MODULATION OF CELLULAR PSEUDOMONAS A SENSITIVITY

THROUGH INDUCED CHANGES IN RECEPTOR EXPRESSION

A Tbesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

JAMES EDWARD LAITHINAITE

In partial fuüülment of requirements

for the degree of

Doctor of Phüosophy

December, ZOO1

O James Edward Laithwaite, ZOO1 uisitions and AOQu-et raphic Services services bibliraphques

The author has granted a non- L'auteur a accordé une licence non exclusive licence ailowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distnibute or sel reproduire, prêter, distribuer ou copies of this thesis in microfim, vendre des copies de cette these sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur fonnat électronique.

The author retains ownershtp of the L'auteur conserve la propriété du copyright in this thesis. Neikthe droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des exûaits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. MODULATION OF CELLULAR PSEUDOMONAS EXOTOXIN A SENSITIVITY THROUGH INDUCED CHANGES IN TOmRECEPTOR EXPRESSION

James Edward Laithwaite Advisor: University of Guelph. ZOO1 Dr. Jonathan LaMarre

Psezidomonas Exotoxin A (PEA) is an intracellular-acting bacterial exotoxin produced by the opportmistic pathogen Pseudornonas aeruginosa. PEA intoxication is a multi-step process in which PEA exploits the cellular machinery of target cells in an effort to reach the cytoplasm in an activated state capable of inhibiting protein synthesis.

We hypothesized that induced alterations in the expression levels of specific componcnts of the cellular machinery exploited by PEA during intoxication represent mechanisms by which cellular sensitivity to PEA can be altered by the host. To test this hypothesis. we

first investigated whether aiterations in cellular sensitivity to PEA cm be induced by diverse cellular events. Using a [3~]leucineincorporation assay to measure inhibition of protein synthesis. we demonstrated that primary rat hepatocytes cultured on a collagen type 1 matrix show markedly decreased sensitivity to PEA in a Ume-dependent fashion.

PEA sensitivity is also markedly decreased in the rat macrophage-like ce11 linr HS-P in a

dose- and time-dependent manner after LPS treatment. We ais0 demonstrated that

normal and transformed murine liver cells exhibit divergent PEA sensitivities. with

transformed cells demonstrating greater PEA sensitivity than their non-transformed

counterparts. PEA intoxication begins when PEA binds the low-density lipoprotein receptor-related protein (LW). PEA toxicity was decreased by a LRP antagonia, the receptor-associated protein, confuming the importance of the LRP in PEA intoxication in these three ce11 types. Induced alterations in cellular PEA sensitivity were positively correlated with changes in funchonal ce11 surface LRP expression, as measured by a?- macroglobulin internalization studies. and LWmRNA levels, as deterrnined by Nonhern blot analysis. The importance of LRP expression in mediating PEA sensitivity was also addressed with conjugate PEA that do not utilize the LRP for cellular entry. We conclude that induced alterations in the expression levels of the LRP are an important mechanism by which cellular PEA sensitivity is aitered by the host. These studies indicate that it is probable that the production of various LRP regulatory factors may be initiated in response to P. aeruginosa, consequently altering cellular and tissue PEA sensitivity during infection. ACKNOWLEDGEMENTS

Jonathan LaMme. my supervisor, for the opportunity to join his lab and for granting me the freedom to explore a wide range of interests. From the onset of my program, he has encouraged me to develop a capacity for understanding the "big picture". and for this 1 am grateful. Gordon Kirby and Rod Memll for serving as members of my advisory comminee and for their guidance and encouragement throughout the course of this study. Everyone associated with the LaMarre lab. both past and present. who are

responsible for creating such a rewarding research environment. 1 am especially indebted

to Sally Bem. who can never be thanked enough for training me and many others who

ventured into Our lab. The Department of Biomedical Sciences and the University of

Guelph for making this study possible. Bill Harris for the oppominity of being a

teaching assistant in Mammalian Physiology under his patient supervision. Allm King

for his sense of humor. friendship and embracing the challenge of being my intenm

supervisor during Jonathan's sabbatical leave. Carlton Gyles for enhancing my

understanding of cellular microbiology by answering countiess questions and serving on

my comprehensive and examination cornminees. Jyogi Yamate for his generosity and

fnendship. Pauline and Jemy fiom the Co-op coffee shop who nourished me not only

physically with food, but also emotionally with their cheerfùlness and joking. The

administrative and technical staff for their help, advice and encouragement. In particular

1 wish to express my gratitude to Carol Ann Higgins and Kim Ben for their assistance

and fnendship. The members of THE TMNEC TANK: Jim Petrik*. Jim Gilmore*.

Rakpong Petkarn*. Jason Raine*. Spencer Greenwood, Dean Bens, Andrew- Drake and Jim Greenaway (*Founding Members), who collectively entertained me every day with thought provoking discussions and humor. Speciai thanks to Jim Petrik for his invaluable

friendship. Fellow graduate students: Ali Ashkar, Harpreet Kochhar, Claire Plumb, Ali

Farmand. Lindsay Roach, Hesem Dehghani and Amanda Starr, who provided support

and encouragement. Financial suppon fiom the Ontario Graduate Scholarship program.

the University of Guelph. the Beredaro and Ingram families, the department of

Biomedical Sciences, the Canadian Cynic Fibrosis Foundation, and the Ontario

Veterinary College-Graduate Student Association is gratefully acknowledged. My

fmily for always being there for me with theu love, encouragement and support. 1 am

forever indebted to my wife Suzan for her love, understanding, and patience and for the

sacrifices she has endured to allow me to chase my dreams - I love you. 1 would like to

dedicate this thesis to my mother and father. Pauline and Edward. 1 will be forever

graeful for their love and kindness; words cannot express what they have done for me. DECLARATION OF WORK PERFORMED

1 declare that with the exception of the items below, al1 work reported in this thesis was performed by me.

The isolation of primary hepatocytes from rat livers was fiequently carried out in collaboration with Dr. Sally Benn and Alison Allan. The LRP cDNA probe and 7s probe used in nonhern bloaing experiments were prepared by Dr. Sally Benn. Dr. Jonathan

LaMarre assisted in the iodination of a2M*. TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... i ... DECLARATION OF WORK PERFORMED...... III

TABLE OF CONTENTS ...... iv .. LIST OF TABLES ...... vri ... LIST OF FIGURES ...... wi~

LIST OF ABBREVIATIONS ...... x

INTRODUCTION ...... 1

REVIEW OF LITERATURE ...... 4 Intracellular-Acting... Bacterial Toxins ...... 4 Enzymatic activities...... 5 Structure...... -8 Intoxication...... 9 Pseudomonas Exotoxin A ...... 12 Pseudornonas aeniginosa as a human pathogen ...... 12 Toxicity and role in infection ...... 13 ...... Intoxication . 16 Intemalrzation...... 19 Activation ...... 20 Tnfficking ...... --77 Translocation ...... -7- J Substrate inactivation ...... 25 Low-Deasity Lipoprotein Receptor-Related Protein ...... 26 Receptor-mediated endocytosis...... -26 The low-density lipoprotein receptor gene family ...... 77 Structure...... 28 Synthesis...... -29 Receptor recycling ...... 30 Ligands and physiological roles ...... 31 ?') Chy [omicron remnant catabolism ...... JJ Proteinase cataboiism...... -34 Exploitation of LRP by pathogens ...... -35 Regdation ...... 36

RATIONALE ...... -38 CHAPTER 1: MATRIX INDUCED DOWN-REGULATION OF THE LOW- DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN DECREASES PRIlMARY HEPATOCYTE SENSITIVITY TO PSEUDOMONAS EXOTOXIN A ...... -40 Introduction...... -40 Materials and Methods ...... -42 Primary rat hepatocyte culture...... 42 Liver perfusion ...... 42 Hepatocyte isolation ...... 44 Hepatocyte culture ...... -44 Cytotoxic assessrnent of PEA-treated hepatocytes ...... 45 [3~~eucineincorporation assay ...... 45 Alamar Blue assay ...... 46 Microscopic evaluation...... 46 Receptor-associated protein protection studies ...... 47 Receptor-associated protein isolation ...... -47 Receptor-associated protein protection ...... 49 Ligand intemalization studies...... 49 Data analysis...... -50 Results ...... -50 Prirnary hepatocyte PEA sensitivity ...... 50 Effect of RAP on primary hepatocyte PEA sensitivity...... 54 Functional LRP expression in primary hepatocytes ...... 57 Susceptibility of primary hepatocytes to TGFgagPEA37...... 57 Discussion ...... 60

CHAPTER 2: ENHANCED MACROPHAGE RESISTANCE TO PSEUDOMONAS EXOTOXIN A IS CORRELATED WTH DECREASED LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN EXPRESSION ...... -66 Introduction ...... 66 Materials and Methods ...... -68 Ce11 cuihue ...... A8 Pseudomonm exotoxin A cytotoxicity assay ...... A9 RAP and LPS protection ...... 70 RNA isolation and nonhem blot analysis...... 71 Preparation of inserts ...... 71 Total RNA isolation ...... 71 Gel electrophoresis ...... -72 Capillary tramfer ...... 73 Probe preparation ...... 73 Hybridization...... -74 . * Ligand intemaltzation studies...... 75 Data analysis...... -76 Results ...... 1.1...... -76 HS-P PEA sensitivity...... -76 PEA sensitivity is decreased by LPS ...... 79 HS-P macrophages are protected hmPEA by RAP ...... 84 Expression of HS-P LW...... 84 Discussion ...... -87

CHAPTER 3: DIVERGENT PSEUDOMONAS EXOTOMN A SENSITIMTY IN NORMAL AND TRANSFORMED LIVER CELLS IS CORRELATED WTH LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN EXPRESSION ...... -93 Introduction ...... -93 Materials and Methods ...... 96 CeII culture ...... -96 C poxicity assay ...... 96 RAP protection studies...... 97 Ligand internalization...... 97 Data anal y sis ...... -98 Results ...... -98 PEA sensitivity ...... 98 Effect of RAP on PEA sensitivity ...... 100 Effect of differential LWexpression on ligand imemalization ...... 100 Susceptibility of cells to TGF-a-PEA37 and PEAGld7...... 103 Discussion ...... 106

GENERAL DISCUSSION ...... 109 lnduced Modulation of Cellular PEA Sensitivity ...... 109 LRP: PEA's portal of entry ...... Il3 hltering LRP Expression: A Cellular Mechanism for Modulating PE.4 Sensitivity...... -115 Therapeutic Intervention...... 119 Limitations of the Study ...... -120 Recommendations for Future Research ...... 122

SUMMARY AND CONCLUSIONS ...... 125

LITERATCIRE CITED ...... -127

APPENDICES ...... 147 LIST OF TABLXS

Ligands for the LRP and potential disease implication of aitered LRP-mediated intemalization of LRP ligands...... 32

ID jo values for prirnary hepatocytes in extended culture on collagen type I and MatrigelTM...... x- -

III Alterations in cellular PEA sensitivity and LRP expression in various cell types induced by a wide range of cellular events...... 1 1 I

vii LIST OF FIGURES

Schematic representation of the process of PEA intoxication...... 18

Cell rnorphology of primary hepatocytes cultured on collagen type i afier exposure to PEA ...... 51

Viability of primar): hepatocytes cultured on collagen type 1 exposed to PEA ....57

Protein synthesis inhibition by PEA in primary hepatocytes culnired on a collagen type 1 matrix ...... -53

Protein synthesis inhibition by PEA in primary hepatocytes cultured on MatrigelTM...... -56

Effect of RAP-GST on PEA-mediated cytotoxicity in primary hepatocytes ...... 58 . . Pnmary hepatocyte a2M* intemalizauon...... 59

Conjugate PENTGF-a toxin (TGF-a-PEA37) sensitivity of pnmary hepatocytes ...... 61

Dose-dependent cytotoxic activity of PEA on HS-P macrophage-like cells ...... 77

Time-dependent cytotoxic activity of PEA on HS-P macrophage-like cells ...... 78

Dose-dependent effect of LPS treatment on PEA-induced cytotoxicity in HS-P cells ...... 80

Time-dependent effect of LPS treatment on PEA-induced cytotoxicity in HS-P ceils ...... 81

Effect of LPS treatment on dose-dependent PEA induced HS-P cytotoxicity .....82

Effect of LPS treatment on HS-P cytotoxicity induced by short duration exposure to PEA ...... 83

Effect of RAP-GST on PEA-induced cytotoxicity in HS-P cells ...... 85

Effects of LPS on LRP mRNA expression in HS-P cells ...... 86

Effect of LPS on HS-P a2M* internalization...... 88

Protein synthesis inhibitory activity of PEA on BNL CL2 and BNL IME A.7R. 1 liver cells...... 99 19 . Effect of RAP-GST on PEA-mediated protein synthesis inhibition in BNL IME A.7R. 1 iiver cells ...... i01

20 . Ligand intemalization studies with a?-macroglobulin (a2M*)...... 102

2 1 . TGF-a-PEA37sensitivity of BNL CL2 and BNL 1ME A.7R. 1 liver cells ...... 104

22 . Sensitivity of BNL CL2 and BNL 1ME A.7R. 1 liver cells to PEAGlu57 ...... 105 LIST OF ABBREVIATIONS azM Alpha-2 macrog 10 bulin a7MS Activated azM ADP Adenosine diphosphate AMP Adenosine monophosphate apoE Apolipoprotein E ATP Adenosine triphosphate CAMP Cyclic AMP CCP Clathrin coated pits cDNA Complementary DNA CDNDB 1-choior-2,4-dinitrobenzene CF C y& Fibrosis CHO Chinese hamster ovary CNS 1 Cytotoxic necrotizing factor 1 CT C-terminus DEPC-A Diethy lpyrocarbonate-A DNA Deoxyribonucleic acid DT EBSS Earles' balanced sait solution EF2 Elongation factor 2 EGF Epidermal growth factor EGFR EGF receptor EGTA Ethylene glycol tetraacetatic acid EHEC Enterohemorrhagic Escherichia coli ER Endoplasmic reticulum FCS Fetal Cdf Sem Gb3 Glycolipid globouiaosyl ceramide GDP Guanine diphosphate GST Glutathione S-tramferase GTP Guanine triphosphate HB-EGF Heparin binding EGF-like growth factor HBSS H&' balanced salt solution HEPES Hydroxyethyl piperazine ethane HL Hepatic lipase HSPG Heparan sulfate proteoglycan ID50 Inhibition dose 50 m-Y iPTG Isopropyl beta-D-thiogalactoside kb Kilobase kDa Kilodaltons LB Luria-Bertani LDso Lethd dose 50 LDL Low-density lipoprotein LDLR LDL recrptor INTRODUCITON

A living multicellular organisrn represents a unique environrnent for bacterial habitation. Compared to a non-living environrnent, a host organism is both warm and nutrient nch and thus. at first glance, appean to offer bacteria an ideal habitat. However. host organisms do not allow bacteria to establish residence and exhaust valuable resources without a fight. and have developed extensive anti-microbid strategies to resist bacterial infection (Kaufmann, 1999; Nahm et al., 1999). In rum. pathogenic bactena have developed products. known as vinilence factors, to resist the anti-microbial strategies of the host and to establish and sustain an infection (Saiyers and Whia. 1994).

Thus, bacterial mechanisms of virulence and host mi-microbid stmegies have been CO- evolving. resulting in a continually escaiating msrace between bacteria and their hosts

(Nesse and Williams. 1993).

Pathogenic bacteria have been the focus of an intense research effort aimed at elucidating bacteriai mechanisms of virulence. ffiowledge acquired fiom this research has lead to the development of a variety of effective strategies with which to prevent and combat bacterial iiifections. such as vaccination programs and antibiotic therapies.

However. despite our efforts to develop and implement these stategies. bacterial infectious diseases are ni11 responsible for extensive morbidity and rnortality worldwide.

Unfortunately, this grave situation is likely to wosen in the near fiintre, as the incidence of new, reemerging and antibiotic resistant bacterial infections is escaiating at an alarming rate (Satcher, 1995). As the post-antibiotic era approaches. it is impcrative that we intensiQ our research efforts aimed at understanding the mechanisms by which bacterial pathogens cause disease so that an adequate knowledge base can be established fiom which novel therapeutic and prophylactic strategies can be developed.

A recurring theme of bacteriai mechanisrns of wulence is the exploitation of noxmai host ce11 processes and components, often at the expense of the host, for the bacteriurn's advantage (Finlay and Cossart, 1997). Nowhere is this phenornenon better exemplified than by intracellular-acting bacteriai . A variety of gram-positive and gram-negative bacteria produce such exotoxins, including Pseudomonus aemginosu. Corynebacterium diphtheriae, Vibrio choferae, Shigella sp. and Bordetella pertussis, which produce

Pseudmonar exotoxin A (PEA), Diphtheria toxin PT), Cholera toxin (CT).

(Sa)and Pemissis toxh, respectively (Salyers and Whitt, 1994). During the process of intoxication. intracellular-acting bacterial exotoxins utilize numerous components of the target cell's notmal machinery to generate an enzymaticaily active toxin hgment and deliver it to its intracellular substrate (Henderson et al., 1999). For exampie. various exotoxins "hijack" specific ce11 surface recepton to facilitate exotoxin entry into host cells by receptor-rnediated endocytosis (RME). Since exploitation of RME is an escient method used by exotoxins to enter hr 4 cells, the expression levels of these "toxin receptors" may represent a cntical factor in determining cellular toxin sensitivity, and consequentiy establish which tissues are damaged following toxin exposure during bacterial infections.

P. aeruginosa has emerged as an important cause of various nosocorniai infections in compromised individuais, including burn victims and cystic tibrosis patients

(Bodey et al., 1983 3 Gilligan, 199 1). P. aeruginosa pathogenicity is cornplex, involving an impressive battery of vinilence factors. including the exotoxin. PEA. PEA catalyzes the ADP-ribosylation of elongation factor 2 (EF2), leading to inhibition of protein synthesis and ce11 death (Iglewski et al., 1977). Like other intracellular-acting exotoxins,

PEA intoxication is a complex process that relies on the active participation of target cells to generate an active toxin fragment and deliver it to the cytoplasm to inactivate EF2.

The fim step of intoxication involves PEA binding to the low-density lipoprotein

receptor-related protein (LRP), followed by intemaiization via RME (Kounnas et al.,

1992). Cells expressing high levels of this receptor are extremely sensitive to the cytotoxic effects of PEA (Mucci et ai., 1995). This study was undertaken to Mer

investigate the molecular mechanisms that govem cellular sensitivity to PEA.

Specifically. 1 wished to determine whether sensitivity to PEA can be altered in and by

host cells and to identi& the cellular mechanisms responsible for this alteration. WVIEW OF LITERATURE

Intracellular-Acting Bacterial Toxins

A wide range of pathogenic bactena both gram-negative and gram-positive. synthesize and secrete toxic proteins that are termed exotoxins. Exotoxins serve as pnmary virulence factors. playing a centrai role in the establishment of infections. dissemination to deeper tissues. evasion of host defenses and transmission to new hosts. by causing hoa ce11 dysfunction, damage and lysis (Patrick and Larkin. 1995). Indeed. the major sy mptoms assoc iated wi th diseases caused by Corynebacterium diphtheriae

(diphtheria). iïbrio cholerae (cholera). Ciosrridium borulinlrm (botulism). CIosrridium terani (tetanus). and enterohemorrhagic Escherichia coli (EHEC)(bloody diarrhea and hemoiytic uremic syndrome) are caused by the exotoxins produced by these bactena

(Salyers and Whit~1994: Mims et ai.. 1998).

The bacteriai exotoxins cmbe divided into three groups according to their site of action. The first class of bacterial exotoxins. known as type 1 toxins. act at the plasma membrane. where the) interfere with cellular signaling by interacting with transmembrane receptoa. This group includes the stable toxins of E. coli (Nair and

Takeda 1998) and the produced by S~aphyIococnrs aurelis and

Streptococnrs pyogenes (Michie and Cohen. 1998). Type II toxins. the second class of

bactenal exotoxins. act directly on the plasma membrane. This group includes enzymes.

such as phospholipases (Songer. 1997), that attack the components of the plasma

membrane. Other members of this group are the pore-forming toxins of the thiol-

activated cholesterol binding cytolysin family (Tweten, 1995) and the RTX toxins

(Welch, 1995). The damage caused by type II toxins disrupts the Uitegrity of the plasma membrane, eventually Ieading to cell lysis. Type IiI toxins are bacterial exotoxins that cross the plasma membrane and act inside the cell. Their action is more subtle than membrane-damaging toxins. involving enymatic modification of specific cytoplasmic subsuates (Middlebrook and Dorland, 1984). Intracellular-acting exotoxins are among the most potent toxic agents to be identified; for example botulinum toxins are lethal to humans at a dose of only 0.1 ngkg of body weight (Payling-Wright, 1955). The potency of intracellular-acting exotoxins has been atuibuted to their cataiytic activity and their ability to gain access to their cytoplasmic substrates. The targeted intracellular substrates are vital components of the cellular machinery involved in various cellular processes. such as protein synthesis. cposketetal rearrangements and intracellular signaling. Toxin- mediated disruption of these cntical cellular processes, by the inactivation of targeted substrates. causes extreme alteration in cellular physiology and often, ce11 death.

Enzymatic activities

Inüacellular-acting bacterial exotoxins can be subdivided into classes according

to their enzymatic activity. The first class of exotoxins catalyzes the transfer of the ADP-

tibose moiety fiom the oxidized focm of nicoiinamide-adenine dinucleotide (NAD') to

eukaryotic proteins (Knieger and Barbier. 1995). In al1 cases, the substrates for ADP-

ribosylating toxins are nucleotide-binding proteins, such as heterotrimeric G proteins and

low molecular weight GTP-binding proteins. The addition of a bulky ADP-ribose moiety

is a post-translational modification that modulates protein function. Diphtheria toxin (DT),

produced by C. diphtheriae, was the first bacterial toxin in which ADP-ribosylating

activity was identified, and has served as the prototype for the snidy of other toxins in this class (Coilier, 1975). DT catalytically attaches an ADP-ribose moiety to a modified histidine residue, diphthamide, on elongation factor 2 (EF2). EF2 participates in the elongation step of protein translocation and inactivation of this essential ribosomal cofactor leads to a complete inhibition of protein synthesis and subsequent ce11 death.

Like DT. Pseudomonas Exotoxin A (PEA), secreted by the opportunistic human pathogen P. ueruginosa. possesses ADP-ribosylating activity and inhibits protein synthesis by inactivating EF2 (Iglewski et ai.. 1977).

The second class of exotoxins possesses N-glycosidase activity. Members of this uoup are the Shiga toxins (Stxs), dso called verotoxins. produced by Shigella C dysenteriae (Stx) and EHEC (StxUStx2) (Tesh and O'Brian. 199 1). These toxins cleave a single adenine residue fiom the 28s rRNA of the 60s ribosomal subunit and prevent elongation factor 1mediated binding of t-RN4 to the ribosomal complex (Sauena et al..

1989). Although Stxs and DT have different enzymatic mechanisms (depurination verses

ADP-ribosylation) and substrates (the 18s rRNA versus EF2) their inhibitory effects on protein synthesis are identical.

The third class of exotoxins is composed of the of C. botrtliniim and

C. tetani and possess proteolytic activity. Each is a metalloprotease that attacks a single protein: as a goup these toxins cleave four specific proteins (VAMP-1 and -2, syntaxin and SNAP-25) (Tonello et al.. 1996). The targrted proteins are involved in the docking of vesicles to the synaptosomal membrane during exocytosis-mediated

netuonansmitter release. Neurotoxins prevent neurotransmitter release and subsequently

impair nem transmission. Botulinurn toxins cause a flaccid paralysis by blocking the fhction of peripherd nerves. Tetanus toxin aEects the nerves of the central nervous system, causing rigid paralysis and muscle spasms.

Deamidation has recently been identified as a mechanism of action for an additional class of exotoxins. Cytoxic necrotizing factor 1 (CNS 1), produced by certain pathogenic svains of E. coli, specifically deamidates a glutamine residue on Rho. Cdc42 and Rac (Schmidt et ai., 1997; Lem et al., 1999). CNS 1-mediated deamidation permanently activates these small GTPases of the Rho family, which induces large-scale reorganization of the actin cytoskeleton into stress fibers and stimulates DNA synthesis but inhibits ceil division, leading to the production of multinucleated cells (Boquet et al..

