibitory effects on RNase L+/+ cells, however, at the concentration of
25µM, SP600125 only suppressed RNase L+/+ the cell migration to 60% of the control
level. The inhibitory effect was not due to cell toxicity (>98% cell viability as
determined by trypan blue dye exclusion, data not shown). It is also consistent with
previous result that fibronectin induced more JNK activation in RNase L-/- cells. The
result implied that RNase L mediated inhibition of migration was due to suppression
of JNK activation.
3.3.6 RNase L is able to interact with integrin β1 and FAK
The functional stimulatory β1 integrin antibody partially restored cell locomotive
ability, impling that the inhibitory effect of RNase L may, at least in part, regulate
integrin activity. Tyrosine phosphorylation of FAK is elevated in the absence of
RNase L. These observations suggested that RNase L negatively regulated integrin
89 and FAK signaling. These results prompted us to examine the possibility that RNase
L might interact with β1 integrin and FAK. Integrin β1 and FAK protein were
immunoprecipitated from the DU145 cell lysates with anti-integrin β1 and anti-FAK
antibody, and then the FAK and integrin β1 immunoprecipitations were subjected to
immunoblotting with RNase L antibody, and vice versa. The results showed that the
endogenous integrin β1 and FAK interacted with RNase L (Figure 3.7a,b).
To map the domain of RNase L interacting with FAK, flag-tagged full-length human
RNase L, C-terminal truncated mutant RNase L1-335 , and N-terminal truncated mutant
RNase L386-741 were transfected into Hela M cells. The cell lysates were subjected to
immunoprecipitation with antibody to FAK, followed by immunoblotting with
antibody to flag. The results showed that full-length RNase L, the C-terminal
truncated mutant RNase L1-335, containing only the nine ankyrin repeats domain,
interacted with FAK. The N- terminal truncated mutant RNase L386-741, which contains the ribonuclease domain, was not able to interact with FAK (Figure 3.7c)
To further confirm the interactions of RNase L with FAK and integrin β1. GST fusion proteins encoding full length human RNase L, N-terminal half of RNase L mutant, RNase L1-335 , and C-terminal half mutant, RNase L386-741 were purified from
E. coli, and were incubated with Du145 cell lysates. Consistent with above
observations, the N-terminal half and the full-length RNase L bound to FAK and
integrin β1. In contrast, the GST itself, and the C-terminal half GST-RNase L386-742
were not able to pull down FAK and integrin β1(Figure 3.7d)
90
GST-integrin β1-CD, which encodes the cytoplasmic domain of integrin β1a [gift
from Dr. Jun Qin], purified from E. coli, was incubated with the Du145 cell lysate.
The results showed that RNase L was pulled down by GST-integrin β1-CD, but not
GST alone (Figure 3.7e). These results showed that RNase L interacted with FAK
and integrin β1.
3.3.7 RNase L can be phosphorylated by FAK in vitro.
Because RNase L interacted with FAK, we examined whether RNase L could inhibit
FAK kinase activity. Du145 cells were serum starved overnight, and cell lysates
prepared. The FAK protein was immunoprecipitated with antibody against FAK.
Purified RNase L was added to FAK protein in FAK kinase buffer and incubated at
30°C for 10 min. In vitro kinase assays showed that RNase L was unable to inhibit
FAK autophosphorylation. However, FAK phosphorylated RNase L in a dose
dependent manner. (Figure 3.8)
3.3.8 Activation of RNase L by 2-5A inhibits cell migration
RNase L was first characterized as a protein that functions in the IFN mediated
antiviral response. The only known RNase L activator is the 2’5’-linked
oligoadenylate (2-5A). To determine the effect of 2-5A on cell migration, we
transfected different concentrations of natural 2-5A mix into Du145 cells. Cell
91 migration was performed in transwell chambers. As shown in Figure 3.9a, 2-5A
inhibited cell migration in a concentration dependent manner. 10 µM 2-5A mix
reduced cell motility more than 4-fold. 10 µM natural 2-5A only caused a small fraction of cell apoptosis around 9 h in Du145 cells. Because the migration assays take about 6 h, it is unlikely that the inhibition of cell migration by 2-5A is due to apoptosis. Activation of RNase L by 2-5A also induced protein synthesis inhibition
(Figure 2.2e). However, It has been reported that hapotactic migration requires no
new protein synthesis (Aznavoorian, Stracke et al. 1990). This is consistent with our
observation that 2 µg/ml of cycloheximide had no effect on cell migration. 10 µg/ml
of cycloheximide, which is sufficient to inhibit protein synthesis and induce apoptosis, only reduced cell migration to 80 percent of the control cells. This results indicates that 2-5A induced cell migration inhibition is not dependent on protein synthesis inhibition (Figure 2.9c). The mechanism by which 2-5A induced cell
migration inhibition needs to be determined.
3.3.9 2-5A promotes integrin relocation to cell membrane.
To investigate the possible mechanism of 2-5A induced inhibition of cell migration,
immunofluorescence staining showed that 2-5A did not change the localization of
RNase L (data not shown). However, in the presence of 2-5A, fibronectin or collagen-
1 induced integrin migration to plasma membrane was increased, compared to the
mock transfection counterparts (Figure 3.10). Since integrin is an abundant protein,
how 2-5A affects the localization of integrin is unclear.
92 3.4 DISCUSSION
3.4.1 RNase L is a negative regulator of cell haptotactic migration
RNase L is a uniquely-regulated endonuclease, activated by 5’-phophorylated 2’5’- linked oligoadenylates (2-5A), that functions in the anti-viral and anti-proliferative activities of the IFNs. Recently, RNase L was suggested to function as tumor suppressor based on its mapping to the hereditary prostate cancer 1 (HPC1) allele and its role in mediating apoptosis in response to several types of external stimuli
(Silverman 2003). In this paper, we identified a novel function of RNase L as a negative regulator of cell migration. Three separate types of experiments showed that
RNase L has inhibitory effects on cell movement. First, RNase L-/- showed a two fold increased haptotactic migration toward different ECM, such as fibronectin, laminin, vitronectin and collagen-1. Second, over-expression of RNase L in NIH 3T3 cells reduced cell movement about two to three fold. Third, knock-down of RNase L by siRNA increased cell migration up to two fold. In contrast, overexpression of RNase
L in NIH3T3 cells or knockdown of RNase L by siRNA in Du145 did not affect cell adhesion (Figure3.2). RNase L , as an inhibitor of cell migration, was consistent with previous observation that RNase L is a tumor suppressor. It is interesting that RNase
L-/- mice are viable. The reason might be that although RNase L is a ubiquitously expressed protein, it remains at a very low level, if any, during embryonic development. RNase L is abundant in the thymus (Zhou, Paranjape et al. 1997).