1998).

A new class of exotoxins that possess UDP-glycosl- activity aiso

targets the Rho family. These toxins use UDP-glucose as a substrate to attach a glucose

moiety to their target. The two large clostridial exotoxins of Chsrridiun dificile add a

single glucose moiety to a specific threonine residue on Rho. Rac and Cdc42. This

modification inactivates these Rho proteins and leads to cellular rounding because of a

breakdown in the actin cytoskeleton (reviewed by von Eichel-Streiber et al.. 1996).

Members of the final class of exotoxins possess adenyiate cyclase activity and

inciude the edema factor of the complex produced by Bacillus anthracis

and exotoxîns produced by Bordetella species (Hanna. 1998; Ladant and Ullmm, 1999).

Once inside the cytoplasm the enzymatic domains of these toxins convert cellular stores

of ATP into CAMP. Increasing the cellular concentrations of this important secondary

messenger perturbs the cellular signaling pathways of targeted host cells. Structure

Inûacellular-acting bacterial exotoxins are also referred to as AB toxins. These toxins were so named following the observation that they have h~ofunctionally separable components. The B component mediates binding to specific host ce11 receptors, while the

A component contains the enzymatic activity. Intracelldar-acting bacterid exotoxins can be classified into three general families based on their AB structure. The fint and simplest structural family is the .48 family. Exotoxins that belong to this farnily, such as

DT, PEA and the neurotoxins, are composed of a single polypeptide chah containing both the A and B components as separate structurai domains (Middlebrook and Dorland.

1984). The second structural family is comprised of exotoxins with the general structure of ABs. where individual A and B components are Iocated on separate protein subunits. which assemble together to form the holotoxin. In the Stxs and CT. the pentameric B- dornain is composed of five identical monomers that associate in a ring-likc rnanner around a single enzymatic subunit (Frasier et al., 1994; Zhang et al.. 1995). In contrast. the ring-like B oligomer of has a more complex structure. composed of one copy of S2. S3 and Sj and two copies of S4 (Stein et ai. 1994). The large clostndal exotoxins belong to the final structural family and are best described as ABX exotoxins.

These single-chained toxins contain a multivalent receptor-binding domain, comprised of multiple copia of a structural motif that is a demonstrated receptor ligand (von Eichel-

Stroeiber et al., 1996). Intoxication

Intracellular-acting bacterial exotoxins are ody able to exert their toxic effects if they gain entry into the cytoplasm of host cells. In addition, since exotoxins are secreted by bactena as proenzymes. they have to be proteolyticaily processed to unrnask their rnzymatic activity (Gordon and Leppla 1994). In an effort to meet these two requirements, generating an enzymatically active toxin fragment and delivering it to its

intracellular subsme. exotoxins circurnvent various components of the mget cell's

normal machinery in a process called intoxication (Middlebrook and Dorland. 1984).

This process relies on the active participation of the target cell. and because it resernbles

the process of viral infection, exotoxins have been referred to as "molecular parasites"

(Ogata et al.. 1990). The ability to exploit a target cell's machinery and processes resides

with the exotoxin's own protein structure. It appears that imitation of natural host

proteins, a phenornenon known as molecular "mimicry", allows exotoxins access to

various cellular processes that are essential for successful intoxication. Generally. the

process of intoxication has four major steps: (i) internalization: (ii) processing; (iii)

translocation: and (iv) substrate modification.

As a means of gaining entry into hosr cells exotoxins "hijack" specific ce11 surface

receptors and are internaiized by receptor-mediated endocytosis (RME). In this way.

exotoxins readily traverse the plasma membrane by exploiting a cellular process that is in

place to eficiently and selectively intemalize ligands fiom the extracellular space

(Mukherjee et al.. 1997). Exotoxins utilize both forms of RME, clathrin-dependent and

clathrin-independent. Toxins use a diverse set of ce11 surface structures as toxin

receptors. including proteins and glycolipids. For example, DT binds to the heparin bhding epidemal growth factor-like growth factor precursor (pro-HB-EGF) (Naglich et

al.. 1992) and Stxs bind to the globo-senes glycolipids, such as glycolipid globotriaosyl

ceramide (Gb,) (Lingwood. 19%).

During intoxication full-length exotoxins are cleaved by target host proteases.

creating active toxin fragments that later translocate across an intemal membrane to the

cytoplasm (Gordon and Leppla 1994). The eukaryotic enzyme responsible for the

activation of a number of bacterial toxins, including Stxs. DT and PEA, is fkin (Gordon

and Leppla, 1994). Furin is an ubiquitously expressed senne protease that processes a

diverse range of host proteins during their transpoa dong the constitutive secretory

pathway. It is believed that the acidic nature of the early endosorne, where exotoxins are

located following uptake by RME. permits toxins to adopt an altered conformation that

increases their susceptibility to enzymatic processing. In addition to proteolytic

processing. exotoxins require reduction of disuifide bonds to acquire full biological

activity (Gordon and Leppla 1994).

As a consequence of entering the endocytic pathways, via RME, bacterial toxins

are handled in a similar manner to physiological ligands and are targeted for destruction

within late endosornes and lysosomes. Only a srnail percentage of intemalized toxins

seem to avoid this fate and hvo mechanisms have been proposed to explain how this

occurs. The fim mechanism involves an activated toxin fragment escaping from its

transport cornpartment before being delivered to a late endosome. For example, the early

endosome is the cornpartment from which the activated fiapent of DT escapes into the

cytoplasm, thus avoiding transport to the late endosome (Umata and Mekada, 1998). The

second mechanism utilized by exotoxins for avoidig proteolytic destruction involves bypassing wcking to the lysosome and alternatively travelling to the endoplasmic reticulum (ER) (Hazes and Read, 1997). Vesicular trafic is extensive between late endosomes and the trans-Golgi network (TGN),involving the delivery of hydrolytic enzymes to late endosomes and recycling of the mannose-6 phosphate receptor back to the TGN. In addition. retrograde transport fiom the Golgi apparatus to the ER has been well documented for proteins containing specific ER retrieval signals (Mukherjee et al..

1997). PEA. CT. Stx and Pemissis toxin have been detected in the Golgi apparatus and the ER. The discovery of KDEL-iike ER retrieval signals in the C-terminus of CT and

PEA suggests these toxins traEc to the ER following binding to the KDEL receptor

(Hazes and Read. 1997). In exotoxins that lack a known ER retention sequence. such as

Sn. the mechanisrns utilized for transport from the TGN to the ER are unknown

(Johannes and Goud, 1998).

It has been speculated that exotoxins to the ER because it presents a favorable environment for transport to the cytoplasm (Hazes and Read. 1997). There is recent evidence indicating that host proteins travel from the ER to the cytoplasm. in a

process cailed retrotranslocation. One interesting hypothesis speculates that exotoxins exploit the retrotranslocation pathway involved in the ER-degradation process by

mimicking improperly folded host proteins (Hazes and Read. 1997). Altematively. as

previously mentioned, exotoxins such as DT. enter the cytoplasm hm the acidic

endosomal comparunent. The DT translocation process is not completely understood but

involves a pH-induced conformational reanangement in DT structure. Bodi subunits of

the altered toxin interact with the endosomal membrane and with the aid of the B

hgment, the A hgment is transiocated into the cytoplasm (Lesieur et al., 1997). Pseudomonas Exotoxin A

Pseudomonas aeruginosa as a human pathogen

Pseudomonas uemginosa is a gram-negative. rod shaped, motiie, aerobic bacterium that is ubiquitously found in the environment (Vasil. 1986). P. aenrginosa is the quintessential opportunistic human pathogen, rarely a problem in penons with intact host- defense systems, but with a remarkable ability to cause senous disease in compromised individuals (Bodey et ai.. 1983). Particularly susceptible are hosts jeopardized by extreme youth or age. severe trauma cancer and those afTected with the genetic disease cystic fibrosis (CF). Infections caused by P. aenrginosa are diverse in nature and cm affect vimially any part of the body, including infections of the urinary tract, gastrointestinal tract. respiratory tract epicardium, as well as eye and ear Section. bone and joint infection. and bacteremia (Anehand Cross, 1993). The specific clinicai condition depends not only on the initiai site of infection. but dso on the underlying condition of the host (Wood. 1976).

P. aerziginosa infections cm range fiom chronic conditions, such as pulmonary infections of CF patients (Gilligan, 199 1), to acute conditions as seen in invasive bacteriai septicernia of individuals suiTering from severe thermal injuries (Holder. 1993). As a demonsaation of its oppoministic na-. P. aeruginosa has ken reported to be one of the four most common gram-negative pathogens found in nosocorniai infections (Weinsteh 1998). Due to a highly selective outer membrane, P. aemginosa possesses a natural resistance to commody wdantibiotics (Hancock, 1986; Hancock, 1997), a circurnstance which severely hampers patient treatment and contributes to the high monality rates associated with infected individuals (Bodey et al., 1983; Bryan et al., 1983; McManus et al.. 1985; Gilligan, 1991). The ability of P. aeruginosa to induce a wide variety of ovenvhelming disease States can be attributed to its Unpressive repertoire of Wulence factors. Endotoxh, exopolysacchande (slime, alginate) and pili are known cell-associated Wulence factors.

Secretory products that play a rote in virulence are proteases (alkaiine protease, elastase). toxins (exotoxin A. cytotoxin. exoenzyme S), pigments and hemolysins @hospholipase C)

(Vasil. 1986). These virulence-associated factoa act both individually and in concert to produce infection and disease. Despite possessing a number of Wulence factoa that are present in true pathogens. it is interesthg that P. aeruginosa is merely an opportunin. It appean that this versatile bacteriun lacks the ability to initiate a persistent colonization or invade tissues in humans with intact nonspecific defense mechanisms and only a senous disniption in these defenses allows P. aerziginosa the opportunity to invade the body.

However. once given the opportunity, P. aemginosu is a formidable adversary and is responsible for extensive patient morbidity and mortaiity (Bodey et al., 1983: Govan and

Glas. 1990).

Toxicity and role in infection

Pseudornonas Exotoxin A (PEA) was first discovered ad purified fiom P. aeruginosa by Liu and CO-workers(Liu et ai.. 1961. Liu et al., 1973). PEA is the most

potent toxic substance, on the basis of weight. produced by P. uerziginosa, with an LDso

value for mice of 2.5 pg/kg (Gill, 1982). This value exceeds the lethal dose of P.

aeruginosa (LPS)by 10 000 times (Pollack, 1983). PEA is lethal to a

number of mammais including mice, rats, dogs, rabbits and rhesus monkeys (Pollack, 1983).

The mon substantial histopathological fïnding, following incavenous injection of purified PEA is necrosis of the liver, although minor hemorrhaging of the lungs and kidney necrosis is also observed. PEA's versatility as a toxin in vivo is matched in vitro as demonstrated in a study by Middlebrook and Dorland (1977). establishing the wide range of ce11 lines, across a number of species. that are susceptible to the toxic effects of PEA.

The fm work to indicate the mode of action of PEA was conducted by Pavlovskis and Shackelford (1971). The authon provided evidence that PEA mediates its cytotoxic effect by inhibiting protein synthesis. Iglewski and colleges confimed this fmding utilizing an in vitro mode1 system and. in addition, discovered that PEA inhibits protein synthesis in a manner identical to DT (Iglewski and Kabat, 1975; Iglewski et al.. 1977). Both DT and

PEA enzymaticaily catalyze the transfer of the ADP-ribose moiety of NAD' to EF2. a criticai component of the translational rnachinery responsible for synthesizing proteins within eukaryotic cells. EFZ controls the translocation of peptidyl-tRN.4 fiom the ribosomal

A (aminoacyl) site to the P (peptidyl) site (Lodish et al.. 1995). Without functional EF2 the ribosomal complex stalls with no way of travershg dong the mRNA: protein synthesis is effectively anested and eventually the ce11 perishes.

A clear indication of PEA's pathoiogical potential resides in its ability to potently inhibit eukaryotic protein synthesis. However. the precise role that PEA plays in P. uenginosa infections remains il1 defined. Unlike pathogenic organisms such as C. diphtheriae, where a single factor has been show to be accountable for the infectious disease diphthena, P. aenginosa utilizes a multitude of diverse virulence factors. and the

contribution of a single factor has been difficdt to ascertain. Despite this, a nurnber of

important studies indicate that PEA contributes to the pathogenicity of a varief~of P.

aemginosa infections. Investigations have been conducted comparing nontoxigenic mutants with parent strains in a variety of experimental model systerns. The overall trend in the literature indicates that mutant strains are less Mentthan toxin producing mains, although results from individual experiments are strikingly dichotomous, which has been attributed to the use of different animal systems. Additional evidence supporthg PEA's importance in P. aemginosa infections is inferred fkom the observation that 90% of al1 P. aeruginosu isolates are capable of producing PEA (Vasil et al., 1984) and patients suf3ering from P. aeruginosa infections often produce antibodies against PEA (Pollack et al., 1976; Cross et al., 1980). It has been noted that the odds of nwival increase significantiy when individuals generate high titers of antitoxin early in bacteremic P. aeruginosa infections (Pollack and Young,

1979; Cross et ai .. 1980). Numerous experiments have shown that passive administration of antibodies directed against PEA is protective against PEA induced darnage ( Paviovskis et al.. 1977; Snell et al.. 1978; Pavlovskis et al., 198 1).

Several lines of evidence indicate that the liver is an in vivo target of systemic PEA.

Utilizing a mouse model, Saelinger and colleagues showed that P. aeruginosa multiplying within burnt tissue is able to produce PEA and. derentering the circulation, targets the liver where it reduces both protein synthesis and the quantity of fiuictional EF2 (Saelinger et al.,

1977). Iglewski and coworkers produced sirnilar redts by adrninistering purifiied PEA to mice, showing that the damaged liver became completely devoid of functional EF3

(Iglewski et al., 1977). It has been proposed that PEA-mediated liver damage may be beneficial to P. aemginosa during infection by increasing the level of systemic iron an essential bacterial growth factor nomally tightly sequestered by the host and by reducing the production of essential liver generated serum proteins with known antimicrobial properties (Holder, 1993).

In addition to its role in mediating liver dysfunction and damage, evidence is accumulating to indicate that PEA may have additional roles during P. ueruginosa infections. PEA suppresses the murine immune system in a dose dependent fashion (Holt and Misfildt. 1984). As a partial explanation of this phenornenon PEA has been found to reduce the numbes of circuiating neutrophils in vivo (Miyasaki et al.. 1995) and inhibit the ability of bone marrow stem cells to proliferate (Stuart and Pollack 1982). PEA is cytotoxic to neutrophils in virro (Bishop et ai. 1987) and alters their ability to make free oxygen radicals (Fontan et al.. 1995). Macrophages and their irnrnediate precursors are likewise susceptible to the cytotoxic effects of PEA (Pollack and Anderson, 1978: Holt and Misfildt. 1984). Furthemore. macrophages treated with low dosages of PEA have a decreased ability to engage in phagocytosis (Pollack and Anderson, 1978) and show altered cytokine secretion levels. including an aiteration in the production of turnour necrosis factor (TNF). interleukin- 1. and interferon-y (NF-y)(Misfeldt et al.. 1990;

Staugas et al.. 1992). PEA also has inhibiton, effects on the functional abilities of B and

T cells (Michalkiewicz et al.. 1989; Vidal et al.. 1993). PEA-induced modulation of the

immune system may represent an additional mechanism by which PEA enhances the

virulence of P. aeruginosa infections.

Intoxication

The PEA gene. to-d. exists as a single copy on the chromosome of P. aenrginosa.

Regdation of r0.d is highly complex and involves multiple environmentai signais9 including iron concentration, temperature, oxygen levels and bacterial population size (Wick et al., 1990; de Kievit and Iglewski, 2000). Like other bacterial toxins, including Stx and

DT, PEA is maximally produced when iron concentrations are low. After undergoing a processing step to remove a 25 amino acid signai sequence, PEA is secreted as a 66-kDa proenzyme. containhg 6 13 amino acids (Gray et al., 1984). For PEA to be cytotoxic it muçt reach the cytoplasm of a target ce11 in an enzymatically active state. PEA accornplishes this by employing the machinery of the target ce11 in a process known as intoxication.

The intoxication of mamrnalian cells by PEA occurs by a multi step process involwig (i) binding of PEA to a specific ce11 surface receptor and intemakation by RME.

(ii) processing of the proenzyme to produce the active form of the toxin. (iii) intracelldar nafficking and (iv) translocation of the active toxin hgment into the cytopiasm and finally.

(v) the ADP-ribosylation of EF3 (Figure 1). The ability of PEA to e.xploit the normal rnachinery of the target ce11 during the intoxication process is due to the structure of PEA.

Analysis of the three-dimensionai X-ray crystaliographic structure of PEA has revealed that this AB toxin contains three distinct structural domains (Allured et al..

1986). Evidence based on a number of genetic studies suggests that each structural domain has a separate role in the process of intoxication. Domain 1 has been mer divided into two subdomains. 1, and Ib. Domain 1, (amino acids 1-252) has been show to be responsible for ce11 recognition (Hwang et al., 1987). Domain Ib (amino acids 365-

404) is physically located near domain 1, although they are separated fiom one another in

the primas, sequence. The function of domain Ib has yet to be identified. Domain II

(arnino acids 253-364) is believed to be involved in the activation and translocation seps

of intoxication (Chaudhary et al., 1988; Ogata et ai., 1990). While the domain Figure 1. Schematic representation of the process of PEA intoxication. (1) Native PEA binds to ce11 surface receptors and enten cells via RME. (II) Native PEA is processed

within the endosomal cornpanment to generate the 37-kDa active toxin fragment. (III)

The majority of internalized PEA is delivered to lysosomes for destruction. however,

sorne activated PEA hgments avoid this fate and instead move to the endoplasrnic

reticulum (ER) via the Golgi network. (IV) Activated PEA fragments translocate across

the ER membrane into the cytosol. where they (V) inactivate elongation factor 2 (EF2),

by enzymatically anaching an ADP-ribose moiety (star). Lysosome

Act ive l nactive responsible for the catalytic ADP-ribosylation of cytosolic EF2 is domain III (amino acids 405-613) (Gray et al., 1984). The individual steps in the intoxication process are presented below, with emphasis on the components of the cellular machinery that are exploited by PEA for its own agenda of inhibiting protein synthesis.

Internalizarion

To inhibit protein syntheis. PEA must journey fiom the extracellular environment. where P. aenginosa produces PEA. to the cytoplasm, the cellular location of EF2. The initial step of the intoxication process involves PEA gaining access to the intenor of a host ce11 by crossing the plasma membrane. PEA bhds to ce11 suface receptors, which then cluster within coated pits before being internalized by RME (FitzGerald et al.. 1980). PEA binding is specific and saturable. suggesting that a specific receptor is responsible for PEA intemalization (Manhan et al.. 1984). An attempt to isolate, puri@ and characterize the

PEA receptor was carried out by Thompson et al. (1991). The authon identified a large glycoprotein isolated from mouse LM ce11 fibroblasts that ~pecificallybound PEA. This

PEA-binding protein was Iater found to be present in moue liver, heart, kidney and spleen

(Foristai et al.. 1991). Kounnas et al. (1992) later demonstrated that the PEA-binding protein and the low-density lipoprotein receptor-related protein (LW)were identical.

The LRP is a multifunctional endocytic receptor that mediates the binding and intemalization of an ever-growing list of physiologically important ligands (see section on

LRP below). The receptor-associated protein (RAP) CO-purifieswith the LRP (Ashcom et al., 1990) and hctions as an antagonia for al1 identified LRP ligands (Willnow. 1998).

RAP prevents PEA binding and abolishes its biological activity in LM mouse fibroblasts, providing additional evidence that the LRP is the receptor involved in binding and intemalization of PEA (KOU~MSet al., 1992). Fitzgerald et al. ( 1995) provided conclusive evidence that functional LRP is required for PEA mediated toxicity by demonstrating that

Chinese hamster ovary (CHO) cells deficient in LRP were resistant to PEA. Similady.

Willnow and Hen (1994) isolated LW-deficient embryonic fibroblasts by using PEA as a

selective agent.

Investigations by Hwang et al. (1987) and Guidi-Rontani and Collier (1987)

indicate that domain 1, of PEA is responsible for receptor interactions. Non-cytotoxic

recombinant PEA proteins containing domains II. Ib and III. but lacking domain 1,. failed

to block PEA cytotoxicity. while non-cytotoxic proteins containing domain 1, inhibited

the toxic effects of native PEA. Mutant proteins, which lacked domain I,, were 200-fold

less toxic than native PEA when injected into mice. Replacement of ~~s~~by Glu, using

site directed mutagenesis. revealed that this arnino acid, located in domain I,, is critical

for receptor binding (Jima et al.. 1988). This mutant was less efficient in binding to

cells. accounting for its 50- to 100-fold lower cytotoxic rffect. Analysis of the crystal

structure of PEA has revealed a major concavity on the surface of domain I,, which

contains L~S"and is likeiy responsible for receptor interactions (Allured et al.. 1986:

Jimo et al.. 1988).

Activation

Full-length PEA requires intracellular processing to express full toxic activity.

E~ymaticcleavage foilowed by disuifide bond reduction separates PEA into two non-

overlapping fragments of 28- and 37-kDa (Ogata et al., 1990; McKee and FitzGerald, 1999). The 37-kDa carboxyl-terminal hgment, containing ai! of domain III and the majority of domain II. later translocates to the cytoplasm where it can ADP-ribosylate EF2. Cleavage occurs between and ~l~~~~within an aginine nch loop at the beginnuig of domain II

(Ogata et ai., 1992). This loop structure, fomed by the disulphide bond between and cys2*', is thought to reside on the surface of PEA and be readily accessible to protease attack.

The characterization of the in vivo enzyme responsible for PEA cleavage was initiated in a study by Fryling et al. (1992). The PEA processing activity was found to be associated with a sub-cellular membrane fraction correspondhg to the early endosome or the plasma membrane. This protease was later purified fiom beef liver and identified as furui (Chiron et al.. 1994). Furin is an ubiquitously expressed serine protease that plays an important role in the constitutive secretory pathway of mammdian cells (Nakayama 1997).

Furùi is responsible for the processing of a number of proproteins such as LRP, idinpro- receptor and transforming growth factor-beta (TGF-P) (Herz et al., 1990; Bravo et al.. 1994:

Dubois et al.. 1995). Other bacterial exotoxins such as anthrax toxin, DT and Stxs also utilize fùrin for their intracellular activation (Gordon and Leppla 1994). Furin's localization within the TGN and at the cellular nuface is consistent with the hypothesis that PEA processing occun within early endosomes. Furin cleaves PEA at an optimal pH range of

5.0 to 5.5 (Chiron et al., 1994). It has been speculated that the acidic nature of the early endosome causes a conformational change in PEA (Farahabkhsh et al.. 1987), making it more susceptible to enzymatic attack. The reduction in PEA cytotoxicity resulting fiom

agents that disrupt cellular pH gradients is believed to be partly the resuit of a reduced

efficiency in toxin processing (Middlebrook and Dorland, 1984; Corboy and Draper, 1997). Strong evidence suggesting that huin is required for PEA intoxication foilowed the isolation of a mutant CHO cell line, RPEAO, that displayed resistance to PEA and a number of RNA vinises (Innocencio et al., 1993). This resistance was determined to be the result of an inability of RPE.40 cells to express furin and following tradection with a finexpression vector, RPEAO cells regained PEA and viral sensitivity (Moehring et al..

1993).

Traflckhg

Following binding to the LRP and intemalkation by RME. PEA localizes within early endosomes. Normally LRP ligands are destroyed following endosomellysosorne fùsion. A number of toxin molecules, however, mut somehow avoid this fate. It has been proposed that the relatively low binding affinity of PEA allows it to dissociate fiom the

LWTwithin the acidic endosorne, and empioy an altemate intracellular pathway, thus bypassing the lysosome (Zdanovsky et al.. 1996). It is suspected that PEA utilizes retrograde cransport dong the Golgi network to the ER.