Accordingly, the RNase-/- L mice showed remarkably enlarged thymuses. A
93 deficiency of RNase L in the thymus possibly enhanced thymocyte migratory ability and defect in apoptosis. However, the spleens of RNase L-/- mice were normal in size, indicating RNase L effects in migration and apoptosis might be cell type dependent.
3.4.2 JNK is involved in RNase L mediated inhibition of cell migration
JNK activation plays essential roles in organogenesis during mouse development, by regulating cell proliferation, survival or apoptosis and in immune responses by regulating cytokine gene expression. Recently, JNK was shown to be involved in the regulation of cell migration and cytoskeletal integrity (Nishina, Nakagawa et al. 2003;
Xia and Karin 2004). In several systems, cell migration required basal JNK activity
(Javelaud, Laboureau et al. 2003). Consistent with this, fibronectin-induced JNK activation is significantly enhanced in RNase L deficient mouse embryo fibroblast cells. Furthermore, JNK inhibitor, SP600125, significantly suppresses RNase L-/- cell migration, while other inhibitors, such as the ERK inhibitor PD98059, PKC inhibitor
GF109203X, AKT kinase inhibitor AKTi, and P38 kinase inhibitor SB203580, were not able to inhibit the RNase L-/- cell migration. This is the first time that JNK activation by ECM has been shown to be inhibited by RNase L. JNK is a common downstream target of multiple signaling pathways controlling cell movement. JNK has been shown to exert its influence on cell movement by phosphorylating paxillin and interacting with microtubules (Nagata, Puls et al. 1998; Huang, Rajfur et al.
2003). FAK, Rac, cdc 42, Src kinase, and MEKK1, which are crucial for cell movement, also activate JNK (Minden, Lin et al. 1995; Christerson, Vanderbilt et al.
94 1999; Oktay, Wary et al. 1999; Ridley, Allen et al. 1999). The question of how RNase
L inhibits JNK activation is still not clear. We hypothesis that RNase L interact with
one of the JNK upstream component, e.g. FAK, and prevents downstream signaling
(Figure 3.11).
It is interesting that RNase L is both an inhibitor and an activator of JNK depending
on the stimulus. When activated by 2-5A, RNase L dimerizes and is able to induce
ribosomal RNA cleavage, thus inducing JNK activation. Activation of JNK is also
required for RNase L induced cell apoptosis. This is not contradictory with an RNase
L function as an inhibitor of integrin-induced JNK activation. First, RNase L activity
induced cleavage of ribosomal RNA, which in turn induced JNK activation might be
through a different pathway from integrin-induced JNK activation. Second, 2-5A
induced RNase L dimerization also might release the RNase L inhibiting effect on
JNK activation.
3.4.3 RNase L interacted with integrin β1 and FAK
Recently, there has been rapid progress in elucidating the integrin-FAK downstream
signaling pathways. But how FAK is activated and regulated by integrin is still not
clear. Our studies indicate that RNase L may have a previously unidentified role as a
negative regulator of FAK activation. First, we found that integrin induced FAK
activation is more potent in RNase L-/- cells than wildtype counterpart cells. Second,
overexpression of RNase L in NIH3T3 cells significantly reduced FAK activation by
95 integrin. We further found that RNase L is able to interact with integrin β1 and FAK.
This interaction was identified by co-immunoprecipitations and was subsequently confirmed by pull-down assays. Interaction of RNase L with FAK and integrin β1
was mediated by the N-terminal domain of RNase L. The N-terminal domain of
RNase L consists of a nine ankyrin repeat domain. Several ankyrin repeat containing
proteins such as Shark (SH2-domain, Ankyrin Repeats tyrosine Kinase) (Ferrante,
Reinke et al. 1995), ILK ( Integrin linked Ser/Thr kinase) (Persad, Attwell et al.
2001), and GGAPs (GTP-binding and GTPase-activating proteins) (Xia, Ma et al.
2003), are involved in cell adhesion, migration cytoskeletal rearrangement, and
morphogenesis. It will be interesting to know if overexpression of the ankyrin repeat
of RNase L can inhibit the enzymatic activities of Shark, ILK, or GGAP.
Interaction of RNase L with FAK and integrin β1 seems to be independent of cell adhesion, since fibronectin induction did not alter the interaction of RNase L with
integrin and FAK. It will be interesting to know whether interaction of RNase L with
FAK was dependent on the major autophosphorylation site, Y397 of FAK. Since
interaction of RNase L with FAK is independent of cell adhesion, it is more than
likely that interaction between RNase L and FAK is independent of Y397
autophosphorylation.
Although RNase L is able to inhibit FAK activation by integrin, the in vivo
mechanism still remains unclear.To examine whether RNase L can inhibit
96 autophosphorylation of FAK, purified FAK from E. coli should be used to perform in vitro kinase assay.
Our lab also found that RNase L was associated with filamin A, a component of cell movement (data not published). Since morphology of RNase L-/- is different from wildtype counterpart cells, and overexperssion of RNase L in NIH 3T3 also changed cell morphology, RNase Lmight be involved in cytoskeleton rearrangement.
Interaction of RNase L with filamin A might also contribute to its inhibitory effect on cell migration (Figure 3.11)
3.4.4 2-5A induce inhibition of cell movement.
When binding to 2-5A, RNase L is activated and induces ribosomal RNA cleavage, thus inhibiting cell translation. 2-5A induced inhibition of migration is concentration dependent and cellular RNase L level dependent (Figure 3.9). It is interesting that 2-
5A inhibited migration is independent of protein synthesis inhibition, since cycloheximide, at 10 µg/ml, which is sufficient to fully inhibit translation, was unable to inhibit cell migration. This is consistent with previous reports that migration requires no new protein synthesis (Aznavoorian, Stracke et al. 1990). 10 µM of 2-5A mix only induced a very small fraction of apoptosis until at 6 h in DU145 cells.