This hypothesis is based on a number of observations. First, early biochernical studies using biotinyl-PEA derivatives in conjunction with avidin-gold colloids, indicate that following internalization PEA is consistently found within Golgi bodies (Moms et al., 1983). Second. Yoshida et al. (1991) found that the Golgi body dimpting agent,

Brefeldin A, is capable of Uihibiting PEA toxicity. And fmally, the discovery that the carboxyl teminal sequence of PEA. Arg-Glu-Asp-Leu-Lys (REDLK), resembles the

characteristic ER retention sequence (KDEL), which has been shown to be important in

retaining newly synthesised proteins within the ER (Chaudhary, et al., 1990). Removal of these residues resulted in a protein with decreased toxicity. Since these mutants exhibited normal ce11 binding. activation and enzymatic activity it was postulated that the reduction in cytotoxicity was the result of a reduced ability to translocate to the cytoplasrn. However. when the native carboxyl-terminai sequence was altered to the consensus ER retention sequence, KDEL, the mutant PEA was more toxic than native

PEA (Kreitman and Pastan. 1995). It is now postuiated that the carboxy terminus of PEA intencts with the KDEL receptor (ERDZ). helping to transport the toxin fiom the Golgi to the ER. Recently. Hessler and Kreitman (1997) proposed that lysine613is removed by a plasma carboxypeptidase prior to PEA binding the LRP. The authors sugpst this is an essential step in the intoxication by PEA, based on earlier observations that the ERD7 receptor binds peptides containing REDL. but fails to bind those containing REDLK.

Translocation

Very littie is known regarding the in vivo translocation process that allows the active fragment of PEA to escape from a membrane-enclosed compartment into the cytoplasm.

The number of active toxin fragments that actuaily arrive in the cytoplasm is extremely low and has hampered progress in this area of research. It has been speculated that the highly

acidic environment of the endosome plays a crucial rolc in the process of translocation.

Mutant eukiryotic cells unable to decrease endosomal pH levels, due to a defect in

ATPase/K pumps. are resistant to the cytotoxic effects of PEA (Menon et al., 1983).

Sensitivity is also reduced following treatment with agents known to Uicrease endosomal pH

levels (Corboy and Draper. 1997). In normal susceptible cells it is likely that the acidic

environment of the endosome causes PEA to adopt an altered conformation, exposing hydrophobie residues that can then interact with the interna1 membraw of this cornpartment

(Farahbakhsh, et al., 1987).

An essential step in the translocation process must involve binding and insertion into an innzicellular membrane. PEA binding and insertion into membranes has been shown to increase dramatically as the pH is lowered, with an optimum range between pH 4.0 and 5.0

(Zalman and Wisnieski, 1985; Sandvig and Moskaug, 1987). Taupiac et al. (1996) and

Alami et al. (1998) have confhed that exposure to an acidic environment is a prerequisite to PEA translocation and also provided evidence that translocation of PEA requires energy. supplied by the hydrolysis of ATP.

Cytotoxicity is greatly diminished when the fim half of domain II (residues 254-

263) is removed fiom PEA (Hwang et al., 1987). This altered toxin still possesses native

PEA cell-surface binding and enzymatic activity, suggesting that domain II is important in

PEA translocation. Prior et al. (1992) confied the importance of dornain II's role in

translocation by demonstrating that a conjugate toxin, composed of the soluble enzyme

barnase and domain II of PEA, couid be successfblly transported to the cytoplasrn.

The N-terminus of the 37-kDa fragment of PEA is thought to intetact with an

unknown intracellular component (Zdanovsky et al., 1993). Mutations at 'frp? eu'^ and

Tjdg9cause a loss in toxic activity, yet have no effect on toxin binding, processing or

enzymatic activity. The authors suggest that, following processing and prior to

translocation, the N-teminus of the 37-kDa fragment interacts with an intracellular

component. This association is sanuable and represents the rate-lunithg step in the

intoxication process. The nature of this component is unknown at this time, but the

followiog have been proposed: a chaperone responsible for unfolding the active PEA fragment, a binding protein that transports the toxin to the ER or Golgi, or a constituent of the cellular machinery responsible for translocation (Zdanovsiq et al., 1993).

Subs~rateinactivation

Mer txanslocating across the ER and entering the cytoplasm, the 37-kDa PEA fragment interacts with its substrate. EF3. and cofactor. NAD'. PEA is a mono-ADP-

ribosyl transferase. which catalyses the following reaction (Iglewski et al.. 1977):

NAD' + EF2 = ADP-ribose-EF2 + Nicotinamide + K

PEA attaches the ADP-ribose moiety to EF2 at amino acid 715, which is a post-

translationally modified histidine residue. known as diphthamide (Iglewski et al.. 1977).

In addition. PEA possesses NAD* glycohydrolase activity and in the absence of EF2.

PEA causes the hydrolysis of NAD' to nicotinamide and ADP-ribose (Leppla et al..

1978). The significance of this minor enzymatic activity on host cells during intoxication

is presently unknown.

The etizymatic activity of PEA is located in the carboxy-terminus of the protein

(Gray et al.. 1984: Hwang et al.. 1987). Deletion analysis on the terminal ends of PEA

demonstrates that the entire structure of domain III is required for the stable expression of

eqmatic activiry (Hwang et al.. 1987: Siegall et al.. 1989). Analysis of the crystal

structure of PEA has revealed that domain III contains an extended clefi that is thought to

house the active center of PEA (Allured et ai., 1986). Biochemical and genetic studies

have been used to identifi individual amino acids important in substrate binding and in

carrying out the catalytic reaction. A number of deletion and substitution studies have

identified eluS3 as a critical residue for enzymatic activity (Carroll and Collier. 1987; Lukac et al.. 1988). GIU"~ is located in domain III and its side chah extends into the clek where it is thought to contribute to NAD' binding. ~~r~~~is thought to be involved in interactions with EF2 (Lukac and Collier, 1988; Wick and Iglewski, 1988).

EF2 is a 95-kDa single chah polypeptide that has intrinsic GTPase activity.

belonging to the guanine nucleotide binding protein family (Kohno et al., 1986). EF2 is an essentiai factor of the protein synthesis machinery and participates in the elongation

step of translation (Lodish et al., 1995). Specifically. the energy released from the

hydrolysis of GTP to GDP by EF2 is used to carry out the translocation reaction, which is

the movement of the ribosomal complex dong the mRNA to reposition the peptidyl-

tRNA from the ribosomai A site to the P site (Lodish et al., 1995). EF2 is inactivated by

the PEA-mediated attachent of ADP-ribose to the unique diphthamide residue (Iglewski

et al., 1977). The presence of an enzymatically activated PEA fragment in the cytoplasm

npidly inactivates the cellular stores of EF2. Consequently, without functional EF2. the

ribosomal complexes cannot carry out the translocation reaction, stall on the message

they are mslating, and cellular protein synthesis is irreversibly arrested. PEA-mediated

inhibition of protein synthesis is detrimental to host cells and pnerally leads to ce11

death, with both necrosis and apoptosis being observed (Morimoto and Bonavida. 1992;

Brinkrnann et al., 1995).

Low-Density Lipoprotein Receptor-Related Protein

Receptor-mediated endocytosis

Receptor-mediated endocytosis (RME) is a process in which extracellular

macromolecuies gain entry to the intracellular environment. RME plays an important role

in many ceilular processes (Mukherjee et al., 1997). For exarnpie, it is involved in the uptake of various nutrîents (iron, cholesterol), regdation of growth factor receptor expression (epidermal growth faftor receptor) and clearance of accumulahg waste proteins fiom the extracellular environment. Various infectious agents and toxins have evolved mechanisms to exploit RME in an atternpt to enter host cells (Stahl and

Schwartz, 1986). RME ofien occurs at specialized regions of the plasma membrane called clathrin-coated pits (CCP), which are invaginated basket-like structures that significantly concentrate specific cell-surface receptors. In general, cell-surface endocytic receptors specifically bind extracellular rnacromolecular ligands and move to

CCP. CCP are transient structures. and following formation. are rapidly pinched off fiom the plasma membrane. forrning vesicles that enter the endocytic pathway (Schmid. 1997).

A common fate for intemdized 1igand:receptor complexes involves rapid dissociation within the acidic environment of the early enodosome. followed by recycling of the receptor to the ce11 surface and ligand transport to the lysosome for degradation. By this mechanism. cells acquire essential nutrients and remove and degrade potentidly darnaging waste molecules from the extracellular environment (Mukhejee et al.. 1997).

The low-densitv lipoprotein receptor family

The low-density lipoprotein receptor (LDLR)gene family (Hussain et al.. 1999) is comprised of a group of endocytic receptors that mediate the intemalization and

degradation of a wide range of smifnirally dissimilar ligands. The most widely studied

member of the family is the LDLR which has a central role in lipoprotein metabolism

(Willnow, 1999). 0th family members that are expressed in mammaiian tissues include

the apoE receptor 2 (Kim et al.. 1996), the very iow-density lipoprotein receptor (Takahashi et al., 1992), the LW(Hen et al, 1988) and gp330/megalin (Saito et al..

1994). The LDL gene family is highly conserved with additional family members expressed in the nematode Caenorhabdiris elegam (C. elegans receptor). chicken

(chicken vitellogenin receptor) and Drosophilu melonogaster (Y 1 protein) (Yochem and

Greenwald, 1993; Bujo et al., 1994; Schonbaurn et al., 1995). Al1 family membea are type4 membrane proteins containhg a single plasma membrane spanning domain. a short carboxy-terminal cytoplasmic tail and a large ligand-interacting extracellular dornain. Ligand interaction is mediated by clusters of cysteine-nch complement-type repeats that are approximately 40 amino acids in length and contain three intemal disulphide bonds. ïhe extracellular domains of these receptors also contain epidermal growth factor (EGF)precursor homology domains that are critical for pH induced ligand dissociation. which occurs within endosomes. The EGF precursor homology domains are comprised of cysteine-rich EGF-type repeats separated by a cysteine-poor YWïD motif spacer sequence (Brown er al.. 1997). An O-linked glycosylation domain is found in certain receptoa and may serve to extend the extracellular domain away fiom the PM

(Hussain et al.. 1999). The cytoplasmic tails of family members contain short arnino acid motifs that mediate receptor clustering and intemalization at CCP. In the case of the Y 1 protein. it is a dileucine motif and for al1 other family members it is the NPXY sequence

(Hussain et al.. 1999).

Structure

LRP was fim cloned by Herz et al. (1988) as the second member of the LDLR

farnily. The LRP is expressed on the ce11 daceof a ..vide range of ce11 types. including hepatocytes, macrophages, neurons and smooth muscle cells. Tissue sites where LRP expression is high include the brain and Iiver (Feldman et al., 1985; Moestmp et al..

1992). LRP is a large endocytic receptor and, unlike other farnily members, its mature form is expressed on the ce11 surface as a heterodimer. It is synthesized as a 600-kDa single chain polypeptide that is processed during transport to the ce11 surface by funn within the trans-Golgi cornpartment (Hea et al., 1990). The 5 15-kDa a-chah and the

85-Da P-chah remain associated through non-covalent interactions. nie P-chah contains the transmembrane domain, which anchoa the receptor in the plasma membrane. and two copies of the NPXY intemalization signai sequence within its short cytoplasmic tail. The a-chain is expressed entirely in the extracellular space and is responsible for ligand binding. This subunit is comprised of 8 EGF precursor homology domains that separate four ligand binding domains containhg clusten of 2. 8. 10 and 1 1 complement-type repeats. The presence of 3 1 ligand-binding repeats provides the LRP with an impressive array of domains for ligand binding and helps to clarie how this

receptor is capable of interacting with such a wide range of structurally diverse

macromoiecules (Hen et al.. 1988: Hussain et ai.. 1999).

Svnthesis

LRP biosynthesis is complex, involving interactions with the 39-kDa chaperone-

like protein RAP (Williams et al.. 1992) and proteolytic processing by funn (Henet al.,

1990; Willnow et ai., 1996a). The human LRP gene is Iocated on chromosome 12

(Hilliker et al., 1992) and is approximately 92 kb in size containing 89 exons (Van

Leuven et al., 1994). LRP is transcnbed as a 15 kb mRNA transcript that is translated by ribosomes on the ER (Herz et al., 1988). Newly translated LRP is thought to interact with RAP, preventing the LWfiom interacting with recently synthesized LWligands within the ER lumen (Bu et al., 1995). After transport from the ER to the cis-Golgi compartment the LRP:RAP complex dissociates, an event thought to be mediated by the decrease in pH encountered upon entenng the Golgi apparatus. The acidic environment likely prevents ligand binding to the LRP in the absence of RAP during the subsequent period of transport to the ce11 surface (Willnow. 1999). RAP contains an ER-retention sequence and retums. via retrograde Wicking, to the ER after receptor uncoupling (Bu et al.. 1995). Mile in the ms-Golgi compartment. LRP is processed by the senne protease Mnto form the mature heterodimer. Furin cleaves the LRP between amino acids 3925 and 3926 at a consensus hirin cleavage sequence (RXRR) (Herz et al., 1990:

Willnow et al.. 19963). The reason for the processing event is not yet apparent. since unprocessed LRP is still functional (Ko et al.. 1998). Mer processing. the mature receptor is transported to the ce11 surface.

Receptor recvcling

LF2P:ligand complexes are intemalized at CCP and are then transported to the early endosomal cornpanment. Here. receptor:ligand complexes dissociate due to the low pH of the endosomal lumen. which is thought to induce a conformationai change in the LRP. possibly mediated by the EGF-precursor homology domains (Brown et al..

1997). At this point ligand and LRP follow different itineraries. LW is transported back to the PM after a brief transit through the Golgi apparatus. LRP molecules are highiy resistant to the harsh acidic conditions of the endosomal compartment and undergo multiple rounds of intemalkation and recycling in theu lifetime (Brown et al., 1997).

Intemalized ligands move dong the endosomal pathway, eventually reaching lysosomes, where they are degraded into constituents (amino acids, cholesterol) that cm be utilized by the cell.

Ligands and physiolopical roles

A key feature of the LRP is its ability to bind and intemalize a wide range of strucnirally and functionally diverse ligands (Table 1) (Strickland et al., 1995; Hussain et al. 1999: Willnow. 1999). Through the binding of these ligands the LRP plays a role in the removal and catabolism of nurnerous macromolecules from the extracellular space.

In this way, LRP acts as a scavenger receptor clearing the extracellular space of accumulating waste ligands. By regulating the abundance of ligands in the extracellular space. LRP influences various physiologicai processes, such as lipoprotein metabolism. protehase metabolism. tissue remodeling, and cellular migration (Strickland et al., 1995;

Gilemann. 1998). The removal of LRP-specific ligands from the circulation is mediated prirnarily by the liver (Chu and Pino, 19941, which is comprised of hepatocytes and resident macrophages (Kuppfer cells), both of which express LRP at high levels

(Feldman et al., 1985; Moestrup et al., 1992). Dysreguiation and nibsequent inappropnate LRP expression interferes with the normal elimination of ligands from the extracellular space and has been linked to a number of pathological processes, including atherosclerosis and Alzheimer's disease (Table 1) (Mahley and Ji. 1999; Ulery and

Stickland. 2000; Yu and Cooper, 2001). Table 1. Ligands for the LRP and potential disease implication of aitered LRP-mediated intemaiization of LWligands.*

------Potential implications of altered LRP- Ligands for the LRP mediated intemalization Lipoprotein metabolism apo-E Accumulation of lipoproteins and lipases. Lncreased oxidative changes to lipoproteins, P-VLDL which are re-directed to scavenger receptors Chylomicron rernnants for uptake by macrophages. Leading to Lipoprotein lipase foarn ce11 formation and atherosclerosis Hepatic lipase lesions. Proteinase metabolism PA. Pro-uPA, uPA uPA:PAI. tPA:PAI uPA:protease nexin 1 Excessive local proteinase activity. Protease:atM Leading to alteration in coagulation and Protease:a i-antitrypsin fibrinolysis, and increased destruction of Protease:C -inhibitor the extracellular matrix. Protease:protein C inhibitor Protease:antithrombin III Thrornbin:heparin cofactor II Tissue factorlTFP 17

Excessive growth factor and cytokine Growth factor and cytokine metabolism activity. Leading to exaggerated a2M:cytokine complexes inflammation, excessive matrix deposition a2M:growth factor complexes and inappropriate ce11 division.

------Pathoiens and toxins Malaria circumsporozoites Alteration in the efficiency of Minor group rhinovirus intemaiization, may represent a mechanism Pseudomonas Exotoxin A by which the host can alter cellular Sapotin sensitivity. (This issue is examined in this A thesis study).

Others RAP Thrombospondin-1 and -2 Excessive matrix deposition B-amyloid precursor protein Alzheimer's disease Lactoferin

* Modified fiom Strickiand et al., 1995; Gliernann et al., 1998; Hussain et al., 1999 Chylomicron reminunt catabolisrn

In vivo experiments by Wilhow. Hen and colleagues using transgenic and knock- out mice displaying reduced LRP expression have been instrumental in demonstratinp the role of LWin chylomicron remnant clearance (Willnow et al.. 1994: Willnow et al..

1993). Noteworthy. is the study by Rohlman et al. (1998) using the CreAoxP recombinase system to speci fically inactivate the LRP gene in mouse hepatocytes in vivo.

While inactivation of hepatic LRP had no effect on chylomicron remnant catabolism in wild type mice. because these animais have functional LDLR inactivation of hepatic

LRP in LDLR knock out rnice resulted in a substantial increase in systemic levels of c hylomicron remnants. Collectively. these experiments indicate that the LDLR and the

LRP both participate in the clearance of chylomicron remnants from the circulation by the liver.

In the secretion-recapture model. chylornicron remnants enter the sinusoidal space and are enriched by hepatocyte-secreted apo-E and possibly hepatic lipase (HL). which aid in enhancine binding of chylornicrons to the LRP (Brisiegel et al.. 1989: Kounnas et

al.. 1995). In addition. lipoprotein lipase (LPL) can also mediate uptake of remnants by

binding to both lipoproteins and the LRP (Beisiegl et al.. 1991). Heparan sulfate

proteoglycans (HSPG)have recently been recognized as important macromolecules in

LRP rnediated uptake of remnants. HSPG can bind to apo-E. LPL and HL and are

beiieved to play a role in sequestering chylornicrons in the space of Disse. making hem

available for LW-mediated intemaiization (Mahiey and Ji. 1999: Yu and Cooper. 2001). Proteinase catabolism

The LRP binds and intemalizes a wide range of proteinase and proteinase:inhibitor complexes and is likely important in preventing their accumulation in the extracelldar space and subsequent inappropriate proteinase-induced activation of the coagulation, fibrinolytic and complement cascades (Strickland et al., 1995; Giliemann.

1998). A wide range of proteinase-serpin complexes are thought to be removed from the extracellular space by the LRP. although the clearance pathway is ofien complex. involving multiple ligands and receptors. For exarnple, it has been demonstrated that the

LRP has a role in the removal of the serpin plasminogen activator inhibitor-l (PAI-1) in complex with either tissue-type plasminogen activator (@A) or urokinase-type plasrninogen activator (@A) (Nykjaer et al., 1992). The LW also mediates the uptake of pro-uPA. uPA and tPA (Kounnas et al., 1993; Warshawsky et al.. 1994) and it is thought that plasminogen activators also contribute to the binding of PAL1 complexes to the

LRP. At the ce11 surface uPA is primady found bound to the uPA receptor (uPAR) and

PAI-1 is able to form a cornplex with uPAR bound uPA. LW mediates the internalization of the PM-I :uPA:uPAR complex via interactions with PM-1 and uPA

(Conese et al.. 1995). This complex regulation of ce11 surface uPA expression may be crîtical for tissue remodeling, particularly during mi_mtion by maiignant cells (Chapman.

1997).

In addition to clearing the circulation and extracellular compartment of

proteinase:serpin complexes, the LRP also mediates the uptake of a2M:proteinase

complexes (Kristensen et al.. 1990). a2M is a 720-kDa homotetramer glycoproteîn found

in the human semm at high concentrations and inactivates endogenous proteinases, such as proteinases involved in coagulation and fibrinolysis, and exogenous proteinases, such as bacterial proteinases, by a unique trapping mechanism. Attack of the "bait region" by a proteinase initiates a pronounced conformational change in azM resulting in the entrapment and inactivation of the proteinase. nie proteinase induced conformational change also unrnasks sites on each subunit of a2M that are recognized by the LRP

(Anderson et ai.. 1995). Upon formation. orzM:proteinase complexes are rapidly removed fiom the circulation with the principal site of clearance being the liver (Chu and Pizzo.

1994).

Exploi farion of L RP by pathogens

Invasion of host cells is an essenrial stage of infection for nurnerous viral. bacteriai and protozoan pathogens. Various pathogens have evoived mechanisms to bind to cellular receptors and exploit RME in an effort to gain access to the interior of host cells. Exploitation of RME is also a rnechanism by which intracellular-acting to'rins cross the plasma membrane of tarpt cells during the initial stage of the intosication process. A number of pathogens as well as bacterial and plant toxins utilize the LWas a gateway for envy into target cells (see Table 1). Consequently. cells and tissues that express high levels of LRP are targeted by these pathogens and toxins. For exampie. as previously discussed. the intracellular-acting bacteriai exotoxin PEA exploits the LWas a portal of entry into host cells (Kounnas et al.. 1992). Systemic PEA targets the liver. causing severe hepatic damage and dysfunction. PEA-mediated liver darnage is a

consequence. at leaa in part. of hepatic cells expressing hi& levels of the LRP.

Similady, circdating malaria sporozoites are targeted to the liver where they rapidly and selectively invade hepatocytes by a receptor-mediated mechanism. The circurnsporomite

protein, which covers the entire surface of the sporozoites, interacts with the LRP

(Shakibaie and Frevert, 1996). The circurnsporozoite protein also interacts with HSPG

and therefore, intemalkation of sporozoites may be mediated by both HSPG and the

LW,in a manner analogous to that seen with chylomicrons.

Repulation

Studies utilizing a wide variety of ce11 types demonstrate LRP expression,

distribution and function are subject to modulation by various cellular events, including

cellular differentiation, cancerous transformation (Y mamoto, et al., 1997) and exposure

to extracellular signaling factors. Cellular mechanisms for modulating LRP expression

include redistribution of functional receptors fiom intracellular pools to the cell surface,

and alterations in LWsynthesis. Hormones, growth factors. cytokines and bacterial

products modulate LRP levels by either or both of these mechanisms. Epidermal growth

factor, insulin. and nerve-growth factor cause a rapid and pronounceci increase in ce11

surface LW expression in vascular smooth muscle cells, adipocytes, and nerve ceils,

respectively (Descamps. et al., 1993: Weaver et al.. 1996; Bu et al.. 1998). In the

hepatocyte derived HepG2 ceil line, dexamethazone treatment increases functional LRP

expression two-fold, which correlates with an increase in the steady-state levels of LRP

mRNA (Kancha and Hussain, 1996). LRP expression was induced during differentiation

of trophoblasts into syncytiotrophoblasts. which was suppressed by CAMP(Gafiels et al.,

1992). Macrophage-colony stimulating factor induced differentiation of monocytes to

macrophages induces enhanced LRP expression (Hussaini et al., 1990; Watanabe et al., 1994). Macrophage LWexpression is rapidly dom-regulated at the functional binding, protein and mRNA levels after exposure to LPS and MF-y (LaMme et al., 1993). MF-y induced macrophage LRP dom-regulation can be diminished by pre-treatment with

TGF-P (Hussaini et al., 1996). In addition. regulation of LRP expression in the breast cancer ce11 line T-47D cmbe increased by estrogen (Li et al., 1998).