Therefore, the inhibition of migration by 2-5A was not due to cell apoptosis. We hypothesized that RNase L, activated by 2-5A, cleaved the ribosomal RNA, which caused ribosomes to release a signal which in turn suppress cell migration.
97 250 Ctr LN VN FN CN-I Ctr LN VN FN CN-I
200 RNase RNase L-/- L+/+
150
cells/field 100
50
0
Ctr LN VN FN CN-I Ctr LN VN FN CN-I -/- +/+ RNase L RNase L B
600
500
400
300
cells/field 200
100
0
Ctr FN LN Ctr FN LN NIH3T3-neo NIH3T3-RNaseL
C NIH3T3-RNaseL NIH3T3-neo
Lane: 1 2 3 4
RNase L
98 D 350
300
250
200
150 Cells/Field
100
50
0 1 Ctr FN LN Ctr FN LN Ctr FN LN
Du145 Du145-RNaseL-MSM Du145-RNase Li
E Du145-RNase Li Du145 Du145-RNaseL-MSM
RNase L
Actin
Figure 3.1 RNase L inhibited cell haptotactic migration. (A), RNase L+/+, or RNase L -/- MEF cells, (B) NIH 3T3
stably expressing RNase L (NIH3T3-RNase L or empty vector (NIH3T3-neo), and (D) prostate cancer cell line
Du145 or stably expressing RNase L SiRNA (Du145-RNase Li), or stably expressing of mismatch mutant
(DU145-RNase L-MSM) were migrated toward fibronectin (FN), laminin (LN), vitronectin (VN), or Collagen-I (CN- I). Migration assays were conducted as described in Materials and Methods. (C,E) The human RNase L level of
NIH3T3-RNase L, NIH3T3-neo, Du145, Du145-RNase Li and Du145-RNase L-MSM cells were determined by
western blot analysis.
99 A
120
100
80
60
cell number(X10-3) 40
20
0
Ctr FN LN Ctr FN LN
NIH3T3-neo NIH3T3-RNaseL B
120
100
80
60
cell number(X10-3) 40
20
0
Ctr FN LN Ctr FN LN Ctr FN LN Du145 Du145-RNaseL- Du145-RNase Li MSM
4) Figure 3.2. RNase L did not affect cell adhesion on fibronectin and laminin. Cells (1X10 ) respectively, (A), NIH 3T3 stably expressing RNase L (NIH3T3-RNase L) or empty vector (NIH3T3-neo), and (B) prostate cancer
cell line Du145, or stably expressing RNase L small interference RNA (Du145-RNase Li), or stably expressing
of mismatch (DU145-RNase L-MSM) were plated into FN, or LN pre-coated 96 wells, as described in Materials
and Methods . Data were summarized from three independent experiments and standard deviation shown.
100 A
400 350
300 d
250 cells/fiel 200 150
100
50
0 FN - + + + - + + + - + + + BLK - - + - - - + - - - + - INTβ1 ---+ - - - + - - - +
Du145 Du145-RNaseL-MSM Du145-RNase Li B 1000 900 800 700 600 500 400 cells/field 300 200 100 0 1
FN + + + + STIM INTβ1 - + - + NIH3T3-neo NIH3T3-RNaseL
Figure 3.3 RNase L inhibit β1 integrin mediated haptotaxis. (A), Du145, Du145-RNase Li, and Du145 RNase L-
MSM were pretreated with function-blocking β1 integrin antibody or β3 integrin antibody for 30 min before plating into migration chambers. (B) NIH3T3-neo and NIH 3T3-RNase L were pre-treated with functional stimulatory β1 integrin antibody. Migration assays were conducted as previously described.
101
A
NIH3T3-RNase L NIH3T3-neo
FN - + - + 1 2 3 4
p-FAK
FAK
p-ERK1 p-ERK2
B
RNase L+/+ RNase L-/-
FN - + - +
1 2 3 4 p-FAK
FAK
Figure 3.4 RNase L inhibited adhesion dependent FAK tyrosine phosphorylation. (A), NIH3T3-neo and
-- NIH 3T3-RNase L, (B) RNase L and RNase L MEF cells were starved for 24 hours, and then plated to
dishes coated with 20µg/ml fibronectin. After 1 h incubation, cells were collected and immunoprecipitated
with FAK, followed by immunoblot with phosphotyrosine (p-Tyr-100) Ab.
102 A
RNase L+/+ RNase L-/- Time, h 0 1 2 0 1 2
1 2 3 4 5 6 P-c-jun
p-P54-JNK p-P45-JNK
Actin
B
RNase L-/- RNase L+/+
Ctr LPS IL1 TNFα Ctr LPS IL1 TNFα
1 2 3 4 5 6 7 8 p-P54-JNK p-P45-JNK Actin
Figure 3.5. RNase L inhibited adhesion dependent JNK activation and c-Jun phophorylation. (A),
RNase L+/+ and RNase L-/- MEF cells were starved for 24 hours, and then plated to dishes coated with
20 µg/ml fibronectin, after the indicated incubation time. Cell lysates are subjected to western blots probed with phospho c-Jun, phospho JNK1/2, and Actin antibody according to manufacturer’s protocol.
+/+ -/- (B) RNase L and RNase L MEF cells were treated with LPS (100 µg/ml) for 1 h, or IL1 (20 ng/ml) for 1 hour, or TNFα (20 ng/ml) for 15 min.
103
250
+/+ RNase L 200 RNase L-/-
150
100
50
0
SP600125( µM) - 1 10 25
Figure 3.6. The JNK inhibitor, SP600125 suppressed cell migration in RNase L deficient cells.
-/- +/+ RNase L cells and RNase L cells were starved for overnight, then cells were preincubated with the JNK inhibitor, SP600125 at the indicated concentration for 30min. Migration toward fibronectin were
described in Materials and Methods.
104
A IgG Cell extract RNase L IP IgG Cell extract FAK IP 1 2 3 4 5 6
FAK RNase L
RNase L FAK
B IgG Cell extract Integrin β1 IP RNase L IP IgG Cell extract 4 5 6 1 2 3 Integrin β1 RNase L
RNase L Integrin β1
C
flag-RNase L386-741 + flag-RNase L1-335 + flag-RNase LR667A + flag-RNase L +
vec + 1 2 3 4 5
FAK:IP * Flag:IB * FAK
105 D GST-RNase L1-335 GST-RNase L
GST GST-RNase L∆N385 1 2 3 4 FAK
Integrin β1
GST-RNase L
GST-RNase L386-741
GST-RNase L1-335
GST
E GST GST-integrin β -CD 1a RNase L
GST-integrin β1a-CD
GST
Figure 3.7. RNase L interacts with integrin β1 and FAK. (A) Du145 cell lysates were subjected to immunoprecioitation analysis with FAK , integrin β1 antibody,, or the mouse IgG, followed by immunoblot
analysis with anti-human RNase L antibody, or vice versa (B). (C), Hela M cells were transfected with flag-
tagged wildtype RNase L and mutants. Cell lysates were subjected to immunoprecipitation with FAK
antibody, followed by immunoblot with anti-Flag antibody. (D). GST, GST-RNase L1-335, GST-RNase
L∆N385 , and GST-RNase L proteins was purified as described in materials and methods. The indicated
proteins were incubated with Du145 cell lysates for overnight at 4 °C, followed by immunoblot analysis
with FAK. (E). GST-integrin β1a-CD(cytoplasmic domain) or GST, were incubated with DU145 cell lysate, followed by western blot with RNase L Ab.
106
ATP - + + +
RNase L(ng) - - 20 200
1 2 3 4 P-FAK
FAK
P-RNase L
RNase L
Figure 3.8. RNase L can be tyrosine phosphorylated by FAK in vitro. FAK was immunoprecipitated from serum- starved Du145 cells. 20 ng or 200 ng of purified RNase L was mixed with FAK in kinase reaction buffer and incubated on ice for 30min. After adding
ATP, the reaction mix were incubated at 30°C for 10min. The tyrosine phosphorylation were detected by specific phospho-tyrosine antibody, p-tyrosine-100 (cell signaling).
107 A
18 0
16 0
14 0
12 0
10 0
80
60
40
20
0
2-5A mix (µM): - 0.5 2 5 10 15 2 10 CHX(µg/ml)
300
250
200
150 cells/field
100
50
0
2-5A mix (10µM): - + - +
Du145-RNL-msm Du145-RNLi
108 C
400
350
300
250
200 Cells/field
150
100
50
0
2-5A mix (10µM): - + - +
NIH3T3-neo NIH3T3-RNase L
Figure 3. 9. Activation of RNase L by 2-5A abolishes cell haptotaxis. (A), Du145 cells, (B), Du145-
RNL-msm and Du145-RNLi, (C), NIH3T3-neo and NIH3T3-RNase L were serum-starved for
overnight, then transfected with 2-5A or Cycloheximide (CHX) at the indicated concentration for 2
hours. Migration assays were performed as previously described.
109 2-5A mix control
CN-1
FN
Figure 3.10. 2-5A transfection promoted integrins migration to plasma membrane. Du145 cells were transfected with or without 10 µM 2-5A for 2 h, and plated into slide chamber coated with 10 µg/ml
Collagen –I (CN-1) or fibronectin (FN) for 1 hour . Cells were then stained with monoclonal integrin beta1
ab. Ctr, control.
110
virus
Ds RNA
2-5A synthetase Integrin
? RNase L RNase L 2-5A
F-acin ? RNase L
FAK Rac Src
P130Cas Ribosomal RNA Crk
JNK ?
Paxillin Mitonchondria
F-actin Apoptosis
Migration
Figure 3.11. RNase L mediated inhibition of integrin induced FAK and JNK activation
111 CHAPTER 4. PMA INDUCES RNASE L
PHOSPHORYLATION AND PREVENTS 2-5A INDUCED
APOPTOSIS
4.1 INTRODUCTION
RNase L is a ubiquitously expressed latent endoribonuclease involved in mediating
the anti-viral and pro-apoptotic activities of the interferon induced 2-5A system.
Double stranded RNA is an intermediate product during viral infections which
activates 2-5A synthetase (2’-5’ oliogoadenylate synthetase OAS). Activated OAS converts ATP to PPi and a series of short 2’-5’ linked oliogoadenylates, collectively
referred as 2-5A[px(A2’p)nA, x = 1 to 3, n=2 to ≥4 ] (Figure 1.2), which in turn
activates RNase L. Upon activation, RNase L cleaves cellular rRNA /mRNA 3’ of
UpUp and UpAp sequences, leading to the inhibition of protein synthesis. It has also
been reported that in virus infected cells, viral RNA was preferentially degraded
compared to cellular RNA (Li, Blackford et al. 1998; Li, Blackford et al. 2000). In
addition, RNase L can exert an important role in mRNA stability, since two IFN
regulated gene, ISG 43 and ISG15’s mRNA half lives have been significantly
extended in RNase L-/- fibroblasts (Li, Blackford et al. 2000). Activation of RNase L
by 2-5A also induced JNK activation, which led to apoptosis by an undetermined
mechanism. Apoptosis initiated by 2-5A and IFN has been shown to be defective in
JNK1-/-JNK2-/- mouse embryonic fibroblasts (Li G, et al 2003). Apoptosis induced by
112 RNase L is mitochondria dependent (Rusch, Zhou et al. 2000), and is inhibited by overexpression of Bcl-2 (Castelli, Hassel et al. 1997).
Evidence has been accumulated that in addition to the antiviral function of RNase L, there exists other unidentified functions of RNase L. First, RNase L null mice showed enlarged thymuses at early ages compared to wildtype mice, suggesting that RNase L may be involved in T cell development. In addition, thymocytes and lymphocytes isolated from the spleen of RNase L-null mice were resistant to apoptosis induced by staurosporine (Zhou, Paranjape et al. 1997). Second, there is delayed rejection of skin allografts in RNase L null mice suggesting that RNase L is involved in cellular immunity (Silverman, Zhou et al. 2002). Third, recently, the interferon antiviral pathways and prostate cancer genetics converged on RNase L. The presence of germline mutations in RNase L within hereditary prostate cancer(HPC) families and the loss of herterozygosity in tumor tissues suggest a novel role for the regulated endoribonuclease in the pathogenesis of prostate cancer (Silverman 2003)
Although there is evidence that RNase L might have unidentified functions, there is little evidence showing how RNase L can be regulated besides binding to 2-5A. It has been reported that RNase L was co-precipitated with cytoskeleton sediments (Tnani,
Aliau et al. 1998), indicating that RNase L might interact with the actin cytoskeleton.