In surnmary. the LWis a large rnultifunctional endocytic receptor belonging to the LDLR farnily. The LRP mediates the clearance of accumulating waste ligands from the extracellular space by ME. LRP ligands are cleared from the circulation by the liver. which is a tissue site that expresses high levels of LRP. LRP expression is subject to regulation by a wide range of cellular events, such as exposure to extracellular signaling molecules. Based on the diverse range of ligands that bind to the LRP. the LRP has been implicated in a variety of important physiological processes, such as lipoprotein and protease catabolism. In addition. the LRP is the portal of entry for a number of pathogens and their products. including the subject of this thesis study, the intracelluiar- acting bactenal exotoxin PEA. RATIONALE

PEA is an intracellular-acting bacterial exotoxin that is a of the opportunistic pathogen P. aeruginosu. PEA rnediates its cytotoxic effect by inhibiting protein synthesis via the enzymatic inactivation of EF1 (Iglewski and Kabbat. 1974:

Iglewski et al., 1977). It is known that host tissues and cells display a wide range of sensitivities to PEA-mediated cytotoxicity (Pavlovskis and Shackelford. 1974:

Middlebrook and Dorland. 1977; Saelinger et al., 1977). However. whether cells have the ability to alter their sensitivity to PEA has not been determined. Therefore, the objectives of this study were to determine whether alterations in cellular sensitivity to

PEA can be induced. and if so. to determine the molecular mechanisms that might be responsible for this novel cellular phenomenon.

Cellular sensitivity to PEA is determined, at least in part, by the efficiency with which a ce11 cames out PEA intoxication. which is the multi-step process in which PEA exploits the cellular machinery (receptors. proteases) of the target ce11 to transport a generated enzymatically active toxin fragment to EF2. The constitutive level of expression of the cellular machinery exploited by PEA during intoxication govems the rficiency of the process of intoxication and. in turn. cellular PEA sensitivity (Fendrick et al.. 1992: innocencio et al.. 1993. FitzGerald et al., 1995). However. it is not known whether induced alterations in the Ievel of expression of the cellular machinery exploited by PEA during intoxication can alter cellular sensitivity to PEA. The working hypothesis in this thesis investigation was that induced alterations in the expression levels of specific components of the cellular machiner). exploited by PEA during ,ntoxication are mechanisms by which cellular sensitivity to PEA can be altered by the host. To begin to test this hypothesis we examined whether induced or constitutive alterations in celiular sensitivity to PEA are positively correlated with alterations in the expression levels of a specific component of the cellular machinery exploited by PEA. the LRP. We chose to investigate the LRP for two reasons. Fust because of its importance in mediating the efficient intemalization of PEA the constitutive levels of expression of LRP are a cntical factor in governing cellular sensitivity to PEA (Mucci et al., 1995). Secondly. cellular LRP expression is not static, but instead, is altered in a variety of ceIl types by a wide range of cellular events, suggesting the potential to act in the regulation of cellular PEA sensitivity. CHAPTER 1: MATRIX INDUCED DOW-IUCGULATION OF THE LOW- DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN DECREASES PlUMARY HEPATOCYTE SENSITMTY TO PSEUDOMONAS EXOTOXIN A

Introduction

Pseudomonas aeruginosu is a gram-negative. opportunistic pathogen that can cause life-threatening infections in a compromised host, particularly individuals suffenng from extensive thermal injuries (Holder. 1993). In this patient group, P. aeruginosa

Frequently causes septicemia with a high mortaiity rate (Heggers et al.. 1992: Richard et al.. 1994). due in part to the invinsic resistance of P. aeruginosa to antibiotic therapy

(Handcock. 1986). Several factors have been impiicated in P. aenrginosa virulence. including the 66-kDa secretory toxin. Pseudornonas exotoxin A (PEA). Utilizing a mouse model. it has been demonstrated that PEA is released into the circulation from P.

PEA causes cellular injury by irreversibly arresting eukaryotic protein synthesis

(Iglewski and Kabbat. 1974), which ultimately leads to cell death. Like other

intracellular-acting exotoxins. such as Diphtheria toxin. Shiga toxin and Ricin. PEA

intoxication is a complicated, multi-step process that involves binding to ce11 surface

receptors (Manhart et al., 1984). internalization by receptor-mediated endocytosis (M)

(FitzGeraid et ai ., 1%O), enzymatic activation (Fryhg et al., 1992), intracellular traficking (Chaudhary et ai., 1990), translocation to the cytoplasm, and fmaily ADP- ribosylation and inactivation of elongation factor 2. a critical component of the cellular protein synthesis machinery (Iglewski et ai., 1977). Intoxication begins when PEA binds

to a ce11 surface receptor and enters target cells by RME. The PEA receptor responsible

for this initial stage of intoxication has been isolated fiom mouse liver (Fomstal et al.,

1991) and fibroblasts (Thompson et al., 1991), and identified as the low-density

lipoprotein receptor-related protein (LW)(Kounnas et al., 1992).

The LRP is a large muitifunctional endocytic receptor that is a rnember of the

growing farnily of low-density lipoprotein receptors (Gilemann, 1998). Mann LRP is

expressed on the ce11 surface as a heterodimer cornposed of a-(5 15-kDa) and P-(85- kDa)

chahs that are noncovalently associated (Heaet ai., 1990). The P-chah anchors the

receptor in the plasma membrane and mediates receptor intemalization through two

NPXY intemalization motifs located within its cytoplasmic tail. The ashain is

responsible for binding an extraordinary assortment of structurally and Functionally

diverse ligands. including proteinase inhibitos, proteinase inhibitor complexes. a?-

macroglobulin (azM)growth factor complexes, lipoproteins. infectious agents and toxins

(Gilemann. 1998). The receptor-associated protein (RAP)is a 39-kDa chaperune protein

that binds to the LRP with high afinity and is believed to assis in LRP biosynthesis and

maturation by preventing premature ligand-receptor interactions within the endoplasmic

reticulum (Bu et al., 1995; Wilinow et ai., 1996). The antagonistic properties of RAP

have been exploited experimentaily to prevent binding between the LRP and its ligands,

including PEA (Kounnas et al., 19%). Accumulating experimentai evidence indicates

that the LRP is involved in numerous physiologicai processes, including proteinase and lipoprotein catabolism. tissue remodeling, and cellular growth regdation (StncWand et al.. 1995; Hussain et al. 1999; Willnow, 1999).

It has been demonstrated that cellular LRP expression is subject to modulation by various extracellular signaling molecules (Descamps et al.. 1993; Bu et ai.. 1996; Li et al.. 1998). state of cellular differentiation (Gafvels et al.. 1992) and transformation

(Gonias et al.. 1994). The LRP represents the sole identified portal of entry for PEA, and as such. the level of functional LRP expressed on the ce11 surface is a critical determinant of cellular PEA sensitivity (Mucci et al.. 1995). Due, in part, to different cellular LRP expression levels. marnmalian tissues and cells display a wide range of sensitivities to

PEA (Iglewski and Kabat. 1974; Pavlovskis and Shackelford. 1974; Middlebrook and

Dorland, 1977; Saelinger et al., 1977). Specifically, the observation that the liver is the principal site of injury following systemic exposure to PEA is largely attributable to high levels of LRP expression in hepatocytes and Kuppfer cells (Feidman et al., 1985;

Moesmp et al.. 1992). Wr hypothesize that inducible changes in cellular expression of

LRP represents an important mechanism by which hepatocpe sensitivity to PEA can be regulated by the host. In order to test this hypothesis we have examined LRP expression and PEA sensitivity in primary rat hepatocytes.

Materials and Methods

Primary rat hepatocyte culture

Liver pe@ion

Primary rat hepatocytes were isolated fiom 200 to 250 g male Sprague-Dawley

rats (Charles River. St. Constant, PQ) by a collagenase (Gibco/BRL, Burlington, ON) perfusion method, adapted fiom Thonon et al. (1994). Rats were housed at the Central

Animal Facility, University of Guelph. with constant access to both rodent chow and water. On the day a perfusion was performed. animals were transported to the laboratory. weighed and anesthetized using an intrapentoneal injection of sodium pentobarbital (0.2 mUlOO g of body mass). While anesthetized. the rat's abdominal area was shaved and rinsed wirh 70% ethanol in preparation for surgery. The skin and the musculature were then removed from the abdominal area. exposing the viscera of the peritoneal cavity. The intestines were reflected to fully expose the liver and its blood vessels, the portal vein and the inferior vena cava. A loose 2-0 silk ligature was placed around the infenor vena cava.

The infenor vena cava was then nicked, using iris scissors, postenor to the Ioose ligature.

Small diameter polyethelyne tubing was then inserted into the inferior vena cava through the smail nick. and fastened into place by tightening the pre-positioned ligature. A penstaltic purnp, set at 10 muminute. was used to introduce a blanch solution (Appendix

1). consisting of Hank's Balanced Salt Solution (HBSS) (without cao2 and hig-') containing 1mM EGTA and IOmM HEPES (pH 7.4). to clear the liver of blood and divalent extracellular cations. To allow for outflow of the perfusion solutions From the liver. the portal vein was severed. Afier 10 minutes the blanch solution was replaced with pre-oxygenated ( 15 minutes) pemision medium ( Appendis 1) containing Williams'

E medium. 10 mM HEPES (PH 7.4) and 0.5 rng/ml collagenase. The liver was perfused

with this solution at a 80w rate of 20 mYmh for 15 min. After cutting through the diaphragm. the superior vena cava was clamped with hemostats to ensure that perfusion

medium was exiting the liver solely via the portal vein. Hepatocyte isokution

mer the perfusion was complete, the liver was excised and transferred to a tissue culture containment hood were it was disaggregated with scissors and forceps and filtered through sterile mesh gauze into 50 ml tubes. The suspension was centrifuged at 50 x g

(600 rpm) for 3 minutes to pellet the cells. The cells were then resuspended in 25 ml of attachment medium (Appendix I, Williams' E medium supplemented with 10% fetal calf sem(FCS). 50 units of penicillinfml, 50 pg of streptomycin/mi. 2 mM of L-glutamine.

10 mM of HEPES, M of dexamethasone and 20 units/L of bovine insulin) and added to 24 ml of a density gradient mixture composed of 2 1.6 ml Percollm and 2.4 ml of 1OX

HBSS (Appendix 1). The ce11 suspension and density gradient were mixed by inversion and centrifuged for 10 minutes at 50 x g (600 rpm). The nonparenchymal and nonviable cells were removed with the supernatant. leaving a pellet of viable parenchymal cells. which were then washed in 50 ml of fresh attachment medium. Trypan blue (0.5%) exclusion was used to confirm that cellular viability was at least 90% pnor to plating.

Hepatocyre culture

Hepatocytes were cultured on either collagen Type 1- (10 pglcm'. Vitrogen.

Coilagen Corporation. Paio Alto. CA) or MatrigelTM-coated(IO @m', Coilaborative

Research, Bedford, MA) tissue culture plastic. Matrix coated tissue culture plastic was

prepared 24 hours prîor to plating and incubated at 37OC and 5% CO?. Isolated

hepatocytes were cultured at a concentration of 4x10'' cells/well in 96-well plates

(cytotoxicity studies) and 2x 10' cells/well in 24-well plates (intemalization studies).

Foliowing a two-hour penod in attachment medium, celis were culnired in serum-fke Williams' E medium (Appendix 1), containing 35 mM L-proline and 10 mM pyruvate in addition to the supplements added to attachent medium as described above. Medium. semm and supplements were al1 obtained fiom GibcoBRL. Ail tissue culture plastics was purchased fiom Santedt Inc., St. Leonard, PQ.

Cytotoxic assessrnent of PEA-treated hepatocytes r3weucine incorporarion crssay

PEA and PEA conjugate toxins used in these studies were kindly provided by Dr.

David FitzGerald (National Cancer Institute, National Institutes of Health. Bethesda,

MD). Primary hepatocyte PEA sensitivity was detemined by assaying the inhibition of

protein synthesis. At various tirnes in culture (2, 24 and 48 houn). pnrnary hepatocytes

cultured on a collagen mavix in 96-well plates were treated with various concentrations

of PEA in 100 pl of serum free medium. Cells were exposed to PEA for 2 houn at 37°C.

Following challenge. PEA was removed by aspiration and cells were incubated with 100

pl of medium containing c leucin ci ne at 5 pCi/ml (ICN. Montreal. PQ) for 21 houn.

Radioactive medium was then removed, cells were detached using a trypsin solution and

harvested ont0 filter mats. using a 96-well plate hantester (LKB Wallac. Turku. Finland).

Filter mats were then sealed in a bag with 10 ml of liquid scintillation cocktail.

Incorporated radioactivity was determined with a Betaplate Liquid Scintillation Counter

(LKB Wallac). Data are presented as a percentage of protein synthesis compared with

control cells that were not treated with toxin. PEA sensitivity of Matrigelm cultured

hepatocytes was determined in an identical mamer? except that treatment with

MatnspeneTM(Collaborative Research) was included, in addition to trypsin ueaunent, for ce11 detachment. The inhibition dose 50 (IDso)values, the dose of PEA in ng/ml required to inhibit protein synthesis by 50% cornpared to cells receiving no toxin, were determined for collagen and Matrigelm cultured hepatocytes at 2, 24 and 48 hours. For the determination of transforming growth factor-alpha (TGF-a)-PEA37 sensitivity, primary hepatocytes cultured on collagen were challenged with various concentrations of TGF-a-

PEA37 at 37°C for 1 hour and then processed as described above.

AlamarBlue m~~ay

The viability of PEA treated hepatocytes was evaluated by the Alamar BlueTM assay according to the manufacturer's instructions (Biosource International, Montreai.

PQ). At 2. 24 and 48 hours in culture. collagen cultured primary hepatocytes were

treated with various concentrations of PEA for 2 hours at 37OC. Forty eight hours after

toxin challenge. Alamar Bluem was added to the cultures and incubated for 2 houn at

37OC. The absorbance at 540 and 600 nm of each well was measured using a micro plate

reader (Titenek Multiskan MCC. Flow Laboratones, Mississauga ON). Cellular viability

data are presented as a percentage of control cells that were not treated with toxin.

iMicroscopic evaiuiat ion

Freshly isoiated primary hepatocytes were cuitured (1x1 o6 celldplate) in collagen

coated 60 mm culture dishes and at 2, 24 and 48 hours in culture cells were treated with

100 ng/d of PEA for two hours at 37°C. PEA was removed and replaced with fiesh

serum fiee medium. Forty-eight hours after toxin addition. cells were washed nvice with

5 ml of sterile phosphate buffered saline (PBS) and treated with trypan blue (0.5%) to determine hepatocyte viability. Cells were subsequently photographed using a F301 camera attached to a Diaphot phase contmt microscope (Nikon Instruments. Canada).

Receptor-associated protein protection studies

Recepror-associated prorein isolation

Receptor-associated protein-glutathione S-tramferase (RAP-GST) was prepared

from a pGex-21 expression vector. (a kind gifi fiom Dr. D. K. Strickland. at the

Amencan Red Cross. Rockville. MD) fiom bacterial lysates using a GST purification

module following the manufacturer's instructions (Pharmacia Biotech Baie d'urfe, PQ).

Plasmid DNA (10 pl) was added directly to competent XL 1 Blue E. coli (200 pl). The

DNAIE.coli mixture was first placed on ice for 30 minutes, then in a 37OC waterbath for

7 minutes and finally placed back on ice for an additional 2 minutes. îhis mixture (800

pl) was added to prewarmed (37OC) SOC medium (Appendix II) and placed in a shaking

water bath at 37OC for 43 minutes. One hundred ~1 of this mime was then spread over

an arnpicillin (50 pg/ml) Luria-Bertani (LB) agar plate and incubated at 37OC overnight.

In the moming, plates were placed at 4OC until the afiemoon when single bacterial

colonies were picked into 3 ml of LB broth (Appendix II) containing 50 pg/mI of

ampicillin and placed overnight in a 37°C waterbath. Plasmid DNA was isolated fiom

the E. coli. using a Flexi-prep kit as descnbed by the manufacturer (Pharmacia Biotech)

and resuspended in TE beer (Appendex II). The RAP fragment was excised fiom

pGex-2T using EcoR 1 and BamH 1.

To isolate RAP-GST,glycerol stocks of transfonned cells were used to inoculate

3 ml of ZXYTA medium (Apendk III) and were placed in a shaking water bath at 37OC overnight. The next day. cultures were diluted into 2 L of 2XYTA medium and placed in a shaking water bath at 30°C. When the culture reached an of 1.2 to l.j* IPTG

(Appendix III) was added to a fuial concentration of 0.1 mM and placed back into the shaker for 4 hours. The culture was centrifuged at 7 700 ?r g (8 000 rpm in a Bechan

JA2O rotor) for 10 minutes at 4°C to pellet the cells. which were subsequently resuspended in 100 ml of cold PBS. While on ice, cells were lysed using short busts

from a sonicator. Triton X-IO0 was added to the sonicate to a final concentration of 1 %.

The sonicate was then mixed gently, dlowed to sit on ice for 30 minutes and centrifuged at 12 000 x g (10 000 rpm in a Beckman JA20 rotar) for 10 minutes at 4°C.

The supernatant was applied to the matrix of a drained and washed (20 ml of cold

PBS) Glutathione Sepharose 4B RediPack Colurnn (Pharmacia Biotech) and allowed to

flow through. The matrix was then \vashed three times with 20 ml of cold PBS. To elute

the bound RAP-GST fusion protein. the colurnn was incubated for 10 minutes at room

temperature with 2 ml of Glutathione Elution Buffer (Appendix III). The eluate.

containing the fusion protein was then collected 60m the column. This elution procedure

was repeated a total of three times and the eluates pooled.

A GST Detection Module (Pharmacia Biotech) was used to identi& RAP-GST

fusion proteins via a biochemical assay. Bnefly. diluted samples were incubated in a

provided reaction buffer with the GST substrate 1-cholor-2.4-dinitrobenzene (CDNB)

and glutathione (Appendix III). The formation of the GST enzymatic product CDNB-

glutathione was determined by measuring the change in absorbance at 340 nM. Purified

RAP-GST was also detected on Coomassie Blue stained SDS-PAGE gels. RA P-GST protection

Freshly isolated hepatocytes cultured on collagen coated 96-well plates were cotreated with 100 nghl of PEA and 2500 ng/ml of RAP-GST for 1 hour. Cells were then incubated with [3~leucinefor II hours and processed as described above.

Ligand internalization studies

Human a2M (a generous gift from Dr. S. L. Gonias, University of Virginia

Charlottesville. VA) was converted to its receptor-recognized state (a2M*)with 200 nM methylamine HCl. and iodinated with '"1 (Amersharn Life Sciences. Oakville. ON) using

Iodobeads as described by the manufacturer (Pierce Chernicals Company, Rockford. IL.).

For ligand intemalization studies. prirnary hepatocytes were cultured as described above and. at various times in culture (2. 24. 48 hours), were washed in Earle's Balanced Salt

Solution (EBSS) (Gibco/BRL) containing 10 mM HEPES and 1 mg of bovine semm albumin per ml (pH 7.4) (incubation medium), afier which incubation medium containing

1 nM 12S~-a2~*was added for 2 hours at 37°C. Afier ligand removal, hepatocyres were washed with cold EBSS containing 10 mM HEPES @H 7.4) and then detached after treatment with a trypsin solution for 30 min at 4°C. Cells were collected, pelleted. lysed and radioactivity determined with a gamma counter. Protein content was determined by the Bio-RadTMprotein assay (Bio-Rad, Richmond. CA). Non-specific internalization was determined by including a 100-fold excess of unlabeled a2MZ.S pecific intemalization was determined by subtracting nonspecific intemalization from total internalization. Data Anaiysis

Al1 data are presented as the mean f standard error of the mean. Data were analyzed by a Student's t-test; p < 0.05 were considered sipificant.

Results

Pnmarv hepatocvte PEA sensitivity

Prîmary hepatocyte sensitivity to PEA was initially examined by visualizing toxin-induced changes in hepatocyte morphology. Photomicrographs of PEA-treated and untreated hepatocytes culnired on a collagen matrix are shown in Figure 2. Hepatocytes cultured for 2 hours were treated with PEA and afier 48 hours in culture displayed an abnormal. rounded morphology when compared to non-treated cells. which displayed typical cellular morphology for hepatocytes cultured on a collagen matrix. Trypan blue uptake was used to determine that PEA treated hepatocytes with altered cellular morphology had Iost membrane integrity and were no longer viable (data not shown). To confirm that freshly isolated hepatocytes are highly sensitive to the cytotoxic effects of

PEA. the Alamar BlueTMassay was used to measure cellular viability following ueatment with various concentrations of PEA (Figure 3). In addition. since inhibition of protein synthesis is the pnmary action that PEA has on target cells. we investigated hepatocyte

PEA sensitivity by measuring [3~leucineincorporation following toxin exposure (Figure

4). These data indicate that fieshly isolated primary hepatocytes are sensitive to PEA in a

dose-dependent manner. In addition. similar to the situation in vivo. fieshly isolated

primary hepatocyte PEA sensitivity is hi&. indicating that these cells possess al1 of the

required cellular machinery to eficiently process and succurnb to PEA. Figure 2. Ce11 morphology of prirnary hepatocytes cultured on collagen type 1 after exposure to PEA. Freshly isolated nt primary hepatoctyes were cultured on collagen coated dishes and at 2. 24 and 48 hours in culture were incubated in serum-fiee medium containing PEA (100 @ml) for 2 hours. Phase contrast photogaphs of hepatoctyes 48 hours afier toxin treatment are presented (ZOX magnification). Control PEA Figure 3. Viability of primary hepatocytes cultured on collagen type 1 exposed to PEA.

Freshly isolated rat primary hepatoctyes cultured on collagen coated 96-well plates for 2

(open circles). 24 (open squares) and 48 (open triangles) hours were incubated in senun- free medium containing various concentrations of PEA for 2 hours. Forty-eight hours afier toxin treatment. hepatoctye viability was determined using the Aiamar BlueN assay and expressed as a percentage relative to control celis that did not receive toxin. The results shown are fiom a representative experiment that was repeated three times. Each data point represents the mean r standard error of six determinations. PEA (nglml) Figure 4. Protein synthesis inhibition by PEA in primary hepatocytes cultureci on a collagen type 1 matrix. Freshly isolated rat primary hepatoctyes cultured on collagen coated 96-well plates for 2 (open circles), 24 (open squares) and 48 (open triangles) hours and were incubated in senun-free medium containing various concentrations of PEA for

2 houn. Protein synthesis levels were determined by measuring the incorporation of

[3~]leucineinto cellular protein for 71 hours and are expressed as a percentage relative to control cells that were not challenged with toxin. The results show are Erom a representative experiment that was repeated four times. Each data point represents the mean + standard error of six determinations. tn e- cnn

PEA (nglml) In contrast to the situation observed with fieshly isolated primary hepatocytes, cells in extended culture (24 and 48 hours) on a collagen mahwere markedly resistant to PEA. Morphologically, toxin treated and non-treated hepatocytes were similar (Figure

2) and, even at high concentrations of PEA. were still largely viable (Figure 3). nie effect of PEA on protein synthesis inhibition was also decreased in hepatocytes culnired for 24 and 48 hours when compared to fieshly isolated hepatocytes (Figure 4) (Table II).

However. the inhibitory effect of PEA on protein synthesis was greater in hepatocytes cultured for 24 and 48 hours than on cellular viability. as measured by Alarnar Bluem, in these cells.

To assess whether the observed change in PEA sensitivity of primary hepatocytes cultured on collagen is simply the result of time spent in culture or. whether it is a result of matrix-inducible changes. we investigated PEA sensitivity of primary hepatocytes cultured on Matrigel? Results shown in Figure 5 and Table U demonstrate that PEA sensitivity of hepatocytes cultured on this matrix did not change substantiaily over 48 hours. indicating that the change in PEA sensitivity in pnmq hepatocytes is due to cellular changes induced by the collagen matrix and not rnerely a response to time in culture or to processes initiated by the act of tissue dissociation.

Effect of RAP on pnmw hepatocyte PEA sensitivitv

The LRP is the ce11 surface receptor implicated in the binding and internalization of PEA. It has been previously demonstlated that RAP, a naturd LRP aatagonist, prevents PEA binding to the LRP and subsequently decreases PEA cytotoxicity (Kounnas et al.. 1992). To verifi the importance of the LRP in PEA intoxication in primary Table II. IDsovalues for primary hepatocytes in extended culture on collagen type 1 and

Fold Change in Matrix Time (hours) PEA Sensitivity

Collagen type I (n=4) 1

' Çigniticantly different from 2 houn. p < 0.05. using a Sntdmt's t-test. b Significantly different from 24 hours, p < 0.05, using a Student's t-test. ' Significantly different from 2 houn. p < 0.05. using a Student's r-test. Figure 5. Protein synthesis inhibition by PEA in pnmary hepatocytes cultured on

MauigelTM. Freshly isolated rat primary hepatoctyes culnired on MatrigelTM-coated96- well plates for 2 (open circles). 24 (open squares) and 48 (open triangles) hours were incubated in sem-fiee medium containing various concentrations of PEA for 2 hours.