Treatment of cells with PMA can abolish then interactions of RNase L with the actin cytoskeleton. Recently, it was reported that PMA treatment of murine L929 cells,
113 induced RNase L degradation in a dose and time-dependent manner. Degradation of
RNase L was proteasome dependent.(Chase, Zhou et al. 2003).
PKC is a family of serine /threonine protein kinases that regulate various cellular
functions, including proliferation, differentiation and apoptosis. The 10 identified
PKC isoforms are divided into three subfamilies based on their structures and the
cofactors required for optimal activation. Phosphatidylserine(PS), a negatively
charged membrane phospholipid is required for all isoforms. The atypical (aPKC)
isotypes (ζ, λ/ι) are dependent solely on PS for activation. The conventional or
classical PKCs (cPKCs: α, βI, βII, γ ) and the novel PKCs(nPKC, δ, ε, η and θ) are optimally activated by the additional binding of diacyglycerol (DAG), a lipid that is produced from the hydrolysis of membrane inositol phospholipids by phospholipase
C. However, cPKC and nPKC isoforms differs by binding DAG in a calcium – dependent and –independent manner, respectively. An intriguing aspects of PKCs, is that different cells express different PKC isoforms, indicating different PKC isoforms have different functions.
Among the many functions of PKCs, one is regulating cytoskeleton rearrangement by
interacting with many cytoskeleton-associated proteins. RNase L is reported to co-
precipate with actin cytoskeleton (Keenan and Kelleher 1998). We hypothesize that
RNase L is also a substrate of PKC and might be involved in PKC mediated signaling
pathway. Here we report that treatment of human cells with PMA induced RNase L
phosphorylation. Human RNase L is not degraded after being phosphorylated.
114 Pretreatment with PMA suppressed 2-5A mediated apoptosis. We further found that
RNaseL is associated with PKCα.
115 4.2 MATERIALS AND METHODS
4.2.1 Protein interaction assay
Expression of GST fusion proteins encoding full length human RNase L, C-terminal truncated mutant RNase L1-335 , and N-terminal truncated mutant RNase L∆N385 were already described (Dong B et al 1994). Briefly, after inducing protein expression
(1mM IPTG for 3 h, 30 °C), cells were pelleted, washed, and resuspended in PBS-C buffer(Dong B et al 1994), and lysed with a French Press. The lysates were centrifugated at 14000×g for 15 min. The supernatants were incubated with glutathione–Sepharose 4B beads (1-2h at 4 °C). Beads were washed four times with
PBS-C buffer and incubated with prepared cleared Du145 cell lysates overnight at 4
°C. Beads were then washed with cell lysis buffer for three times, once with PBS-C buffer, and then was eluted with 10mM glutathione for 20min at room temperature.
The eluted proteins were subjected to SDS-PAGE analysis and immunoblotting.
4.2.2 RNase L immunoprecipitation
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in a cell lysis buffer [20 mM tris pH 7.35, 1% triton x-100, 150 mM NaCl,1mM EDTA,
1mM EGTA 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate , 1 mM
Na3VO4, 1 mM PMSF and 1µM leupeptin]. Clarified cell extracts were immunoprecipitated with the monoclonal anti-human RNase L antibody.
4.2.3 2D gel electrophoresis
116 RNase L immunoprecipitates were dissolved in rehydration buffer [8M Urea, 4%
CHAPS, 50mM DTT, 0.2% carrier ampholytes, 0.0002% bromophenol blue]. In the
first dimension, iso-electric focusing (IEF) was done with commercially available
preformed immobilized pH gradients (linear pH gradient 3–10, 18 cm). The gels were rehydrated overnight by placing the strips gel-side-down in sample-containing rehydration solution in an IPGphor strip holder (Biorad), applied 50 volts overnight
and covered with mineral oil. IEF was conducted using an IPGphore Isoelectric
Focusing System (Biorad). IPG strips were then subjected to a preprogrammed
procedure: linearly increasing the voltage from 0 - 250V for 0-15 h, then IEF was
performed linear increase from 250 to 6000 V for 3h, maintained at 6000V for 3-5h.
Before the second-dimension, the IPG gel strips were incubated at room temperature for 15 min in equilibration solution (50 mM Tris–HCl, pH 8.8, 6 M urea, 2% SDS,
30% glycerol) containing 1% DTT and then in equilibration solution containing 2.5% iodoacetamide for 15 min. The gels were then submitted to second dimensions, 10%
SDS-PAGE gels, without stacking gels, using the Pharmacia IsoDalt system. The IPG gel strips were embedded on top of the gels. Electrophoresis was conducted at 30 mA/gel for 5 h. The 2D polyacrylamide gels were stained with silver nitrate or subjected to western blotting.
4.2.4 Silver Staining
After gel electrophoresis, gels were stained using the Silver Stain Plus kit(Biorad).
Briefly, after washing the gel using distilled water for several times. Gels were placed in the Fixative Enhancer Solution with gentle agitation for 20 min. Gels were rinsed
117 in 400 ml deionized distilled water three times, each for 10 min with gentle agitation.
Gels were stained with Image Development Reagent for approximately 20 min or until desired staining intensity was reached. After the desired staining was reached, the gels were placed in 5% acetic acid to stop the reactions. After stopping the reactions, the gels were rinsed in high purity water for 5 min. The gels are then ready to be dried or photographed.
4.2.5 Western blot analysis
Equal amounts of proteins were fractionated on SDS-polyacrylamide gel and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline containing 0.05% Tween 20 (PBST) and probed with the indicated primary antibodies. After incubation with secondary antibodies, immunoblots were visualized with ECL detection kit (Amersham
Biosciences). For reprobing the membrane with another antibody, the membrane was stripped in 2% SDS, 0.1% 2-mercaptoethanol and 50 mM Tris-Cl, pH 6.8 and incubated at 50°C for 30 min.
118 4.3 RESULTS AND DISCUSSION:
4.3.1 PMA stimulate RNase L phosphorylation
PMA has been shown to downregulate mouse RNase L level in murine L929 cells
(Chase BI et al, 2003). We attempted to extend this observation to the human ovarian
carcinoma cell line, Hey 1B. Interestingly, PMA treatment did not cause human
RNase L degradation in Hey 1B cells(Figure 4.1a). We further investigated whether
PMA treatment could induce phosphorylation of RNase L. Hey 1B cells were treated
with PMA (10ng/ml) for 30 min. RNase L was then immunoprecipitated and
subjected to 2-D gel electrophoresis. As seen in Figure 4.1b, PMA stimulated more
than 80% of RNase L to migrate towards a lower pH. In contrast, the PKC inhibitor ,
H7, fully inhibited PMA-stimulated migration of RNase L. Here we found that
although human RNase L is phosphorylated after PMA stimulation, human RNase L
is resistant to proteasome dependent degradation , which occurred with mouse RNase
L, indicating human RNase L might have a different function from mouse RNase L in
PMA stimulated signaling pathways.
4.3.2 PMA blocked 2-5A induced apoptosis.
To further investigate whether PMA can affect RNase L function, we performed 2-5A mediated RNase L assays in intact cells by measuring ribosomal RNA degradation.
After PMA treatment, ribosomal RNA damage was slightly attenuated (data not
119 shown). RNase L is able to induce apoptosis after activation by 2-5A (Figure 4.2a lane 7). However, after PMA treatment for 15min, 2-5A was unable to induce apoptosis in Hey 1B cells (Figure 4.2a, lane 4). Pretreating Hey 1B cells with PKC
inhibitor H7, or GF109203X for 1 h before the PMA treatment, suppressed the effect
of PMA on RNase L-induced apoptosis. (Figure 4.2a, lane 10, 12). The PMA induced
inhibition of RNase L-induced apoptosis was confirmed by TUNEL assay. 2-5A
induced 26% TUNEL positive cells, while after PMA treament, 2-5A only induced
about 8% cell apoptosis. The inhibitory effect of PMA was likely due to PKC,
because the PKC inhibitor H7, abolished the PMA effect (Figure 4.2b). These
findings showed that PKC is involved in a cell survival pathway, either by inducing
cell survival gene expression or by phosphorylation of RNase L, preventing its pro- apoptotic activity. By RNA protection assay PMA treatment induced significantly up-
regulated levels of TRAF2 (data not shown), which is an anti-apoptotic protein.
However, the effect of TRAF2 on RNase L induced apoptosis was not investigated.
4.3.3 RNase L interacted with PKCα.
PKC inhibitors effectively blocked PMA induced phosphorylation of RNase L and
inhibition of RNase L-induced apoptosis. Besides, recently, PKCα was shown to
interact with the actin cytoskeleton, FAK, and integrin β1, which are also associated
with RNase L (see chapter 3). We next investigated whether RNase L is able to
interact with PKCα. GST fusion proteins encoding full length human RNase L, a N-
terminal RNase L fragment, RNase L1-335, and a C-terminal fragment, RNase L386-741
120 were purified from E. coli, and incubated with Hey 1b cell lysates with or without
prior PMA treatment of cells. GST-RNase L, and the N-terminal half of GST-RNase
L1-335 were able to pull down PKCα (Figure 4.3 lane 3,4,7, and 8). In contrast, the C- terminal half of RNase L, RNase L386-741, was not able to interact with PKCα (Figure
4.3 lane 5,6). PMA treatment seems did not change RNase L interaction with PKCα
in vitro. These results suggested that PKCα might be able to interact with and
phosphorylate RNase L in vivo. However, further experiments will be needed to
confirm interaction between PKCα and RNase L. Interaction between RNase L and
PKCα might be important in regulating PKC and/or RNase L mediated signaling
pathways.
121 A
PMA(10ng/ml) time, h: 0 0.25 0.5 1 2 1 2 3 4 5
RNase L
B
PH: 8------5
Ctr
PMa
H7+PM
Figure 4.1 PMA stimulated RNase L phosphorylation. (A), Hey 1B cells were treated with PMA
(10 ng/ml) for the indicated time. RNase L levels were determined by western blots. (B). Hey 1B cells were treated with or without PMA.PKC inhibitor H7 was added into medium 1 h before PMA treatment. RNase L was precipitated with RNase L monoclonal antibody , and subjected to 2-D gel electrophoresis, followed by western blotting with RNase L antibody as probe.
122 A PMA(10ng/ml) + + + 2-5A(10µM) time, h 0 2 6 16 2 6 16 2 6 16 616 H7(10ng/ml) + + + GF109203 X + + 1 2 3 4 5 6 7 8 9 10 11 12 Intact PARP
Cleaved PARP B
35
) 30
25 % D C eath ell ( 20
15
10
5
0 123456
2-5A(10µM) - + - + - + H7(10ng/ml) - - - - + + PMA(10ng/ml) - - + + - +
Figure 4.2 PMA treatment suppress 2-5A induced apoptosis. Hey1b cells were pretreated with PMA for 15 minute. PKC inhibitor H7 were added into medium 1 hour before PMA pretreatment. 2-5Awas transfected into cells for 3 hours, and cellls replaced with fresh medium. Apoptosis was determined (a) by PARP cleavage (b)by
FACS TUNEL assay at 24 hours post-treatment.
123 PMA(10ng/ml) - + - + - + - + 1 2 3 4 5 6 7 8
PKCα
GST-RNase L
GST-RNase L386-741
GST-RNase L1-335
GST
Figure 4.3 RNase L interacted with PKCα. GST (lane 1,2 ), GST-RNase L1-335 (lane
3,4), GST-RNase L386-741 (lane 5,6), and GST-RNase L (lane 7,8) proteins were purified
as described in Materials and Methods. The indicated proteins were incubated with
Hey 1B cell lysates for overnight at 4 °C, followed by immunoblot analysis with PKCα.