Protein synthesis levels were determined by measuring incorporation of ['~lleucineinto cellular protein for 21 houn and are expressed as a percentage relative to control cells that were not challenged with toxin. The results shown are from a representative experiment that was repeated three rimes. Each data point represents the mean + standard error of six detenninations. PEA (nglml) hepatocytes, RAP-GST was used as a cornpetitor for PEA cytotoxicity. As indicated in

Figure 6, a W-GST hision protein decreased PEA toxicity in primary hepatocytes.

These results demonstrate that primary rat hepatocytes use the LRP in the process of PEA intoxication and that functional antagonism of the LRP reduces PEA susceptibility in this ce11 type.

Functional LRP expression in primaw hepatocytes

Our findings that PEA intoxication is mediated by the LW lead us to investigate whether down-regulation of functional ce11 surface LRP expression represents a mechanism by which PEA resistance is increased in primary hepatocytes. As shown in

Figure 7 hepatocytes cultured for 24 and 48 houn on collagen intemalize 2.5-fold and

7.3-fold less a2Mt.a LRP sprcific ligand. This result extends our initial report indicating

that steady-state LRP expression is down-regulated at the protein and mRNA level. as

measured by western and northem blot analysis. in pnmary hepatocytes cultured on a

collagen matria. (Schrnoelzl et al.. 1998: Benn. 1999). In addition. these results extend

the correlation between functional LRP expression and hepatocyte PEA sensitivity.

Susceptibilitv of primarv hepatocvtes to TGF-a-PEA37

Although the correlation between decreased PEA sensitivity and decreased LRP

expression is hi& in primary hepatocytes. additionai cellular components utilized by

PEA during the intoxicarion process (processing factors. cellular targets) rnay also be

altered. Such changes rnight enhance or diminish any protective effects mediated by

dom-replation of LRP expression. Therefore we have also investigated primary Figure 6. Effect of RAP-GST on PEA-mediated cytotoxicity in pnmary hepatocytes.

Following two hours in attachment medium, hepatocytes were treated with both PEA

(100 ng/mI) and RAP-GST (2500 ngiml) for 1 hour. Protein synthesis levels were determined by measuring incorporation of [3~]leucineinto cellular protein for 21 hours and are expressed as a percentage relative to control cells that were not challenged with toxin. Each data point represents the mean f standard error of three separate experiments. * indicates significantly different @ < 0.05) when compared to non RAP treated cells using a Student's t-test. O RAP-GST Figure 7. Primary hepatocyte a2Mt intemalization. Pnmary hepatocytes cultured on a collagen tye 1 matrix were washed and incubated at 37OC for 2 hours with 4 nM '"I- a2M*.in the presence or absence of excess unlabeled ligand. After ligand removal, cells were washed. Iysed and radioactivity determined. The results are means k standard enor of tripiicate samples fiom three separate expenments. * significantly different (p < 0.05) from prirnary hepatocytes after 2 hotus in culture using a Student's t-test. ** significantly different (p < 0.05) fiom primary hepatocytes afier 24 hours in culture using a Student's t-test. 2 24 48 Tirne (hours) hepatocyte sensitivity to a conjugate PEA toxin (TGF-a-PEA37) that utilizes the epidermal growth factor receptor (EGFR)for cellular intemalization. By bypassuig the

LRP for toxin entry the importance of LRP levels in mediating PEA sensitivity cm be addressed indirectly. Binding expenments, using '"1-EGF, indicate that prirnary hepatocytes cultured on a collagen matrix for 24 hours no longer express high affuiity

EGF receptors (Wollenberg et al. 1989). Based on these changes in EGF receptor expression. we would expect that primary hepatocyte sensitivity to TGF-a-PEA37 would rapidly decrease over 24 houn in culture. but that additional time in culture would not substantially decrease sensitivity Mer. As show in Figure 8. freshly isolated hepatocytes are initially highly sensitive to this conjugate toxin but sensitivity is rapidly and rnarkedly decreased with time in culture. As predicted. the sensitivities for hepatocytes at 24 and 48 hours is nearly identical while these cells continue to display significantly different sensitivities to native PEA (Figure 4). Collectively thesr results indicate that the different dynamics of change in EGF receptor and LRP expression are responsible for differences in the pattern of change in sensitivity to TGF-a-PEA37 and native PEA.

Discussion

Numerous intracellular-acting bacterial exotoxins exploit eukaryotic cellular processes to effectively deliver a catalytically active toxin hbment into the cytoplasm of cellular targets. Toxins acting in this manner initiate the intoxication process by binding to specific ce11 surface receptors thus allowing efficient transit across the plasma membrane via RME. Becaw of their central role in the process of intoxication. these Figure 8. Conjugate PEMGF-a toxin (TGF-a-PEA37) sensitivity of primary hepatocytes. Ptimary hepatocytes cultured on collagen coated 96-well plates for 2 (open circles). 24 (open squares) and 48 (open triangles) hours were incubated in serum-fiee medium containing various concentrations of TGF-a-PEA37 for 1 hour. Following toxin exposure? cells were incubated with medium containing [3~]leucinefor 21 hours. The graph shown is fkom a representative experiment that was repeated three times. Each data point represents the mean t standard error of six determinations.

"toxin recepton" dictate cellular sensitivity and tissue tropism. 'Ihis is clearly the case for PEA. where the level of expression of the LRP plays a central role in determining cellular PEA sensitivity. as well as dictating which tissues will undergo PEA-mediated damage itz vivo. Since the liver is an important LRP expressing organ (Moestrup et al..

1992). responsible for clearing nurnerous LRP ligands fiom the circulation, it is not surprising that hepatic tissue is a primary target of PEA (Saelinger et al.. 1977). Despite the long-standing recognition that systemic PEA targets the liver. few studies have

investigated PEA susceptibility in cells of hepatic origin. Utilizing in vitro assays to

measure cytotoxicity and inhibition of protein synthesis. we report here that freshly

isolated primary rat hepatocytes are highly sensitive to PEA in a dose-dependent manner.

In addition. the ability of RP-GST to reduce the cytotoxic effects of PEA confirms that

PEA utilizes the LW for intoxication in primary hepatocytes. The observation that

freshly isolated primary hepatocytes are highly sensitive to PEA and capable of

intemalizing the LRP-specific ligand a2M* demonstrates that these cells express high

levels of functional ce11 surface LW. These results confirm earlier studies (Feldman et

al.. 1985). reponing that LW expression in freshly isolated prima* hepatocytes is hi&

and funher strenghens the observed positive correlation between LRP expression and

cellular PEA sensitivity (Mucci et al., 1995). Collectively. our results indicate that

freshly isolated primary hepatocytes are a relevant in vitro mode1 system for examining

issues related to cellular PEA sensitivity.

The extracellular matrix is known to be an important extracellular signaling factor

that regulates nurnerous cellular functions. Collagen is a component of the extracellular

matriv that is known to alter primary hepatocyte gene expression (Benn et al., 1999) and LW expression in particular (Schmoelzi et al., 1998). This decrease in LRP is substantially reduced on MatrigelW. Culture on a collagen matnv markedly decreased primary hepatoctye PEA sensitivity. However, aitered PEA sensitivity was not observed when hepatocytes were plated on a Matrigelm manut. indicating that the reduced toxin sensitivity observed in collagen cultured hepatocytes is matrix-dependent and not merely a temporal effect. These findings demonstrate that cellular PEA sensitivity is not static but. instead. subject to modulation by various extracellular signaling molecules.

Modulation of cellular Shiga toxin sensitivity has aiso been demonstrated. with increased endotheliai sensitivity taking place following treatment with LPS or pro-inflammatory cytokines (Tesh et ai.. 199 1: Rameogowda et al., 1999).

Interestingly our results indicate that hepatocytes cultured on collagen for 24 and

48 hours were more resistant to the cytotoxic effects of PEA than to the PEA-induced inhibition of protein synthesis. Despite gaining entry and inhibiting protein synthesis.

PEA did not induce ceIl death, indicating that sensitivity to these two processes may be partially distinct in prima* hepatocytes. These results are in agreement wvith previous studies demonstrating cellular resistance to PEA-mediated cytotoxicity. even when PEA- mediated protein synthesis inhibition is present (Morimoto and Bonavida 1992;

Brinkmann et al.. 1995). Differences in hepatoctye sensitivity to protein synthesis inhibition and cytotoxicity rnediated by PEA may also occur in vbo as recently demonstrated by Schümann et al. (L998). The authors reported that PEA-mediated liver damage was due to the combined effects of protein synthesis inhibition and the apoptosis- inducing signaling factor tumor necrosis factor (TNF). The authos clearly demonstrated

PEA-mediated protein synthesis inhibition sensitized murine livers to the cytotoxic effects of TNF. Further studies are clearly required to investigate the cytotoxic potential of TNF on PEA-treated primary hepatocytes in an effort to elucidate additional, non-LRP dependent mechanisms by which hepatocytes cultured on collagen matrix may increase their resistance to PEA.

Studies performed by FitzGerald et al. (1995) and Willnow and Hrn ( 1994) were instrumental in confirrning the role of LRP in PEA intoxication by demonstrating that cells harboring defects in the expression of functional ce11 surface LRP are highly resistant to PEA. As predicted in these studies, cells lacking LRP would have a reduced rfficiency in PEA internalization and consequently would display increased toxin resistance. The opposite scenario also appears to be me, as recently established by

Avramogh et al. (1998). who dernonstrated that PEA sensitivity cm be restored in a LW deficient ce11 line by expressing functional chicken LWon the ce11 surface. Because of its importance in determining cellular PEA sensitivity. we investigated levels of LW expression in primary hepatoctyes cultured on a collagen matrix in an aKempt to identi. the mechanism by which these cells reduce their PEA sensitivity. Both LRP espression and fùnction were decreased. strongiy sugpsting the lower levels of LRP expression are likely to be a mong convibuting factor to the decreased PEA hepatocyte susceptibility.

We hypothesize that regdation of cellular LW expression in target cells

represents an important mechanism by which PEA sensitivity can be modulated by the

host. In support of this. we recently demonstrated that LPS-induced dom-regdation of

fùnctional LRP expression correlated with increased PEA resistance in a ber derived

macrophage-like ce11 line (Chapter 2). Due to the high susceptibility of hepatocytes to

the effects of PEA Ni vivo and in vitro. factors that alter LRP expression in the liver may potentially have significance in the host response to P. aeruginosa infection. As demonstrated here, a decrease in LRP expression in piimary hepatocytes correlated with a profound decrease in PEA sensitivity. It is probable that the production of various LRP regdatory factors rnay be initiated in response to P. aeruginosa. consequently altering cellular PEA sensitivity during infection.

Acute liver injury by various insults, including toxins and viruses, results in substantial loss of the hepatocyte cell population by necrosis or apoptosis. In addition, damage of this type alters the quantity and composition of the hepatic extracellular matrix, which is a critical signaling factor controlling hepatocyte fùnction (Burt. 1993).

Alterations to the hepatic extracellular matrix are important determinants for initiating hepatocyte responses to injury. One such response is liver regeneration, where hepatocytes npidly undergo multiple rounds of ce11 division in an attempt to replace dmaged cells. The extracellular matrix plays a role in both initiation and termination of liver regeneration, by influencing hepatocyte differentiation (Martinez-Hernandez and

Amenta, 1995). Regenerating hepatocytes express a different profile of genes than do hlly differentiated hepatocytes. including altered expression of members of the LDL receptor gene family (Bocchena et al., 1993). Although it is not known whether LRP expression is altered during liver regeneration. our in vitro mode1 using primary hepatocytes cultured on a collagen matrix. which mimics the in vivo regenerating hepatocyte in many respects (&!M et al., 1999), suggests that this may indeed be the case. LRP down-regulation in growing hepatocytes during PEA-induced liver damage

rnay provide a nirvi.d advantage to regenera~ghepatic tissue by reducing the

efficiency with which hepatocytes intemalize PEA. CHAPTER 2: ENHANCED MACROPHAGE RESISTANCE TO PSECraOMONAS EXOTOXM A IS CORRELATED WITH DECREASED LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN EXPRESSION

Introduction

Pseudomonas exotoxin A (PEA) is an extracellular virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa. PEA irrevenibly inhibits eukaryotic protein synthesis by ADP-ribosylating cytosolic elongation factor 2. leading to ce11 death (Iglewski et al.. 1977). PEA is secreted as a 66-Da pro-enzyme. which is cmensively modified by target cells in order to generate and deliver the activated 37-kDa rrizymatic Fragment to the cytoplasm (Ogata et ai., 1990). The initial step in the intoxication process involves PEA binding to specific cell surface receptors followed by receptor-mediated endocytosis (RME) (Manhart et al., 1984). A ce11 surface PEA binding protein was isolated from mouse fibroblasts (Thornpson et al.. 1991) and liver cells (Fomstal et al.. 1991) and subsequently identified as the low-density lipoprotein receptor-related protein (LRP) (Kounnas et al.. 1992). The isolation of LRP deficient cells that are highly resistant to PEA confirmed the role of LRP as a cellular PEA receptor that mediates PEA cytotoxicity (Willnow and Hem 1994: FitzGeraid et al..

1995). Recently. Avramoglu et al. (1998) have restored PEA sensitivity in a LRP- deficient CHO ce11 line by expressing functional chicken LRP.

The LW is a large cell-surface glycoprotein belonging to the LDL receptor gene family. The LWis synthesized as a 600 Dapro-receptor. which is post-nanslationally processed into 5 15-kDa and 85-kDa chahs that remain associated through non-covalent interactions (Herz et al., 1990). nie heaw chah is expressed entirely on the ce11 surface and is capable of binding an emordinary range of stnicturaily and functionally diverse ligands including lipoproteins. lipases, proteinase inhibitors, proteinase inhibitor complexes, a-macroglobulin (azM) growth factor complexes, pathogens and PEA

(Kristensen et al.. 1990; Strickland et al., 1990; Hussain et al.. 1991; LaMarre et al..

199 1 ; Bu et al.. 1992; Kounnas et al., 1992; Hofer et al.. 1994: Orth et ai.. 1994; Kounnas et al.. 1995: Shakibaei and Frevert, 1996). The 39-kDa receptor-associated protein

(RAP) CO-purifieswith the LRP and acts as an antagonist for al1 ligands binding to the

LRP. including PEA (Kounnas et ai., 1992). It has been proposed the LRP may play a role in such diverse physiological processes as tissue remodeling, cellular growth regulation and the rnetabolism of lipoproteins and proteinases (Strickiand et al.. 1995:

Hussain et al. 1999: Willnow. 1999).

An important determinant of cellular PEA sensitivity is the constitutive level of functional LRP expressed on the ce11 surface of different target cells. Mucci et al. ( 1995) discovered that a positive correlation exists between LRP expression and PEA sensitivity: cells constitutively expressing low levels of the LRP are highiy resistant to PEA. Due in part to their different LRP expression levels (Moestnip et al.. 1992). mamrnalian tissues and cells display a wide range of sensitivities to PEA (Pavlovskis and Shackelford. 1971:

Iglewski et al., 1977; Middlebrook and Dorland. 1977: Saelinger et ai.. 1977). In particular, the observation that the liver is the rnost cornmon site of damage from systemic PEA (Pavlovskis and Shackelford. 1974; Iglewski et al.. 1977; Saelinger et al..

1977) is largely attributable to hi& levels of cellular LRP expression in hepatocytes and

Kupffer cells (Feldrnan et al.. 1985: Moestmp et al., 1992).

Various signaling molecules such as hormones (Descamps et ai., 1993: Li et al..

1998) and growth factors (Weaver et al., 1996; Bu et al.. 1998) have been shown to alter LRP levels in diverse ce11 types. Macrophage LRP expression is subject to regulation by specific cytokines and bacteriai products. It was previously reported that lipopolysaccharide (LPS) and interferon-y markedly decreased LRP expression at the mRNA. antigen and functional levels in the RAW 261.7 macrophage-like ce11 line and in bone marrow macrophages (LaMarre et al.. 1993: Hussaini et al., 1996). In studies presented in the previous chapter. we established that matrix-inducible changes in LRP expression alter cellular PEA sensitivity in hepatocytes. We next wished to determine wvhether changes in LWexpression induced directly by bacterial factors could aiso dter cellular PEA sensitivity We hypothesize here that inducible changes in cellular expression of LRP represent an important mechanism by which cellular susceptibility ro

PEA is regulated by the host. This should be particularly me for decreases in LRP expression that are induced by signaling molecules expected to be present when the risk of PEA intoxication is hi&. In order to test this hypothesis we have examined the effect of LPS on LRP expression and toxin susceptibility in cells of macrophage origin that are sensitive to PEA.

Materials and Methods

Ce11 culture

An HS-P macrophage-like ce11 line was recently isolated fiom a spontaneous histiocytic sarcoma from the liver of a rat (Yamate et al., 1996) and was chosen for this snidy based on its high sensitivity to PEA and its rnonocyte/macrophage origins. HS-P macrophage-like ceils were culnired in T-75 flasks in RPMI medium, supplemented with

10% heat inactivated fetal bovine senim. 50 units of penicillin per ml and 50 pg streptomycin per ml at 5% CO2, 95% humidity and 37°C. Medium, serum and supplements were al1 obtained hmGibcolBRL. Burlington. ON. Cells were detached using nypsin and passaged every 2-3 days. For experiments. cells were seeded at a density of 4x10~cells/well into 96-well plates (for cytotoxicity assays) or at 2x10~ cells/dish into 60 mm culture dishes (for northem analysis) or at 2x10~cells/well into 24- well plates (for ligand internalization studies) and were incubated overnight before treatments. Al1 tissue culture plastic was purchased from Sarstedt Inc.. St. Leonard. PQ.

Psetrdomonas esotoxin -4 cytotoxicitv assav

HS-P sensitivity to PEA was determined by assaying the inhibition of protein synthesis as described in Chapter 1. Following ovemight incubation in 96-well plates.

HS-P cells were challengeci with PEA in 100 pl of senim fiee medium. Unless otherwise stated. ail experiments involving PEA treatment were perf'ormed at 37°C. Cells were treated either for 24 hours with various concentrations of PEA or with 50 ngml of PEA for various penods of rime. Afier PEA challenge. the toxin-containing medium was removed and cells were incubated with medium containing 3 pCi/ml of [3~]leucine(ICN.

Montreal. PQ) for 21 hours. Radioactive medium was removed. cells were detached using a trypsin solution and harvested ont0 filter mats. Incorporated radioactivity was determined with a Betaplate Liquid Scintillation Counter (LM3 Wallac, Turku. Finland).

Data are presented as a percent of protein synthesis compared with cells that were not challenged with toxin. RAP and LPS protection

Receptor-associated protein-giutathione S-transferase (RAP-GST) was obtained using the pGEX expression vector. (a kind gifi fiom Dr. D.K. Strickland, at the Amencan

Red Cross. Rockville, MD) From bacterial lysates using a GST purification module following the manufacturer*s instructions (Pharmacia Biotech, Baie d'Urfe. PQ). as described in Chapter 1. Afier ovemight culture. HS-P cells were CO-treatedwith 50 ngfml of PEA and various concentrations of RAP-GST for 1 hour. CelIs were then incubated with ['~lleucine for 21 houn and then harvested as descnbed above. HS-P cells were pre-treated for 24 hours with various concentrations of LPS (Escherichia coii 0127:B8.

Sigma. Oakville, ON) or with 100 ngfml of LPS for various periods of time. Following pre-treatment. cells were chdlenged for 2 hours with 100 ng/ml of toxin and then processed as described above. HS-P cells were also pre-treated for 24 hours with 100 n@ml of LPS and then cMlenged for 2 hours with various concentrations of PEA. The

ID5o values. the dose of PEA in ng/ml required to inhibit protein synthesis by 50% compared to cells receiving no toxin, were determined for both non-treated and LPS treated HS-P cells. In short-term pulse expenments. cells were treated for 15 minutes with 1000 ndml of PEA then washed three times with fiesh medium in order to remove

unbound toxin from the ce11 surface. Cells were then incubated with ['~lleucine for 21

hours and hwested as described above. RNA isolation and northem blot analvsis

Preparation of inserts

Plasmid DNA was transformed into competent XLl Blue E. coli and isolated using a Flexi-prep kit as descnbed in Chapter 1. The 400 bp rat LWinsen (kindly provided by Dr. G. Bu. Washington University, St. Louis. MO) was exciseci from pBluescriptSK-LRP using the restriction enzymes EcoRl and BamH1. The 178 bp 7s insert (kindly provided by Dr. Allan Baimain, Onyx Pharmaceuticals. Richmond. CA) was excised frorn pB1uescrîptSK'-7s by restiction digestion using BamH1. Restriction digests were perfomed in the appropriate buffen for 2 houn at 37°C. The genented fragments were separated on 1 % TBE (Appendix II) agarose gels run for 1 hour at 100 volts. The insert band \vas observed under UV radiation. removed fiom the gel with a clean scalpel and placed in dialysis tubing. The insert DNA was then electroeluted at 215 volts for 30 minutes using O.OjX TBE buffer. The DNA was then precipitated with 2 volumes of 100% ethanol and 1/10 volume of 3M sodium acetate (pH 5.2) overnight at -

70°C. The precipitated DNA was pelleted by centrifùgation at -1OC for 45 minutes at 15

000 s g ( 13 000 rprn). The DNA pellet was then washed with 500 pl of cold 70% ethanol and centrifuged at 15 000 x g for 15 minutes at J°C. The insert pellet was resuspended in

50 pl of DEPC-A water (Appendix IV) and quantified using a LN-Spectrophotometer at

360 m.

Tom! RV.4 isolation

HS-P cells were cultured in 60 mm culture dishes as described above and then ueated with 100 ndml of LPS in serum fiee medium. At specified times, cells were washed a single tirne in ice-cold PBS, scraped directly into 1 ml of Trizolm reagent

(Gibco/BRL) and then transferred to a 1.5 ml rnicrofbge tube. Chloroform (200 pl) was added and the samples were mixed by inversion and then refrigerated at J°C for 30 minutes. nie samples were centrifuged at 15 000 x g (13 000 rpm) for 15 minutes at 4°C

(Biohige 13R Bavter Scientific. Osterode, Germany) to facilitate phase separation. The upper phase was removed. placed in a clean 1.5 ml tube and an equal volume of cold (-

20°C) isopropanol was added to the ~amples.The ~ampleswere placed at -20°C and the

RNA allowed to precipitate ovemight. The samples were then centrifbged at 7 000 x g for

10 minutes at 4OC to pellet the RNA. The RNA pellet was washed with 500 pl of 70% ethanol and then centrihiged at 7 000 x g for 5 minutes at 4OC. Residual ethanol remaining in the tube was removed using a drawn pipet and the pellet was allowed to air dry. The pellet was redissolved in 16 pl of DEPC-A water and incubated at SOCfor 10 minutes to assist with RNA solubilization. Samples were diluted 1:800 into DEPC-A water and quantitated using ultraviolet spectroscopy at an absorbance of 260 nm. Al1 absorbance measurements used DEPC-A water as a blank. Isolated RNA samples were stored at -80°C.

Gel electrophoresis

Twenty ug of total RNA from cells at each time point were separated by electrophoresis in 0.8% (wh) formaldehyde/agarose gels. Samples of RNA were

prepared for electrophoresis by combining 20 ug of total RNA with 17.5 pl of a master

mix solution. which was composed of 2 p1 SX MOPS buffer (Appendix IV). 3.5 pl 37%

(vh) formaldehyde. 10 pl 100% formamide, 2 pl loading buffer and 0.2 pl ethidium bromide (stock 0.2 mgml). Mer the master rnix was added the samples were denatured in a 65°C water bath for 15 minutes and then immediately placed on ice for 10 minutes.

Samples were loaded and the gel was nui in IX MOPS buffer at 12- 15 volts for 16 houn.

.i\fier electrophoresis the gel was photographed under ultraviolet light to ensure consistent RNA load and to confïrrn the absence of degradation. The gel was then placed in 0.05 N NaOH for 15 minutes to facilitate subsequent -fer of the RNA to a nylon membrane (Hybond N. Amersham Life Sciences. Oakville, ON) by capillary transfer in

5X MOPS buffer. After 48 hours of capillary transfer. the membrane was UV cross- linked at 120mJicm2 using a FB- WXL- 1000 W Crosslinker (Fisher. Scientific.

Xepean. ON). Membranes were stored in plastic wap. in the dark and at room temperature.

Probe prepararion

"P-labelled cDN.4 probes were prepared by combining 50 ng of insen cDNA with DEPC-A water to a final volume of 42 pl. The cDNA insen was then boiled for 5 minutes and added to a RediprimeOQ random primer labeling mixture (Arnenham Life

Sciences) containing dAV. dTTP. dGTP. random prirners and Klenow enzyme. Fi@ uCi of radioactive nucleotide [a-3'~]d~~~(10 uCi/pl. specific activity of 3 000 uCi/mmol. Amersharn Life Sciences) was then added to the DNA Rediprimd mixture and placed at 37OC for 10 minutes to facilitate maximal radioactive nucleotide incorporation. The incorporation reaction was teminated by the addition of 5 pl of 0.2 M EDTA (pH 8.0). The radioactive labeled probe was then applied to the top of a Quick

Spin Column (BioRad Laboratories. Richmond, CA) containing 1.5 mi Sephadex G-50

(Appendix IV). Three hundred pl of TEN @H 8.0) (Appendix IV) was applied to the colurnn and then centrifuged at 700 x g (2 000 rpm) for 2 minutes. One pl was removed from the eluate (approximately 351 pl) and radioactivity was measured using a Delta 300 liquid scintillation counter (Tracor Analpic).

Hvbridiznriun

Following UV crosslinking. membranes were incubated with 5 ml of prehybridization solution containing 0.5% SDS. 6X SSPE (Appendix IV). 20 &ml salmon sperm DNA (denatured by boiling for 5 minutes). 5X Denhardt's reagent

(Appendix IV) and 50% formamide. Prehybridization was performed in 12-inch glas hybridization tubes for 1 hour at 42°C using a Rotating Hybridization Incubator (Mode1

1100. Robbins Scientific. Sunnyvale. C.4). The labelled cDNA probes were added to 5.0 ml of fresh prehybridization solution. boiled for 10 minutes and then placed on ice. The mixture and the mrmbrane(s) were incubated in I --inch glass hybridization tubes for 12-

16 hours at 42'C in a rotating hybridization incubator.

After hybridization membranes were nvice subjected to a low stringency wash for

15 minutes at 42°C in 100 ml of a solution containing 1X SSC (Appendix IV). 1% SDS

and then to a single hi& stringency wash for 70 minutes in 100 ml of 0.1 x SSC. O. 1%

SDS at a temperature of 6j°C. Afier detection the labeled probe was removed from the

membranes using 500 ml of boiling 0.1% SDS applied directly to the membrane that

were then rinsed in 2X SSC. As a control for load. stripped membranes were rehybridized with a radiolabeled probe for murine 7s RNA and subjected to two low stringency washes as described for LRP and then to a higher stringency wash for 20 minutes at 55°C using 100 ml of O.Ix SSC, 0.1% SDS. A GS250 Molecular Imager

(Bio-Rad), located in the Clarke Chalmers Molecular Imaging Facility. Department of

Biomedical Sciences. University of Guelph, with Molecular Analyst Version 2.1 software

(Bio-Rad) was used for signal detection and quantification of northem blots.

Ligand internalization studies

Human a2M was converted to its receptor-recognized conformation with 200 nM methylarnine HCl. Activated uzM (azM*) was radiolabelled with "'1 (Arnersham Life

Sciences) using Iodobeads as described by the manufacturer (Pierce Chernical Company.

Rockford. IL). The specific activity was 1000-2000 cpdng. Ligand uptake studies were conducted as previously described (FitzGerald et al., 1993). Bnefly, HS-P cells were cultured as described above and then treated with 100 ng/ml of LPS for 21 hours. LPS- treated and non-treated cells were then washed in Earle's Balanced Salt Solution (EBSS)

(Gibco/BRL) containing 10 mM HEPES, I mg/ml bovine serum albumin. pH 7.4

(incubation medium) and then 1 nM of "'1-a2~*in incubation medium was added for 2 hours at 37OC. After ligand rernoval. cells were washed in cold EBSS containing 10 mM

HEPES. pH 7.4 and then treated with a trypsin solution for 30 min at 4°C. Detached cells were subsequentiy collected. pelleted by centrifugation. lysed and the radioactivity determined in a y-counter. Protein content was determined by the Bio-RadTMProtein

Assay (Bio-Rad). Non-specific internalization was determined by including a 100-fold excess of uniabeled azM* in the incubation miunire. Specific intemalization was determined by subtracting non-specific intemalization from total intemaiization.

Data Anahsis

Al1 data are presented as the mean + standard error of the mean. Data were analyzed using a Student's 1-test: p < 0.05 was considered significant. Data were also malyzed by an ANOVA with a lem significant diFerence (LSD)cornparison of means.

Results

HS-P PE-4 sensitivity

Using a ['H]lcucine incorporation assay to measure inhibition of protein synthesis the cytotoxic effect of PEA on HS-P macrophage-like cells was detemined. Results from expenments where HS-P cells were treated with various concentrations of PEA for two hours are displayed in Figure 9. and indicate that HS-P cells are sensitive to PEA in a dose-dependent manner. To examine the effect that duntion of tosin exposure has on macrophage PEA c'oxicity. HS-P cells were treated with 50 ngml of PEA for the indicated tirnes (Figure 10). In both cases HS-P cells are clrarly sensitive to PEA. indicating that they possess the required cellular machinery for successful PEA intoxication. including functional ce11 surface LRP. and are a suitable macrophage ce11

Iine to evaluate factors which mi& alter PEA susceptibility. Figure 9. Dose-dependent cytotoxic activity of PEA on HS-P macrophage-like cells. HS-

P cells were cdtured ovemight in 96-weli plates with medium containing 10% sem.

Cells were incubated in sem-free medium containing various concentrations of PEA for

? houn. Protein synthesis levels were determined by rneasuring the incorporation of

[j~lleucineinto cellular protein and are expressed as a percentage relative ro control cells that received no toxin. Each data point represents the mean r standard error of three

separate experiments. PEA (nglml) Figure 10. Time dependent cytotoxic activity of PEA on HS-P macrophage-Iike cells.

HS-P cells were cultured ovemight in 96-well plates with medium containing 10% serum. Cells were incubated in serum-free medium containing 50 ngml of PE.4 for the various time penods indicated. Protein synthesis Ievels were determined by rneasuring the incorporation of ['~lleucineinto cellular protein and are expressed as a percentage relative to control cells that receivec! no toxin. Each data point represents the mean t standard error of threr separate expenments. Time (hours) PEA sensitivitv is decreased bv LPS

We next wished to determine whether treatment of macrophages with LPS modifies their sensitivity to PEA. HS-P cells were pre-treated for 2.1 hours with LPS and then challenged with 100 ngml of PEA for 2 hours. Increasing concentrations of LPS caused a decrease in HS-P PEA sensitivity (Figure 11). The duration of pre-treatment also affected PEA sensitivity: cells exposed to LPS for an increased tirne acquired a ereater resistance to PEA (Figure 12). These results indicate that LPS pre-treatment t decreases macrophage PEA sensitivity in a dose- and time-dependent fashion. To further examine this issue. IDco values. the concentration of PEA required to inhibit protein synthesis by 50% compared to cells which received no toxin. were determined for both

HS-P cells pre-treated with LPS at a concentration of 100 ngml for 2-1 hours and for untreated controls (Figure 13). The IDjo value of HS-P cells pre-treated with LPS was

25.7 = 1.0 nyml. compared to a value of 11.3 = 1.2 @ml for untreated cells. The IDco value for pre-treated cells is significantly different (p < .Oj) from control cells. indicating that LPS pre-treatment increases HS-P PEA resistance two-fold. In order to ensure that

the extent of LRP down-regulation was not offset in this assay by increased receptor

turnover. ive assessed PEA tosicity after brief exposure to a saturating concentration of

PE.4 ( 1000 np'ml) in control and LPS treated cells. As demonstrated in Figure 14. the

rewlts indicate ['~]lrucine incorporation is three-fold higher in LPS treated cells under

these conditions compared to untreated cells. Taken together these results demonstrate

that exposure to LPS confen partial protection from PEA-mediated toxicity in

macrophages. and that the protection conferred is highest when the duration of esposure

tc ce11 surface receptors is short. Figure 1 1. Dose-dependent effect of LPS treatment on PEA-induced cytotoxicity in HS-

P cells. HS-P cells were cultured ovemight in 96-well plates with medium containing

10% FBS. HS-P cells were treated for 24 hours with LPS at the indicated concentrations.

Following LPS exposure. cells were challenged with 100 @ml of PEA for 2 hours.

Following toxin exposure, cells were incubated with medium containing [3~leucinefor

21 hours. Each data point represents the mean k standard error of three separate

erperirnents. * indicates results significantly different fiom untreated cells (p < 0.05)

using an ANOVA with a LSD cornparison of means. LPS (nglml) Figure 12. Time-dependent effect of LPS treatment on PEA-induced cytoto?cicity in HS-

P cells. HS-P cells were cultured overnight in 96-well plates with medium containing

10% FBS. HS-P cclls were treated with 100 ng/ml of LPS for 6. 12. 18 and 24 hours.

Following LPS exposure. cells were challenged with 100 ndml of PEA for 2 hours.

Protein synthesis levels were determined by measuring the incorporation of [%?]leucine into cellular protein and are expressed as a percentage relative to control cells that received no toxin. Each data point represents the mean r standard enor of three separate experirnents. * indicates results significantly different from untreated cells (p < 0.05) using an ANOV.4 with a LSD cornparison of means.. 6 12 18 24 Time (hours) Figure 13. Effect of LPS treatment on dose-dependent PEA induced HS-P cytotoxicity.

HS-P cells were pretreated with 100 ndml of LPS for 24 hours (open squares) or were untreated (solid squares). Cells were then challenged with PEA for 2 houn at the indicated concentrations. Following toxin exposure. cells were incubated with medium containing [%]leucine for 2 1 hours. Each data point represents the mean + standard crror of the separate experirnents. * indicates results sipificantly different from untreated cells (p < 0.05) using an WOVA with a LSD cornparison of means. O 20 40 60 80 1O0 PEA (nglml) Figure 14. Effect of LPS treatment on HS-P cytotoxicity induced by short duration exposure to PEA. LPS rreated (100 ng/ml. 24 hours) and nontreated cells were challenged with 1000 ngml of PEA for 15 minutes. washed and exposed to medium containing leucine ci ne for 21 hours. Each data point represents the mean 5 standard error of rhree separate esperimenrs. * indicaies a significant difference from untreated and treated cells (p < 0.05) using a Student's t-test. O 100 LPS (nglml) HS-P macrophages are protected from PEA bv RAP

The receptor-associated protein (W)can act Ni vitro as a nanval antagonist that prevents binding of al1 known ligands to the LW. It has previously been reponed that

RAP prevents PEA binding to the LWand subsequently decreases PEA cytotoxicity

(Kounnas et al.. 1992). To help determine the role of the LRP in HS-P intoxication by

PEA. cells were incubated with a RAP-GST hsion protein. When HS-P cells were rxposed to 50 ngml of PEA for I hour. RAP-GST diminished PEA cytotoxicity in a dose-dependent manner (Figure 15). These results indicate that HS-P cells utilize the

LRP in the procrss of PEA intoxication and that functional antagonism of the LRP ieads to reduced macropliage PEA sensitivity.

Expression of US-P LW

To ascertain the rnechanism by which macrophage PEA susceptibility is

decreased by LPS. we examined the effect of LPS treatment on the expression levels of

LRP. A decrease in the nurnber of functional receptors. resulting fiom a decrerisr in the

expression levels of LW. represents one potentiai rnechanism by which macrophages

might reduce their sensitivity to PEA. Northem blot analysis revealed that treatment of

HS-P cells with 100 ndml of LPS rapidly and substantially decreased LRP rnRNA levels

(Figure 16A). Analysis of 3 independent expenments revealed that the level of cellular

LRP mRNA decreased to 18.5 * 3.3 % of zero time values 6 hours after LPS treatment

(Figure 168.). demonstrating for the first time that in macrophages LPS-dependent LRP

modulation occurs very rapidly. at the mRNA level. after treatment. Previous results in

QLU laboratory. including studies on the macrophage-like cell line RAW 246.7. indicate Figure 15. Effect of RAP-GST on PEA-induced cytotoxicity in HS-P cells. Following ovemight incubation. HS-P cells were treated with both PEA (50 @ml) and various concentrations of NP-GST. Protein synthesis levels were determined by measuring the incorporation of ['~lleucineinto cellular protein. Each data point represents the mean 5 srandard rrror of three separate experiments. * indicates significantly different (p < 0.05) when compared to non RAP treated cells using a using an ANOVA with a LSD comparison of means. RAP-GST (ng/ml) Figure 16. Effects of LPS on LRP mRNA expression in HS-P cells. HS-P cells were treated with 100 @ml of LPS for the indicated times. (A) Northem blot analysis of total

RNA (20 pg per lane) was performed with a rat LRP (rLRP) cDNA probe. The lower panel shows the results after hybridization with a 7s RNA cDNA as a load control. The results sholrn are from a representative northem blot repeated three timts. (B)Relative intensity of LRP mRNA at O. 0.5. 1. 2. 4. and 6 hours following LPS exposure normalized to 7s and expressed as a percentage of time zero. The results shown are means t standard mors of three separate expenments. Time (hours)

Time (hours) that LRP protein and functional levels decrease concomitantly with LW mRNA

(LaMme et al., 1993). To ver@ that LRP down-regdation occurs at the fùnctional level in HS-P cells afier LPS treatment. intemalization snidies were conducted utilizing the

LRP specific ligand a2M*. Results fiom these experiments (Figure 17) demonstrate that treatment with 100 @ml of LPS for 24 hours reduces HS-P arM* intemalization three and one half-fold compared to untreated HS-P cells.

Discussion

Macrophages constitute an important component of the host defense against bacterial pathogens such as P. aeniginosn. It is therefore not surprising that the macrophage is a target of P. aeruginosa vinilence factors (Speert. 1993). Specifically. it has been dernonstrated previously that PEA is cytotoxic to macrophages (Pollack and

Anderson. 1978) and hampers their ability to carry out critical cellular processes. For example. PEA inhibits the ability of macrophages to engage in phagocytosis (Pollack and

Anderson. 1978) and alters their secretion profiles of various cytokines. including interleulün-1 and tumor necrosis factor (Staugas et al.. 1992). Using an in vitro assay to rneasure the inhibition of protein synthesis we report here that HS-P macrophage-like cells are sensitive to PEA in a time- and dose-dependent marner. similar to other cells of

macrophage origin. Howvever. using our assay we have determined that the macrophage-

like ce11 line RAW 246.7 is approximately IO-fold less sensitive to PEA (data not shown)

when compared ro HS-P cells. indicating that macrophage ce11 lines appea. to have

marked differences in PEA sensitivity. The reasons for these observed differences in

macrophage sensitivity to PEA are likely io be muitifactorial, including variations in the Figure 17. Effect of LPS on HS-P azM* intemdization. HS-P cells were treated with

100 ng/ml of LPS for 24 hours. washed and incubated at 37°C for 2 houn with "'~-az~+

(4nM). in the presence or absence of excess unlabeled ligand. Cells were washed. collected. Iysed and radioactivity was determined. The results shown are means = standard error of triplicate samples from threc: separate experiments. (n = 9). * indicatrs significantiy different (p c 0.05) when compared to control cells using a Student's t-test. O 1O0 LPS (nglml) expression of cellular components (receptoa. proteases) exploited by PEA for efficient intoxication.

PEA intoxication is a complex multi-step process that relies on the efficient participation of the target cell. Therefore. susceptibility to PEA should be based. at Ieast in part. on the number of functional target cellular components available for toxin interaction. The observation that HS-P cells are highly sensitive to PEA, have abundant

LRP mRNA levels and are capable of intemalizing the LRP specific ligand a2M*. suggests that these cells express high levels of the LRP. In addition, the ability of RU'-

GST to block the cytotoxic effects of PEA confirms the LRP dependence of PEA toxicity in this ce11 type. Our initial hypothesis suggested that LPS exposure would act to protect macrophages from PEA through down-regulation of ce11 surface LRP. Northem blot anal ysis revealed that LRP mRNA levels dramatically and quickl y decrease fol lowing

LPS treatment. In addition. functional ce11 surface LRP levels decrease concomitant1y with LRP mRVA as determined by aiM* internalization studies. These results extend

Our initial studies reporting that LPS treatment doun-regulates the quantity of functional ce11 surface LRP in other macrophages (LaMarre et al.. 1993).

LPS. a componrnt of the outer membrane of Gram-negative bacteria is a well- recognized activating agent for macrophages. initiating a series of events which increases their ability to effectively combat invading pathogens (Sweet and Hume. 1996). The primary macrophage LPS receptor is the glycosylphosphatidyIinositol-anchored -elvcoprotein - CD14 (Wright et al.. 1990). however activation can also occur via a CD14- independent pathway. The end result of LPS-induced signal transduction is an altered expression pattern for a variety of genes, including increased expression of pro- inflammatory cytokines and enzymes responsible for generating reactive oxygen and nitrogen species. While many of these changes in gene expression clearly enhance the ability of macrophages to destroy invading pathogens. the role of decreased cellular expression of some genes. particularly those for receptors (Van Lenten et al.. 1985:

Shepherd et al.. 1990: LaiMane et al.. 1993). is far less clear. If. in fact. ce11 surface receptors constitute important portals of entry for pathogens or their products. then the potential advantage of ac tivel y decreasing the number of suc h sites is apparent.

In the present study we have identified one such potential mechanism. Pre- treaunent with LPS significantly decreased macrophage PEA sensitivity in a dose and time-dependent marner. In addition, based on IDso values. we observed that LPS pre- treatment for 24 hours at a concentration of 100 ngiml. decreased toxin sensitivity two- fold. In order to Mher implicate receptor-dependent mechanisms in the obsemed differences in cellular toxin sensitivity. we also investigated cellular toxin susceptibility afier a short duration of exposure to PEA. In this way, cellular receptors should be saturated with toxin and differences in receptor numbers may be more directly refiected by changes in cellular susceptibility than in studies utilizing longer periods of toxin esposure. Our results suggest a 3-fold higher susceptibility of untreated cells verses LPS- stimulated cells. further supponing our contention that receptor levels are positively correlated with toxin sensitivity. This observed decrease in toxin sensitivity correlates extremely well with the functional decrease in LRP-dependent ligand intemalization in this ce11 type. We have also consistently observed that LPS exposure decreased RAW

764.7 PEA sensitivity. however this effect was neither as reproducible nor as extensive as reported here for HS-P cells. It is not yet clear why this is the case, but the relative resistance of RAW cells to PEA described above may play a role in masking any LPS- mediated protection. It shouid also be emphasized here that LPS and cytokine-induced activation does not universally enhance cellular resistance to bacterial toxllis: cellular sensitivity to Shiga and Shiga-like toxins in vascular endothelial cells is increased following LPS or cytokine treatment (Louise and Obrig. 1991; Tesh et al., 199 1; van der

Kar et al.. 1992; Rarnegowda and Tesh. 1996).

Although it is not yet known whether LPS-mediated dom-regulation of LRP occurs in vivo. the protective ef3ect of LPS reported here would have obvious beneficial effects on macrophage viability during PEA challenge. In such a scenario, macrophages. which have diminished levels of the LW,would be relatively protected from PEA because they lack an efficient route for toxin intemalization. The hypothesis that inducible cellular changes in LRP expression confer relative protection against PEA is also supported by the studies on hepatocytes presented in Chapter 1. which demonstrated that matrix-dependent changes in LRP expression correlated with PEA resistance. Since extracellular signaling molecules have the ability to modulate LRP levels. it is plausible that this regulatory mechanism rnay be a factor in determining cellular and even tissue

PEA sensitivity in vivo. Indeed, the results of the present study may suggest an

additional mechanism by which LPS confers enhanced resistance to PEA challenge in

vivo (Zehavi-Willner et al.. 1991). It is probable that the production of various LW

regulatory factors may be initiated in response to P. aenîginosa. thus modulating PEA

cellular sensitivity during infection. It is premature to predict whether such alterations in

cellular PEA sensitivity would ultimately benefit the host or the pathogen. Since it is

suspected that the LWis also utilized for cellular entry by other pathogenic organisms. such as malaria (Shakibaei and Freve~1996) and minor-group rhino vinises (Hofer et al.. 1994). the importance of LRP replation in host resistance may not be resvicted to

PEA susceptibility.

Although the correlation between induced changes in LRP expression and PEA sensitivity is high. it should be emphasized that the LPS-induced decrease in macrophage

PEA sensitivity seen here may be a product of many phenotypic changes to macrophages

induced by LPS. Changes in any of the other steps involved in the PEA intoxication

pathway might readily augment or oppose the protective effect resulting from decreased

LRP expression. Nevertheless. it is clear from these studies that changes in the

expression of cellular receptors which act as portals of en. for pathogenic factors.

constinite a strong potential mechanism of host defense during P. aeniginosu infection. CHAPTER 3: DCVERGENT PSEUDOMONAS EXOTOMN A SENSIT'MTY IN NORMAL AND TRANSFORMED LIVER CELLS IS CORRELATED WITH LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIlY EXPRESSION

Introduction

The low-density lipoprotein receptor-related protein (LW)is a member of the

101~-densitylipoprotein receptor gene family (Willnow, 1999). The LRP is a large (600- kDa) ce11 surface endocytic receptor comprised of nvo non-covalently associated subunits, the a-(515-kDa) and p-(85- ma) chahs (Herz et al., 1990). The P-chah is anchored within the plasma membrane and contains sequences within its cytoplasmic tail that facilitate LW intemalization via clathrin-coated pits. The aîhain is compnsed of numerous epidermal growth factor-like and cornpiement-like domains. which are responsible for ligand interactions. The LRP binds an extraordinaq range of ligands. with over thirty identified to date, including proteinase inhibitors, a?-rnacroglobulin

(azM)growth factor complexes. lipoproteins, infectious agents and toxins (Gilemann.

1998: Hussain et al.. 1999). The receptor associated protein (RAP) is a 39-kDa chaperone protein that interacts with high affinity with the LRP and is believed to aid in LRP biosynthesis and maturation by preventing premature ligand-receptor interactions within the endoplasmic reticulum (Bu et al.. 1995; Willnow et al.. 1996b). Based largely on known LRP ligands, a number of speculative physiological roles for the LRP have been proposed, including proteinase and Iipoprotein catabolism, tissue remodelling and cellular growth regulation (Strickland et al., 1995: Hussain et ai.. 1999). inappropriate expression of the LRP has been implicated in the progression of several pathologicd conditions such as Alzheimer's disease, and atherosclerosis (Ulery and Stickland. 2000;

Yu and Cooper, 2001). In addition, the LRP may play a role in the progression of infectious diseases. acting as a portal of entry for various pathogens (Shakibaei and

Frevert. 1996) and their products. including the bacterial toxin Pseudomonas exotoxin A

(PEA) (Kounnas et al.. 1992).

PEA is a 66-kDa intracellular-acting bacterial exotoxin implicated in the virulence of Pseudomonas aeniginosa. an important opportunistic human pathogen. PEA exerts its toxicity by irreversibiy arresting eukaryotic protein synthesis (Iglewski and Kabat. 1974). which ultimately leads to ce11 death. via the enzymatic attachent of an ADP-ribose moiety and subssquent inactivation of elongation factor2 (Iglewski et al.. 1977). PEA intoxication is a process by which PEA exploits the cellular machinery of target cells during translocation to the cytoplasm in an activated state capable of inhibiting protein synthesis. Due to the complex nature of PEA intoxication the intoxication process has been divided into several steps. which are 1) intemalization by receptor-mediated endocytosis (RiLIE): 2) enzymatic processing: 3) intracellular traff~cking;4) membrane translocation: and 5) substrate inactivation. PEA is compnsed of a single polypeptide chain containing three distinct structurai and functional domains (Allured. 1986).