124 CHAPTER 5. SUMMARY AND FUTURE DIRECTIONS
5.1 SUMMARY:
RNase L is an endoribonuclease that functions in the molecular pathways of interferon (IFN) action against viral infections. Although the pro-apoptotic activity of
RNase L is well known, how RNase L activation during viral infection leads to apoptosis is not clear. Recently, RNase L was considered to be a tumor suppressor based on its pro-apoptotic activity and mapping to hereditary prostate cancer allele 1
(HPC1). While involvement of a viral pathogen in prostate cancer was proposed
(Silverman 2003), an unknown mechanism of RNase L in the suppression of prostate cancer progression still remains possible. This thesis is intended to explore the possible molecular mechanism by which RNase L is involved in anti-viral and anti- tumor activities. In chapter 2, my thesis systematically investigated the RNase L- induced apoptotic signaling transduction pathway during viral infection. In chapter 3, my thesis identified a novel function of RNase L in the regulation of cell migration.
In chapter 4, my thesis demonstrated that RNase L can be phosphorylated by PMA stimulation and interacts with PKCα. All these findings are summarized as follows:
1. 2-5A induced JNK activation is correlated with ribosomal RNA damage in a time dependent manner.
2. Activation of RNase L by 2-5A can induce JNK activation and apoptosis.
3. Inhibition of caspase activation prevented apoptosis in response to 2-5A stimulation of RNase L activity.
125 4. Overexpression of Bcl-2 blocked apoptosis in response to 2-5A, suggesting involvement of mitochondria
5 Virus (EMCV) or dsRNA induction of c-jun NH2-terminal kinase (JNK) and apoptosis are deficient in cells lacking RNase L
6. Pretreatment of cells with a protein synthesis inhibitor potentiated 2-5A induced c-
Jun phosphorylation.
7. Inhibition of JNK activation by JNK inhibitor SP600125, or ablation of
JNK1/JNK2 suppressed RNase L induced apoptosis, indicating that the
JNK/mitochondria pathway is involved in apoptosis induced by RNase L.
8. The above findings suggest that the 2-5A/RNase L system eliminates virus-infected cells through a ribotoxic stress pathway involving JNK.
9. PMA treatment of cells stimulated human RNase L phosphorylation, but did not induce human RNase L degradation. The PKC inhibitor, H7 suppressed PMA induced RNase L phosphorylation.
10. Pretreatment of cells with PMA prevented RNase L induced apoptosis.
11. RNase L interacted with PKCα. The interaction domain was mapped to the N- terminal ankyrin repeats.
12. RNase L induced inhibition of migration induced by fibronectin, laminin, and vitronectin.
13. RNase L inhibits fibronectin induced FAK and JNK activation, but not ERK activation.
14. The JNK inhibitor SP600125, inhibited motility of RNase L-/- embryonic fibroblasts more significantly than wildtype counterpart. In contrast, the ERK
126 inhibitor PD98059, PKC inhibitor GF109203X, AKT kinase inhibitor AKTi, and P38
kinase inhibitor SB203580, were unable to inhibit RNase L-/- cell motility.
15. RNase L induced inhibition of migration is integrin β1 dependent.
16. 2-5A induced inhibition of cell migration is dependent on RNase L.
17. 2-5A induced inhibition of cell migration is independent of protein synthesis inhibition.
18. RNase L is associated with FAK, and integrinβ1. The N-terminal domain of
RNase L is able to interact with FAK and integrin β1.
19. RNase L did not inhibit FAK phosphorylation in vitro, however, RNase L was
tyrosine–phosphorylated by FAK in. vitro.
20. 2-5A induced relocalization of integrin β1 into the plasma membrane.
5.2 FUTURE DIRECTIONS.
5.2.1 Investigate the mechanism of 2-5A induced JNK activation.
The ribonuclease and kinase-like domain were necessary for 2-5A induction of JNK as there was no JNK phosphorylation in ponasterone-treated RNase L-/- cells
containing pINDRNase L EN (Figure. 2.2A, lane 12). There was about a 2-fold more
-/- +/+ JNK activation in RNase L cells containing pINDRNase L compared to RNase L
cells. However, transfection of 2-5A in both cell lines caused a similar level of rRNA
damage (Xiang Y et al, 2002 ), indicating that rRNA damage is not responsible for
the difference in JNK activation by 2-5A in RNase L+/+ cells and RNase L-/- cells with
pINDRNase L. We concluded that the protein-protein interactions mediated by RNase L
127 were also involved in JNK activation. This is further confirmed by recent work in our lab, which showed that camptothecin and TRAIL mediated activation of JNK required RNase L and ribonuclease activity was not required ( Krishnamurthy M et al
2004 ).
Interestingly, RNase L inhibited integrin β1-mediated JNK activation. Inhibition of
JNK by RNase L was independent of its RNase activity, since fibronectin did not induce rRNA cleavage. How JNK can be regulated by RNase L will be a key question for us to understand the function of RNase L. Recently, our lab found that RNase L interacted with filamin A, a component of the actin cytoskeleton. Filamin A is a scaffold protein in the JNK signaling cascade, and is a key protein involved in cell migration. Interaction of filamin with RNase L might be important for RNase L regulation of JNK signaling pathway. In order to clarify that filamin is important for
RNase L to regulate JNK pathway, three experiments are proposed:
1. Knock down of filamin by siRNA, and examining its effect on RNase L induced
JNK activation by 2-5A.
2. Double knockdown of RNase L and filamin by siRNA and examining filamin effect on RNase L inhibition of cell migration.
3. Construction of dominant negative RNase L mutant which is unable to interact with filamin A and examine its effect on 2-5A induced JNK activation.
5.2.2 Investigate involvement of RNase L in PKC signaling pathway.
128 PMA treatment induced mouse RNase L degradation in a PKC and ubiquitin-
dependent pathway (Chase BI et al 2004). We found that human RNase L wasn’t
degraded after PMA treatment. RNase L is phosphorylated by PMA treatment . We also found that RNase L interacted with PKCα by pulldown assay. How RNase L is involved in PKC mediated pathways needs to be determined.