Domain I is responsible for receptor binding, domain II contains the processing cleavage site. as well as sequences implicated in translocation. and domain III contains sequences responsible for PEAts catalytic activity.

Intoxication is initiated when PEA interacts with its cellular receptor and is

subsequently internalized by RME (FitzGerald 1980). The PEA receptor has been

identified as the LRP (Kounnas et d.. 1992) and its importance in the process of PEA

intoxication has been clearly established (Willnow and Herz, 1994: FitzGerald et al..

1995). The LRP represents the sole identified portal of entry for PEA and, as such, the level of functional LWexpressed on the ce11 surface is a cntical determinant of cellular

PEA sensitivity (Mucci et al.. 1995). with cells expressing hi& levels of LRP being highly sensitive to the toxic effects of PEA. Differences in cellular LRP expression levrls are partially responsible for the substantial differences in sensitivity of mammalian tissues and cells to PEA (Pavlovskis and Shackelford. 1974: Middlebrook and Dorland.

1977: Saelinger et al.. 1977). Specifically. the observation that the liver is the principal site of injury following systemic exposure to PEA is largely attributable to high levels of

LRP expression by hepatocytes (Feldman et al.. 1985: Moestrup et al.. 1992).

LRP expression is regulated by a diverse range of extracellular signaling factors. including hormones (Descamps et al.. 1993). the extracellular matrix (Chapter 1 ). cytokines (Hussaini et al.. 1996). and bacterial products (Chapter 2: LaMarre et al..

1993). In addition. LRP expression is dependent upon cellular differentiation (Gafvels et al.. 1992) and oncogenic transformation (Gonias et al.. 1994). In this report. we cxamined the sensitivities of hepatic ce11 lines to PEA and discovered that a uansformed liver ce11 line (BNL IME A7R.I) displayed heightened sensitivity to PEA when cornpared to its normal (BNL CL.?)parental ce11 line. Previously. vie demonstrated that an induced decrease in cellular LRP expression represents a mechanism by which cellular

PE.4 sensitivity can be reduced (Chapters 1 and 2). In this study. we explored whether differences in LW expression induced by cellular ~sfomationis correlated with enhanced PEX sensitivity observed after transformation in these liver cells. Materials and Methods

CeII culture

Normal (BNL CL.?) and chemically transformed (BNL IME A7R.1) BALB/c murine embryonic hepatic fibroblasts were obtained from the Amencan Type Culture

Collection (Rockville. MD). Ce11 lines were cultured in T-75 flasks in Dulbecco's modified Eagle's medium. supplemented with 10% fetal bovine se-, 50 units of penicillin per ml and 50 pg streptomycin per mi at 5% COz, 95% hurnidity and 37OC.

For experiments. cells were seeded at a density of 2x10~cells/well into 96-well plates

(cytotoxicity studies). 2x10' cells/well into 24-well plates (intemalization studies) and were incubated overnight before treaunents. Medium, semand supplernents were al1 obtained from GibcolBRL. Burlington, ON. Al1 tissue culnue plastics was purchased

from Sarstedt Inc.. St. Leonard, PQ.

Cvtotosicitv assay

Sensitivity to various toxins was determined by assaying the inhibition of protein

synthesis. Following ovemight incubation in 96-well plates BNL CL2 and BNL IME

A7R. 1 cells were challenged with various concentrations of either TGF-a-PEA37 or PEA

in sem-free medium at 37OC for one or two hours, respectively. Following challenge.

toxins were rernoved and cells were incubated with medium containhg [3~]leucineat 5

pCi/ml (ICN, Montreal, PQ) for 21 hours. Radioactive medium was then removed, cells

were detached using a uypsin solution and harvested ont0 filter mats. Incorporated

radioactivity was determined with a Betaplate Liquid Scintillation Counter (Lm Wallac.

Turku. Finland). Data are presented as a percent of protein synthesis compared with control cells that were not treated with toxin. PEAGlu57. which demonstrates reduced binding to the LRP but normal catalytic activity (JUMO et al., 1988), was introduced into cells utilizing the Infl~uTh'pinocflic cell-loading reagent according to the manufacturer's instructions (Molecular Probes. Eugene, OR). Briefly, hypenonic loading medium containing 1000 ngml of PEAGIuS7 was added to cells for 15 minutes and then replaced with hypotonic lysis medium for one minute. Cells were then washed in se--free medium. radioactive medium was added and cells were harvested as described above.

RAP protection studies

.%fier ovemight culture BNL IME A7R.l cells were cotreated with 100 ngml of

PEA and 1000 n@ml of RAP-GST for 1 hou. Cells were then incubated with

[j~lleucinefor 2 1 hours and harvested as described above.

Ligand internalization

Following conversion to the receptor-recognized conformation (a&l*) by methylarninr HCl treaunent. cr2M was radiolabeled with "'1 (Amenhm Life Sciences.

Oakville. ON) using Iodobeads as described by the manufacturer (Pierce Chernical

Company. Rockford. IL). Ligand uptake studies were performed as previously described

(FitzGerald et al.. 1995: Chapters 1 and 2). Briefly. cells were washed in Earle's

Balanced Salt Solution (EBSS). and then 4nM of ''S~-ar~*in EBSS was added to cells for 2 hours at 37T. Afier ligand removal, cells were washed with cold EBSS containing

10 mM HEPES and ueated with a trypsin solution for 30 minutes at 4°C. Released cells were subsequentiy harvested into 1.5 ml microfuge tubes and pelleted by centrifugation. Intemalized ligand was inferred fiom the radioactivity in the pellet using a gamma counter. Protein content was determined by the Bio-RadTMprotein assay (Bio-Rad.

Richmond. CA). For non-specific intemalization, a 100-fold excess of cold ligand was included. Specific intemalization was determined by subtracting non-specific intemalization from total internalization,

Data Analvsis

Al1 data are presented as the mean + standard error of the mean. Data were analyzed by a Student's t-test; p < 0.05 was considered significant. Data were also analyzed by an ANOVA with a least significant difference (LSD)cornparison of means.

Results

PEA sensitivity

To assess the sensitivity of liver cells to PEA. the extent of protein synthesis inhibition was determined by assaying ['HJ~eucine incorporation in cells exposed to various concentrations of PEA. Normal (BNL CL.2) and chemically transformed (BNL

1 ME A7R.1) liver cells showed divergent PEA sensitivity (Figure 18). The 50% inhibition dose values (IDs0), the concentration of PEA required to inhibit protein synthesis by 50% compared to cells that did not receive toxin. of BNL CL.l cells was

> 1000 nglml. compared to a value of 10 @ml for BNL IME A7R. 1 cells. indicating that the process of transformation has increased cellular sensitivity to PEA. Figure 18. Protein synthesis inhibitory activity of PEA on BNL CL2 and BNL IME

A.7R.l liver cells. BNL CL.? (open circles) and BNL 1ME A.7R.l (open triangles) liver cells were culiured ovemight in 96-well plates with medium containing 10% senim.

Cells were incubated in serum-fiee medium containing various concentrations of PEA for

2 hours. Protein synthesis levels were determined by measuring the incorporation of

[%]leucine into cellular protein and are expressed as a percentage relative to control cells that received no toxin. Each data point represents the mean = standard enor of five separate experiments. * indicates significantly different (p < 0.05) when compared to

BNL 1 ME A.7R. 1 cells using an ANOVA with a LSD cornparison of means. PEA (nglml) Effect of W on PEA sensitivity

The LRP is the ce11 surface receptor implicated in the binding and internalization of PEA. It has been previously demonstrated that RAP. a natural LRP antagonist. prevents PEA binding to the LRP and subsequently decreases PEA cytotoxicity (Kounnas et al.. 1992). To veri@ the importance of the LRP in PEA intoxication in liver cells. RAP was used as a cornpetitor for PEA cytotoxicity. As indicated in Figure 19 a RAP-GST fusion protein decreased PEA toxicity in BNL ME A7R.1 cells. These results demonstrate that hepatic cells use the LRP in the process of PEA intoxication and that functional antagonism of the LRP reduces PEA susceptibility in this ceIl type.

Effect of differential LRP expression on ligand internalization

To ascertain the mechanism by which PEA sensitivity is increased in BNL LME

A7R. 1 cells. we examined cellular expression Ievels of LRP. Previous binding experirnents (LaMane, persona1 communication) with "?-a2~*.a LRP-specific ligand. demonstrated that BNL CL2 cells have virtually no capacity to bind cc2M*. but BNL

IME A7R.I cells bind a2M* in a specific and saturable mariner. suggesting that these cells express high levels of functional cell-surface LRP. To funher examine this issue. a2M* internalization experirnents were performed. BNL IME A7R. I cells had the capacity to intemaiize six-times more ligand over two hours than BNL CL2 cells (Figure

20). Collectively these data suggest that enhanced sensitivity to PEA in BNL ME

A7R.I ceils is correlated wîth a marked increase in functional ce11 surface LRP expression. Figure 19. Effect of RAP-GST on PEA-mediated protein synthesis inhibition in BNL

1 ME A.7R. 1 liver cells. Following overnight incubation. BNL IME A.7R. 1 cells were treated with both PEA (700 ne/ml) and W-GST (1000 ng/ml). Protein synthesis levels

were determincd by measuring the incorporation of leucine ci ne into cellular protein and

are rspressed as a percentage relative to control ceils that received no tosin. Each data

point represents the mean r standard error of three separate experiments. * indicates

significantly different (p < 0.05) when cornpared to non RAP treated cells using a

Student's t-test. O 1O00 RAP-GST (nglml) Figure 20. Ligand intemalization studies with a?-rnacroglobulin (azM*). Liver cells were incubated at 37°C for 2 hours with '"I-U~M* (4 nM), in the presence or absence of excess unlabeled ligand. Cells were washed. collected. and lysed. and radioactivity determined. The results shown are mrans = standard error of triplicate sampies from three sepante experiments. (n = 9). * indicates significantly different (p c 0.05) when cornpared to BNL CL.? cells usinp a Srudent's t-test. BNL CL.2 BNL IME A7R. Cell Type Susceptibilitv of cells to TGF-a-PEA37 and PEAGlw7

In order to Merimplicate differences in LWexpression in the enhanced PEA sensitivity of BNL IME A7R.1 cells, we designed expenments to introduce PEA into target cells via non-LRP pathways. In this way, PEA sensitivity would not be dependent upon LWexpression and the relative roles of other cellular factors could be assessed. in the first set of expenments. liver cells were treated with increasing concentrations of the conjugate PEA toxin TGF-a-PEA37. in which the LRP binding domain of native PEA is

replaced with TGF-a. This toxin has been shown to enter cells via the epidermal growh

factor receptor (Theuer et al.. 1992). Results are displayed in Figure 21 and demonstrate

that BNL CL.? cells are more sensitive to this conjugate toxin than BNL 1 ME A7R. 1

cells. Differences in cellular conjugate toxin sensitivity may be the result of differences

in EGF receptor expression. These results demonsate that BNL CL2 cells are

susceptible to PEA-mediated inhibition of protein synthrsis when toxin entry occurs via a

non-LRP dependent route.

In an additional experiment designed to bypass LRP-mediated roxin entry. we

used the mutant toxin PEAGluS7. which has a reduced ability to bind to the LRP due to

the replacement of a lysine residue at position 57 that is critical for PEA-LRP interaction.

PEAGluj7 was introduced inro cells utilizing the Influxm pinocytic cell-loading reagent.

which involves rupninng toxin containing pinocytotic vesicles by altering the osmotic

strength of the culture medium. When utilizing rhis method of toxin entry. BNL CL2

ceils were more sensitive than BNL 1ME A7R. 1 cells to PEAGlu57 (Figure 22). These

results indicate that compared to BNL MEA7R.1 cells, BNL CL2 cells are more

sensitive to mutant and conjugate PEA toxins when cellular entry occun via a non-LRP- Figure 21. TGF-a-PEA37 scnsitivity of BNL CL2 and BNL ME A.7R.1 liver cells.

BNL CL2 (open squares) and BNL IME A.7R.1 (open circles) liver cells were challenged with various concentrations of TGF-a-PEAV at 37OC for 2 houn. Following

toxin exposure cells were incubated with medium containing ['~lleucine for 21 hours.

Each data point represents the mean z standard error of three separate espenments. *

indicates significant differences (p < 0.05) between groups using an ANOVA with a LSD

cornparison of means.

Figure 22. Sensitivity of BNL CL2 and BNL 1ME A.7R.1 liver cells to PEAGIuS7.

Hypenonic loading medium containing 1000 nglml of PEAGly57 was added to BNL

CL2 and BNL 1ME A.7R. 1 liver cells for 15 minutes and then replaced with hypotonic lysis medium for one minute. CeIls were then washed in serum-free medium. incubated with radioactive medium and incorporated radioactivity was determined afier 21 hours in culture. Each data point represents the mean = standard enor of three separate

rxperiments. + indiccites significantly different (p < 0.05) when compared to BNL CL2 cells using a Student's t-test. BNL CL.2 BNL 1ME A7R. Cell Type dependent route. This is in contrast to LRP-mediated entry of native PEA in which BNL

1 ME A7R. 1 cells displayed a higher sensitivity to PEA (Figure 17). These studies also indicate that BNL CL2 cells possess al1 of the necessary post-receptor factors necessary for carrying out the process of PEA intoxification.

Discussion

Using an i!7 vitro assay to measure inhibition of protein synthesis, we report here that two liver cell lines consistently demonstrate divergent PEA sensitivities. Compared to untransformed cells (BNL CL.2). transformed cells (BNL ME A7R.1) showed heightened sensitivity to the protein synthesis inhibitory effects of PEA. Due to the central role of the LWin determining cellular PEA sensitivity. we exarnined the level of

Functional LRP espression in BNL CL2 and BNL ME A7R.1 cells to determine whether differences in constitutive LRP expression betwecn these two cell lines might contribute to their different sensitivities to PEA. Resuhs fiom these studies revealed that

BNL CL2 cells do not efficiently bind or intemalize arM*. a LRP specific ligand. suggesting that BNL CL2 cells express low levels of functional LRP on their cell surface. In contrast. BNL 1 ME A7R.1 cells appear to express abundant functional ce11 surface LRP. as demonstrated by the efficient binding and intemalization of a2M*.

Although we have yet to identiQ the genetic lesion responsible for the transformed

pheno-pe displayed by BNL IME A7R.1 cells, it appears that the observed marked

increase in functional LRP levels is likely the result of an increase in steady state mRNA

levels (Benn. 1999). We are currently investigating whether the increase in LRP mRNA

levels observed in BM. IME A7R.1 cells are transcriptionaily or post-transcriptionally induced. Our findings that LWexpression is increased in transformed BNL 1ME A7R. 1 cells are consistent with other reports demonstrating that LWexpression increases der cellular transformation. Lopes et al. (1994) and Yamamoto et al. (1997) have demonstrated that expression of this receptor is elevated in glial ce11 mors. However. it has also been reponed that LRP expression is reduced or absent following malignant transformation (Gonias et ai.. 1994: de Vries et al.. 1996).

Results from the current investigation indicate that a consequence of increased functional LW expression in BNL 1 ME A7R. l cells is a heightened cellular sensitivity to PEA. a finding that funher strengthens the positive correlation between cellular PEA sensitivity and LRP expression (Mucci et al.. 1995). We recently demonstrated that a reduction in hinctional LWexpression following lipopolysaccharide treatment enhanced

PE.4 resistance in a macrophage-Iike ce11 line (Chapter 2). In addition. wè have determined that pnmq hepatocytr PEA sensitivity is diminished in extended culture on a collagen type 1 matris and is correlated with a marked decrease of functicnal LRP

(Chapter 1). Taken together. Our investigations indicate that cellular PEA sensitivity is not a stable feature but rather. subject to modulation by long term cellular events. such as transformation. and in the shorter term. by extracellular signaling molrcules. It is probable that the production of various LRP regulatory factors may be initiated in response to P. ueniginosa. consequently altering cellular PE.4 sensitivity during infection.

Although our evidence suggens that the increased PEA sensitivity of BNL 1ME

A7R.1 cells is likely mediated by increased LRP levels. additional cellular funutions utilized during PEA intoxication may also be altered in these trmsformed liver cells. To address this issue we examined BKCL2 and BNL IME A7R. 1 sensitivity to mutant

(PEAGlu57) and conjugate (TGFa-PEA37) PEA toxins that do not use the LRP for cellular entry. Although these toxïns use different mechanisms for cellular entry. the mechanisms for cellular processing are simiiar. making them ided candidates to evaluate the roles of non-receptor events in the process of PEA intoxication. Our results indicate that sensitivity to these toxins is opposite to that observed for native PEA; BNL CL2 cells e-xhibiting heightened sensitivity to PEAGlu57 and TGF-a-PEA37 compared to

BNL MEA7R. 1 cells. The obsewed differences in cellular TGF-a-PEA37 sensitivity may be the result of differences in EGF receptor expression between BNL CL2 and BNL

I ME A7R. 1 cells. These data clearly suppon the hypothesis that the enhanced PEA sensitivity of BNL IME A7R.1 cells is a direct result of higher levels of LRP. since mutant- and conjugate-toxin sensitivity is actuaI1y higher in the low-LRP expressing ce11 type once the toxin has gained entry into the ce11 by an alternative means. The observation that transformed cells were slightly (but still significantly) less sensitive to toxin when delivered via non-LRP-dependent pathways suggests that cellular transformation may be accompanied by multiple changes in the endogenous pathways which regulate toxin sensitivity. BNL MEA7R.l cells may have alterations in one or more additional cellular components involved in PEA intoxication and these alterations have the potential to oppose toxin-sensitizuig effects mediated by increased LRP expression. GENERAL, DISCUSSION

Pseudornonas Exotoen A (PEA) is an intracellular-acting bacterial exotoxin. implicated in P. aenginosa virulence. PEA irreversibly inhibits protein synthesis. by enzymatically inactivating elongation factor 2 (EFZ). an essential component of the protein biosynthetic machinery (Iglewski and Kabbat, 1974; Iglewski et al.. 1977). PEA intoxication is a complex multi-step process, in which PEA exploits the normal machinery (receptors. enzymes) and processes (RME, enzymatic processing) of target cells to generate and transport an enzymatically active toxin Fragment to its substrate

(EF7) within the cytoplasrn. We believe that PEA intoxication efficiency. and consequently cellular PEA sensitivity, is govemed by the availability of the cellular components exploited by PEA during intoxication. The aim of this thesis study was to test our hppothrsis that induced alterations in the expression levels of specific components of the cellular machinery exploited by PEA during intoxication represent mechanisms by which cellular sensitivity to PEA can be altered by the host. The objectives of this snidy were to determine whether alterations in cellular sensitivity to

PEA can be induced. and if so. to determine the molecular mechanisms that might be responsible for this novel cellular phenornenon.

Induced Modulation of Cellular PEA Sensitivity

Despite the fact that extensive in vitro and in vivo snidies have demonstrated that

mamrnalian tissues and cells display a wide range of sensitivity to PEA-mediated

cytotoxicity (Pavlovskis and Shackelford, 1974; Middlebrook and Dorland, 1977;

Saelinger et al.. 1977). few investigations have examined whether sensitivity to PEA can be modulated (Zehavi-Willner et al., 1991). In this study we have demonsmted that cells possess the ability to modulate their PEA sensitivity in vino. A change in cellular sensitivity occurred in different ce11 types f?om two rodent species (mouse and rat) and was induced by diverse cellular events (Table III). Pre-treatment with LPS decreased

PEA sensitivity in rat HS-P macrophage-like cells in a dose- and time-dependent fahion

(Chapter 1). Sensitivity to PEA also decreased in primary rat hepatocytes in extended culture on a collagen type 1 rnatrix (Chapter 1). Convenely. transfomed murine liver cells were show to be more sensitive to PEA when compared to their non-transforrned counterparts. indicating that the process of transformation has heightened PEA sensitivity in this ce11 line (Chapter 3). In support of Our work. in rirro studies have shown that cellular sensitivity to other inuacellular-acting bacterial exotoxins cm also be modulated by various cellular events. Specifically. cytokine and LPS treatrnents alter sensitivity of a

variety of cell types to Shiga toxins (Stus) (Louise and Obrig, 1991 : Tesh et al.. 199 1:

Ramegowda and Tesh. 1996). In addition. cellular sensitivity to Stu is also increased

following oncogenic transformation (Arab et al.. 1997: Lingwood. 1999).

Our results demonstrate that sensitivity to PEA is not a stable cellular feature.

Instead. cellular PE.4 sensitivity. like other phenotypes. is altered in response to various

cellular stimuli. Therefore. one can infer from Our results. that in vivo cellular sensitivity

to PEA is likely determined by a wide range of factors. such as ce11 type. stage of

differentiation and the components of the cellular microenvironment. including the

extracellular matrix and sipnaling molecules. It is conceivable that changes in the Table III. Alterations in cellular PEA sensitivity and LRP expression in various ce11 types induced by a wide range of cellular events.

Fold change in Cellular Fold Change in LRP Expression Cell type Treatment 1 Time Sensitit...lty r 1 ET* Functional levels mRNA levels 1 ' 2 1 1 1" Primary ' ! Collagen 24 -2 1 -2.5 -10" Hepatocytes t 48 -57 -7.5 -10 a O Macrophages LPS 1 1 1 24 -3- -3 -5 -jb Normal - 1 1" Liver Cells 1 Transformed - > 100 6 3" a Benn. 1999 value is for 6 houn of LPS treatment components of the cellular microenvuonment will modulate cellular PEA sensitivity.

Changes to the cellular microenvironment. including alterations to the extracellular matrix and the concentration of groowth factors and cytokines. are induced by a number of anti-microbial defense mechanisms. including the inflammatory. immune and the acute phase reçponses (Moshage. 1997: Rosenberg and Gallin. 1999). Therefore. it is possible that a consequence of trigering these anti-microbial defense mechanisms in response to

P- oeruginosa or its products. such as LPS, is that cellular sensitivity to PEA is altered.

Alterations in cellular PEA sensitivity may change the extent of PEA-mediated damage and consequently the capacity of the host to effectively eradicate a P. aenrginosa infection. Sirnilarly. alterations in cellular sensitivity to PEA may be induced by an underlying compromising condition and may help expiain why these individuals have increased susceptibility to P. arncginosu infections (Bodey et al.. 1983). For exampie. extensive bum trauma causes drarnatic changes in tissue and systernic physioiogy. including alterations in the expression of inflamrnato~mediators (Youn et al. 1992).

Cellular PEA sensitivity is apt to be modulated by such drastic physioIogica1 changes and may impact the ability of P. ueruginosa to establish an infection.

Our belief that cellular sensitivity to PEA can be modulated h vivo is supponed by the study by Zehavi-Willner et al. ( 199 1) in which they found that LPS pre-treatment protected mice from PEA-mediated lethality. Conversely. in the same study the authors demonstrated that PEA-mediated lethality was increased when LPS was CO-administered with PEA. In addition to LPS. host derived pro-inflanmatory cytokines have been postulated to be critical for PE.4-mediated injury. For example. the protein synthesis inhibitory effects of PEA sensitize hepatocytes to apoptosis signais initiated by Kupffer ce11 derived RIF-a (Schümann et al., 1998). Host-derived cytokines are also important in Stx-mediated damage in individuals uifected with S. ùysenteriae or EHEC and suffering fiom the extra-intestinal complication hemolytic uremic syndrome (HUS)

(Tesh. 1998). It has been proposed that TNF-a and interleukin-1 p, released fiom resident macrophages or infiltrating monocytes, are responsible for sensitizing vascular endothelid cells in the kidney and centrai nervous system to Stxs (Louise and Obng.

199 1 : Tesh et ai.. 199 1: Ramegowda and Tesh. 1996: Tesh, 1998; Rûmegowda et al..

1999). Taken together these findings indicate that intracellular-acting bacterial toxin exploitation involves not only utilizing the cellular machinery necessary for intoxication

(toxin receptors). but also entails altering toxin-mediated darnage by using normal host ce11 responses to extracellular signaling molecules, including those derived from the hon

(cytokines) and fiom bacteria (LPS).