5.2. 2.1 To determine if PKCα is the only protein responsible for phosphorylating
RNase L in response to PMA
We already obtained the prostate cancer cell line C4-2, which stably expresses a small
interfering RNA against PKCα (C4-2/PKCαi). PKCα expression level was only
about 10-20% of the mutant PKCα control cell line (C4-2/PKCα-MM). To determine
if PKCα is responsible for phosphorylation of RNase L, C4-2/PKCαi and C4-
2/PKCα-MM cell line were treated it with PMA for 30 min, RNase L will be then immunoprecipitated, subjected to 2-D gel electrophoresis (as described in Materials and Methods in chapter 4), then followed by immunoblotting with RNase L antibody.
If RNase L can only be phosphorylated by PKCα, there will be little reduction in
RNase L migrating to a higher pH zone.
5.2.2.2 Investigate the effect of PMA treatment on RNase L interactions with the cytoskeleton.
It was reported that RNase L was co-precipitated with cytoskeleton sediments, and when cells were pretreated with PMA, there was no more RNase L in the cytoskeleton sediments (Tnani, Aliau et al. 1998). Recently, our lab found that RNase
129 L was able to interact with filamin A, a component of the cytoskeleton. Interestingly,
PKCα was also found to interact with filamin A. Stimulated PKCα by PMA is able to
phosphorylate filamin A (Tigges, Koch et al. 2003). I hypothesize that PMA
stimulation disrupted the interaction between RNase L and filamin A. PKCα may be involved in the disruption by phosphorylation of both RNase L and filamin. This hypothesis can be tested in the Hey 1B and C4-2/PKCαi cell line.
5.2.2.3 To determine how the PKC signaling pathway is affected by RNase L
PMA stimulation activates many signal transduction pathways, including MAPK,
NF-κB, and AKT. RNase L is a newly identified substrate of PKC. However, how
RNase L affects the PKC signal pathway is not clear. Du145-RNase Li and Du145-
RNase L-MM cell lines will be used to examine how RNase L regulates the pathway activated by PKC.
PKC contributes to many cytokine-stimulated chemotactic pathways. RNase L is a negative regulator of cell migration. We hypothesize that PKC exerts its stimulatory function on cell migration partly by phosphorylating RNase L. Du145-RNase Li and
Du145-RNase L-MM cell lines will be treated with different cytokines, and subjected to the transwell chamber migration assay (chapter 3, Materials and Methods).
Meanwhile, whether phosphorylation of RNase L by PKC changes its interactions with filamin A, FAK, and integrin β1 will be examined.
5.2.2.4 PMA induced gene expression in RNase L+/+ and RNase L-/- fibroblasts.
130 PMA stimulates many genes to be transcribed by induction of MAPK and NF-κB. To
investigate the molecular mechanisms underlying the effects of RNase L on PMA
induced gene expression, RNase L+/+ and RNase L-/- fibroblasts will be subjected to
PMA treatment in a time-dependent manner. Total RNA will be extracted from the
samples. Total RNA from each sample will then be used to generate double stranded
cDNA through reverse transcription, which will be then transcribed into
complimentary RNA (cRNA) in the presence of biotin labeled dUTP and dCTP,
according to Affymetrix protocols. The labeled RNA will be hybridized to DNA
microarrays. Confirmation of the gene expression patterns in oligonucleotide array analysis will be performed by real time-PCR. Understanding the PMA induced differential gene expression in RNase L+/+ and RNase L-/- cells may reveal new signaling pathways which is regulated by RNase L. Furthermore, stimulation of PKC by phorbol ester promotes tumor growth and metastasis through transcription activation. Gene expression profile data may provide potential targets for anti-cancer
therapy.
5.2.3 Investigate the role of RNase L in cell migration
5.2.3.1 Expression of DN-JNK in RNase L-/- cells and examine its effect on
migration.
In the absence of RNase L, fibronectin-induced JNK activation is significantly enhanced. Inhibition of JNK activation by SP600125 dramatically inhibited fibronectin-induced migration of RNase L-/- fibroblasts. To further confirm that JNK
131 is involved in fibronectin-induced migration of RNase L-/- cells, we will stably
express dominant negative JNK (Thr183Ala, Tyr185Phe)(Gupta, Campbell et al.
1995) in RNase L-/- cells and subsequently, subject them to migration assays.
-/- 5.2.3.2 Expression of RNase L and RNase L∆EN in RNase L fibroblasts, and then examine the effect on cell migration.
Interactions of RNase L with FAK and integrin depend on its N-terminal domain. The
N-terminal domain’s effect on cell migration has not been determined yet. To examine if the N-terminal domain is sufficient to inhibit cell migration, the full length human RNase L and RNase domain-truncated muntant RNase L∆EN will be stably
transfected in RNase L-/- cells and then subjected to migration assay.
5.2.3.3 Investigate the effect of RNase L on FAK autophosphorylation
Fibronectin induced FAK activation is two-fold greater in RNase L-/-cells than in
RNase L+/+ cells. Over-expression of RNase L in NIH3T3 cells reduced FAK activation about two-fold, compared to control cell lines (Figure 3.3). These results
indicated that RNase L inhibits FAK activation, yet the mechanism is still unclear. To investigate if RNase L can directly inhibit FAK autophosphorylation, FAK purified
from E. coli will be used to perform the in vitro kinase assay in the presence of
different concentrations of RNase L.
.
5.2.3.4 Examine the effect of RNase L in inhibition of tumor metastasis in nude mice.
132 Prostate cancer is the most common noncutaneous male cancer and one of the least
understood malignant diseases. Identifying key genetic factors involved in the
metastasis of prostate cancer cells is critical. RNase L was initially proposed to be a
candidate tumor suppressor based on its pro-apoptotic activity and identified as the candidate for HPC1. My data suggests that RNase L is a strong candidate as a prostate cancer metastasis suppressor.
To better understand the role of RNase L in prostate cancer progress, prostate cancer cell line DU145, or PC3, in which RNase L level were knocked down by small interfering RNA against RNase L should be generated. The parental cells, or RNase L knockdown cells will be orthotopically injected into dorsal-lateral lobes of prostates as described previously (Stephenson, Dinney et al. 1992). Five to eight mice will be included in each treatment group. The mice wil be then monitored for tumor growth and distant metastases in lymph nodes by palpation. The necropsy will be performed
5 weeks after the implantation, and their prostate glands as well as other organs will be examined thoroughly for primary tumors and metastases. The prostate glands will be dissected out and weighed, and metastases will be confirmed by histology.
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