LRP: PEA's Portal of Entry

Target ceII participation is unquestionably a requirement for successful PEA intoxication. As a result. the ability to carry out this process eficiently is a significant factor in determining cellular sensitivity to PEA. Accordingly, the functional abiiity as well as expression levels of cellular components exploited by PEA have a direct bearing on cellular PEA sensitivity. In fact, cells lacking the capability of expressing these components. whether nanirally or as a result of induced mutations. are highihly resistant to the cytoxic effects of PEA (Fendrick, et al.. 1992, Innocencio et al., 1993, FitzGeraid et al.. 1995). An important target ce11 component mediating PEA sensitivity is the PEA receptor. the LW. The isolation of LRPdeficient cells that are highly resistant to PEA (FitzGerald et al., 1995) and the recent findine that expressing chicken LRP in these mutant cells restores their sensitivity to PEA (Avramoglu et al., 1998), fimly establishes

LW'S pivotal role in determinhg cellular PEA sensitivity. It is not surprising that Mucci et.

al. ( 1995) discovered that a positive correlation exists between hctional LRP expression

Ievels and cellular PEA sensitivity. Intuitively this makes sense, compared to a ce11

expressing only a modes level of functiond LRP, a ce11 displayhg copious amounts of this

receptor would have a heightened ability to intemalize PEA and therefore have a marked

increase in roxin sensitivity. This correlation is Merstrengthened by our fmding that

primary hepatoctyes. macrophages and transfomed liver cells were both sensitive to PEA

and e.xpressed high Ievels of hctional ce11 surface LRP.

These two discoveries. that the LWis the PEA receptor (Kounnas et al.. 1992) and

diat cellular PEA sensitivity and LRP expression are positively correlated (Mucci et al.

1995). have been insrnimental in explainhg previously reported observations concerning

both in vino and in vivo cellular PEA sensitivity; narnely PEA's species range and tissue

tropism. PEA's cross species lethality and cytotoxicity (Pavlovskis and Shackelford. 1974:

Middlebrook and Dorland. 1977) are likely due. at lem in part, to the observation that the

LWis highly conserved between species and is ubiquitously expressed within an organim

(Moestmp et al.. 1992). However. PEA tissue tropism does e'ria. For example. systemic

PEA appears to target the liver. eventually leading to its dysfunction and desmiction

(Saelinger et al.. 1977). Systemic PEA targeting the liver is not surprising since the liver

is one of the primary LRP expressing organs, capable of rapidly and extensively ciearing

the circulation of LWligands. Both hepatocytes and resident macrophages (Kuppfer

cells). the principal ce11 types of the liver. have an abundant ce11 surface LRP population (Feldman et al.. 1985; Moestmp et ai., 1992). Our finding that primary hepatocqs iuid a liver-derived macrophage ce11 line express high levels of functiond LRP and are sensitive to PEA supports this reasoning conceming why the liver is a systemic target of PEA.

The exploitation of LRP-mediated endocytosis by PEA likely allows PEA to be used as a virulence factor by P. aeniginosa in diverse host niches. This characteristic likely contributes to this pathogen's ability to cause a wide range of disease States in multiple hosts (.etensrin and Cross. 1993). A diverse range of hon organisms. plants. nematodes. insects and mammals. have been recently utilized to investigate cornmon virulence mcchanisms used by P. aeruginosa during infection (Rahme et al.. 1997: Duby et al.. 1999: Jander et al.. 2000; Rahme et ai.. 2000). These studies have reveded that

PEA is a consemed virulence factor used by P. aerzghosa to infect evolutionary divergent hosts. Although the mechanism by which PEA acts as a virulence factor in these diverse hosts is unknown. it is interesthg that LRP-like LDLR family members are expressed by some of these hosts (Hussain et al.. 1999). which may by exploited by PEA for cellular entry. thus allowing PEA to act as an intracellular-acting bacterial exotoxin.

Altering LRP Expression: A Cellular Mechanism for Modulating PEA Sensitivity

In this study we have demonstrated that the induced modulation of cellular PEA sensitivity was consistently conelated with a change in cellular expression levels of LRP

(Table III). A decrease in PEA sensitivity in HS-P macrophage-like cells treated with

LPS was associated with a three and a half-fold decrease in functional ce11 surface LW expression. Similarly. the decrease in prirnq hepatoctye PEA sensitivity in extended culture on a collagen type I matrix was associated with a seven and a half-fold decrease in functional LRP expression by 48 houn. And finally, a six-fold increase in functional ce11 surface LRP expression wasassociated with increased cellular PEA sensitivity in a uansfomed hepatic liver ce11 line. ïhese changes in functional cell surface LRP were consistently associated with concomitant changes in LRP mRNA levels.

Induced changes in cellular LWexpression may therefore represent a mechanism by which cellular PEA sensitivity can be controllrd by the host. It has been shown that

LRP expression is altered by a wide range of extracellular signaling molecules. including bacterial products and cytokines. that may be initiated by the presence of P. oenrginosa

(LaMarre et al.. 1993). Therefore, a change in cellular LWexpression induced by P. aerzrginosa is likely to rnodulate cellular PEA sensitivity and rnay represent a mechanism by which cellular sensitivity to PEA could be altered during P. aeruginosa infection.

Decreasing the nurnber of functional ce11 surface LRP receptors on a target ce11 would drcrease the efficiency of intoxication by limiting the uptake of PEA by RME.

Consequently. cellular PEA sensitivity would be decreased. This rnay represent an important host defense mechanism against PEA-induced cellular damage. During exposure to PEA. such a mechanism would be an obvious benefit to cells of the immune system. such as macropahges. attempting to mount a defense to eradicate P. aeruginosu.

In a similar marner. Ranegowda et al (1996) have provided evidence demonstrating that following treatment with a variety of rxtracellular signaling molecules, a macrophage ce11 line (THP-1) becomes less sensitive to Stxs. as a result of decreased expression of the Stu receptor Gb3. Perhaps down-regulation of receptors has evolved as a mechanism by which activated macrophages close the portals of entry to pathogens and their toxin products. This may explain why other promiscuously-binding large endocytic receptors. such as scavenger receptors and the mannose receptor, which are known to bind pathogens and their products. are also down-regulated in activated macrophages (Van

Lenten et ai.. 1985; Shepherd et al., 1990). Down-regulation of receptors that act as portals of entry for pathogens and their pmducts may represent an important component of innate irnmunity. which provides a first line of defense against invading pathogens.

For example. the ability to respond to invading pathogens by reducing the expression of receptors exploited by intracellular-acting bacterial toxins may provide cells involved in the innate immune response. such as macrophages. with a mechanism to decrease cellular toxin sensitivity This would then permit the utilization of immediate effectors mechanisms. such as complement. defensins and phagocytosis. to control invading pathogens while in the presence of bacterial toxins. In addition. increased toxin resistancr rnay provide additional time for cells involved in both the innate and specific branches of the immune system (B and T cells) to generate appropnate adaptive immune responses to eradicate the invading pathogen.

Altemativeiy. increasing cellular LRP expression rnay be an important virulence

mechanism used by P. aeniginosu to increase PEA sensitivity in cells and tissues in

which destruction would aid P. acruginosu swival in the host. It has been demonstrated

that sensitizing cells to the cytotoxic effects of other intraceilular-acting bacterial

exotoxins cm occur by increased expression of the appropriate toxin receptor. For

exampie, expression of the SW receptor. Gb3, is increased in endotheliai cells exposed to

pro-inflammatory cytokines and LPS. which sensitizes these cells to the cytotoxic effects

of Stxs (Louise and Obng. 1991: Tesh et al.. 199 1: Ramegowda and Tesh. 1996: Tesh.

1998: Rarnegowda et ai.. 1999). This receptor-mediated mechanisrn of sensitization is thought to occur in vivo during HUS in Stx ~getedtissues, such as the kidney and the centrai nervous system. K cholercie uses a different method to sensitize host cells to cholera toxin (CT). K cholerae secretes an enzyme (neurarninidase) that creates more

CT recepton (Gui gangliosides) by cleaving other ganglioside receptors on the surface of host cells. Therefore increasing cellular sensitivity to CT by creating an increased level of cell dace Gui ganglioside receptors for CT-mediated cellular entry (Galen et al.,

1992). in addition. other intracellular-acting bacterial exotoxins may use this mechanism of increasing expression of their toxin receptors to increase hoa cellular toxin sensitivity.

For example. expression of the Diphtheria toxin (DT) receptor, the pro-HB-EGF (Naglich et al.. 1992). is known to be increased by RIF-a (Yoshinimi et al., 1993) and may represent an important sensitizing event leading to DT-mediated cardiac damage during diphtheria. Together these findings indicate that increasing the expression of toxin receptors may be a widespread strategy used by bacterial pathogens to increase a host's cellular sensitivity to intracellular-acting bacterial exotoxins.

It is clear that reducing the number of fùnctional toxin receptors would be beneficial to a ce11 during PEA exposure. However, whether induced protection from

PEA-mediated cytoroxicity for specific ce11 types or tissues is ultimately beneficial to the host or to P. aengirzosa is unknown. This issue is likely to be complicated. with an extensive list of host and bacterial factors playing multiple and overlapping roles. For example, dom-regulation in LRP expression in macrophages. induced by the presence of

P. aeruginosa and its products (LPS). may protect these cells from the cytotoxic effects of PEA, by reducing PEA uptake. However, macrophages (Kupffer cells) have been show to play a crucial role in PEA-mediated iiver darnage by producing the pro- inflammatory cytokine TNFa (Schümann et ai., 1998). Therefore, the induced decrease in macrophage sensitivity to PEA may ultimately result in enhanced PEA-mediated liver damage. due to elevated levels of TM-a than would othenvise be present if macrophages had maintained their hi& sensitivity to PEA. and were eliminated by PEA.

Therapeutic Intervention

The greatest benefit in understanding toxin/receptor interactions is the potential for therapeutic intervention in preventing or limiting toxin-mediated damage.

Traditionally this has been accomplished by using anti-toxin antibodies to interfere with toxin:receptor binding (Pavlovskis et al., 1977: Snell et al.. 1978; Pavlovskis et al.. 198 1).

An additional suategy for limiting toxin-mediated damage involves reducing cellular toxin sensitivity by decreasing the number of functional celi surface toxin receptors, thus limiting toxin entry into host cells. In this study we have provided evidence that reducing cellular LWexpression cm decrease cellular PEA sensitivity. A better understanding of the mechanisms controlling cellular LRP expression is required to allow for the potential creation of novel therapeutics designed to dom-regulate LRP espression and thus limit

PEA-mediated damage in individuals infected with P. crencgi~~osu.Therapeutic agents may include those that decrease LRP expression outright or limit the effectiveness of cellular events. such as host-derived signaling molecules, that induce an up-regulation of

LRP expression. We and others (Kounnas et al.. 1992) have aiso show that cellular

PEA sensitivity cm be reduced by converting expressed ce11 surface LRP to a non- functional state by blocking PEA binding sites with the naturai LRP antagonist RAP.

Although not yet exarnined it is possible that administration of RAP wouid prevent PEA- mediated damage in vivo by reducing the quantity of functional LRP molecules in the host. The administration of anti-LRP antibodies that prevent LRP:ligand interactions could also be employed as a method to conven ce11 surface LW to a non-hctional state.

Another approach for blockuig LRP:PEA interactions is the administration of soluble receptors as cornpetitive inhibiton. Possible soluble recepton include engineered LRP molecules containing multiple PEA binding domains. In a similar manner. mimics of the

Sts receptor have been designed as therapeutic agents to neutnlize Stxs (Kitov et al.

2000: Paton et al.. 3000). Lnterestinply. a soluble form of the LRP is expressed in a wide range of species and is able to bind PEA (Grimsley et al.. 1999: Quinn et al.. 1999).

Increased expression of this secretor)' form of the LRP rnay represent an additional mechanism by which PEA-mediated darnage cmbe reduced by the host.

Limitations of the Study

Although the studies presented in this thesis were carefully designed to test our hypothrsis. there are several limitations that may influence the interpretations of the data presented here. One limitation is that internalization of PEA was not measured directly.

Although. we used "'I-~~M*uptake studies to demonstrate significant changes in

functional LRP expression. it is possible. although unlikely. that uptake of PEA \vas

unaffected. '%PEA uptake snidies were in fact attempted on several occasions. which

would have provided this meaningful information. but our efforts to generate functionally

active iodinated PEA proved unsuccessful. Alternative approaches using fluorogenic

labeled toxin may prove usehl in future studies. as has been accomplished with Sa

(Rameogowda et al.. 1999). In this study we have clearly demonstrated that induced alterations in LRP expression are positively correlated with changes in cellular PEA sensitivity. Although important. since it suggests a mechanism by which PEA sensitivity can be altered. we have not completely isolated the direct contribution that changes in LRP expression had on modulating PEA expression relative to other cellular changes induced simultaneously. lnduced changes in cellular PEA sensitivity are likely the result of multiple changes in the host ce11 and thus may collectively mask the contributions made by changes in individual components utilized by PEA during intoxication, such as the LRP. Using conjugate PEA toxins that do not use the LRP for cellular entry the case that changing

LRP expression levels plays a role in modulating PEA sensitivity was clearly strengthened. However. use of additional conjugate toxins and other approaches are warranted to further examine this issue. One approach might be to compare induced changes in cellular PEA sensitivity between cells that have been transfected with an LW expression vector to their parent cells. Such an approach would be effective because cells harboring the LRP espression vector would display high levels of LRP bcth before

and after treatment. while the parent cells would have altered LRP expression following

treatment. By holding LRP levels constant in the plamiid-transfected ce11 line. it might

be possible to determine the contribution made by the other cellular factors on

modulation of cellular PEA sensitivity and conseqwntly the importance of changes in

LW expression in the parent ce11 line could be inferred.

Since PEA is implicated in the virulence of P. aenrginosa in a wide range of

species it wouid have been beneficial to examine if modulation of cellular PEA

sensitivity occurs in additional species. In retrospect. it wouid have been particularly informative if we examined this issue using hurnan cells, because P. aeniginosa is an important opportunistic human pathogen. Since LRP expression is regulated in human cells. for example in monocytes (Handschug et al.. 1998: Vidon et al., 200 1). it seems

likely that cellular PEA sensitivity in humans could be modulated by this mechanism.

An additional limitation of our work is the fact that in our examination of the

modulation of macrophage PEA sensitivity by LPS pre-treatment we only used LPS From

E. coii. Additional data conceming the effect of LPS from P. aeniginosa would be

useful. since dunng PEA challenge the bacterial product likely to be an important

macrophage activator is LPS from P. aeniginosa. Interestingly. P. aeniginosa LPS is

less toxic cornpared to LPS from enteric Wlymembers. due to die composition of its

lipid .4 moiety (Kulshin et al.. 199 1: Goldberg et al.. 1996). This may have important

implications in the host's ability to down-regdate LRP and protect against PEA uptake

during P. aeniginosa infection: namely an inability to induce resistance to PEA during P.

neruginosu infection due to the relatively lower toxicity of P. aeniginosa LPS.

Recommendations for Future Research

In ritro investigations have determined that PEA entry into target cells is

mediated by the LRP (Kou~aset al.. 1992). However. merinvestigations are needed

to ven@ the importance of the LRP in PEA intoxication in vivo. Addressing this issue

has been hampered by the inability to generate a LRP "knock out*' by traditional

methods, due to the fact that LRP expression is necessary for embryonic development

(Willnow and Hem 1991). However. the recent creation of a mouse strain with deficient

hepatic LRP expression. using a Cre/Lox approach. has made available a valuable animal model with which to begin to address the importance of the LRP in PEA intoxication in vivo (Rohiman et ai., 1998). In addition to addressing the role of LWin PEA-mediated liver damage. investigations using this animal model may also identi@ secondary sites of

PEA-mediated damage and the conaibution that PEA-induced Iiver darnage ha in PEA- induced lethality.

We detemiined in this study that cellular sensitivity to PEA cm be modulated. and suggest that such an event may have consequences during P. aeruginosa infections. by altering PEA-rnediated darnage. A current challenge is to estend our in vilro findings by explonng whether induced alterations of cellular PEA sensitivity can occur in vivo. A report by Zehavi-Willner et al. (1991), in which it was reported that LPS treatment altered PEA-mediated mouse lethality. suggests that our findings are highly relevant. but clearly further mdies are warranted. It would be informative if host-derived signaling molecules. such as cytokines, present during P. aeruginosa infection, were exarnined to determine their effect on dictating cellular PEA sensitivity in vivo. Of particular relevance would be an investigation of TNF-a, since studies by Schümann et al. ( 1998) indicate that TNF-a plays a critical role in PEA-mediated liver damage. In addition to identiSing the cellular events that cm modulate cellular PEA sensitivity in vivo. it is important that the molecular mechanisms controlling this process are also investigated.

The results From the current study indicate that investigating the cellular alterations in

LRP expression would be an excellent staning point.

The LRP's attractiveness as a portal of entry is demonstrated by the wide range of pathogens and their products that have evolved to exploit this receptor as a means of entering host cells (Kounnas et al., 1992; Hofer et ai., 1994; Shakibaei and Frevert, 1996). Because of LW'S promiscuity in ligand interactions, it is likely that additional pathogens that are internalized by the LWmay be discovered in the funue. Therefore. the consequences of altering LWexpression may not be restricted to modulating cellular

PEA sensitivity. as examined in this study, but may also have implications in modulating ceilular sensitivity to infection and intoxication of several pathogens and toxins that exploit the LRP for cellular entry. An examination of whether or not this occun is clearly warranted.

Because of the limited number of studies that have addressed the ability of cells to modulate their sensitivity to PEA. there are many cellular mechanisms to be evarnined that may participate in this cellular phenornenon. Altering the level of LRP is one such mechanism that we have identified in this study, but it is possible that changes in the expression levels of any of the other cellular components which interact with PEA during intoxication also modulate PEA sensitivity. One such cellular component is the processing enzyme tUnn. which PEA exploits to generate an enzymatically active toxin fragment (Chiron et al.. 1994). Recently. it has been dernonstrated that fin expression is regulated by TGF-P (Blanchene et al.. 1997). Since furui is exploited by numerous intracellular-acting toxins and viruses for processing events required for intoxication and infection. changes in furin expression wouid likely have an impact on the cellular sensitivity to various toxins and vinises (Gordon and Leppla. 1994; Nakayama et al.

1997) and should clearly be examined in this context. SU!MMMtY AND CONCLUSIONS

The results of this investigation demonstrate that primary rat hepatocytes cultured on a collagen type 1 matrix markedly decrease their sensitivity to PEA in a time- dependent fashion. Compared to freshly isolated hepatocytes. those in culture for 24 and

48 hours were 2 1-fold and 57-fold less sensitive to PEA. respectively. Pretreaunent with

LPS significantly decreased PEA sensitivity in the rat macrophage-like cell line HSP in a dose- and time-dependent manner. in addition, LPS pretreatrnent for 24 hours at a concentration of 100 ng/ml decreased toxin sensitivity two-fold. We have aiso demonstrated that normal and transformed murine liver cells exhibit divergent PEA sensitivity: with transformed cells demonstrating greater PEA sensitivity (3100-fold) than their non-transformed counterparts.

Since PEA intoxication begins when PEA is intemalized by the LRP. we iniriated studies to investigate whether alterations in LRP expression were correlated with the obsrrved changes in cellular PE.4 sensitivity and thus represented a mechanism by which cellular PEA sensitivity can be modulated. When CO-administeredwith PEA. the natural

LRP antagonist R4P diminished PEA-mediated inhibition of protein synthesis in primary hepatocytes. HSP macrophage-like cells and transformed liver cells. These results demonstrate the importance of the LRP in PEA intoxication in these ce11 types.

Intemalization studies conducted with the LRP-specific ligand a2M* indicate that induced modulation of cellular PEA sensitivity is positively correlated with aitered cellular expression of functional ceil surface LRP. Cellular LRP mRNA levels were altered concomitantly with hctional cell surface LRP expression. demonstnting that the change in functional LRP levels may be attributable to changes in cellular LRP mRNA levels.

In addition, the importance of LRP levels in mediating PEA sensitivity were addressed indirectly by an assessrnent of cellular sensitivity to conjugate and mutant

PEA-toxins that do not utilize the LWfor cellular entry. These studies demonstrated that cellular conjugate and mutant toxin sensitivity was different than that observed for native toxin. indicating not only the importance of LRP expression in mediating PEA sensitivity. but dso revealing that changes in the expression of additional cellular components used by PEA during intoxication may also contribute to alteration in cellular

PEA sensitivity.

In conclusion. cellular sensitivity to PEA is not a stable feanire but rather. subject to modulation by long term cellular evrnts. such as transformation. and in the shoner tenn. by e'ruûcellular signaling molecules. Furthemore, changes in cellular PEA sensitivity were consistently associated with altered functional ce11 surface LRP expression and demonstrate that regulation of LRP expression is an important mechanism by which cellular PEA sensitivity can be moduiated. It is probable that the production of various LRP regulatory factors may be initiated in response to P. aeniginosa.

consequently altenng cellular and tissue PEA sensitivity during infection. Induced changes in cellular PEA sensitivity theoretically could be utilizcd as either a defense

mechanism by the hon or as a virulence strategy by P. nemginosa ro either lima or

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Blanch 2.5 ml HEPES (10 mM) 2.5 ml EGTA 20 ml 10X Hanks' balanced salt solution without ~a"or Mg '- 2.5 ml Penicillinistreptomicin 222.5 ml sterile wter

Perhsion Medium 147.5 ml William's E pH 7.4 2.5 ml (10 mM)HEPES 0.5 mgml Hepatocyte qualifieci collagenase

Attachent Medium 435 ml William's E pH 7.4 5.0 ml ( 10 mM) HEPES 5.0 ml (2 mb1) L-glutamine 100 pl (50 C/L) insulin 50 ml Fetal bovine sem 5 .O ml Penicillinistreptomycin 4.9 pl(IO-' M) Drxamethasone

Serum Free Medium 285 ml William's E pH 7.1 3.0 mI(10 mM)HEPES 3 .O ml (2 mM)L-glutamine 3 .O ml ( 10 mM) pyruvate 3.0 ml (0.35 mM)proiine 60 pl (50 Un,insulin 3 .O ml PeniciIlin/streprornycin 2.9 pl ( 1 O" M)Dexamethasone

Percoll densitv centrifugation solution 2 1.6 ml percoll 2.4 ml IOX Hanks' balanced salt solution \cithout ca2- and Mg2-

William's E medium 10.8 g of William's E commerciaily prepared powder (GibcolBRL) was dissolved in 95Oml of ddH20. 2.2 o of sodium bicarbonate was added to the medium and the pH adjusted ro 7.4. The meàium was then serile filtered. Hvchoxv ethvl piperazine ethane (HEPES) 119.3 g of HEPES (ICN) was dissolved in 450 ml of dm20and the pH adjusted to 7.4 using sodium hydroxide. The total volume was brought- to 500 ml and sterile filtered in10 50 ml aliquots.

L-pro line 0.405 g of L-proline was dissolved into 100 ml of Wil amasE medium pH 7.4 and sterile fi ltered.

Sodium pyruvate II g of ppvic acid !vas dissolved into 100 mi of Wil iams E medium pH 7.4 and sterile filtered- Materiais and solutions used in plasmid pnparatioos

P I (resuspension buffer ) 1 L 6.06 g Tris base 3.73 g EDTA.2H20 pH ro 8.0 100 mg base A

P2 (lvsis buffer) 1 L 8.0 g NaOH pellets 50 ml SDS (20%)

P3 (neutratization buffer) I L 294.5 g potassium acetate pH to 5.5

Luria-Bertani (LB) Medium 10 g of bacto-tryptone. 5 g of bacto-yeast extract and 10 g of Nacl were dissolved in 950 ml of ddH?O. The pH was adjusted to 7.0 and the total volume adjusted to I L. The solution was sterilized by autoclaving at 12 1OC for 25 minutes.

SOC Medium 10 g of baco-iryptone. 5 g of bacto-yeast extract and 0.5 g of NaCI were dissolved in 950 ml of ddH20. 10 ml of KCl(Z50 mM) was added to the solution and the pH was adjusted to 7.0. The volume of the solution was then adjusted to 1 L with ddH20 and the solution was autociaved at 1X°C for 75 minutes. Following autoclaving. 20 ml of sterile 1 M glucose was added. followed by 5 ml of stenle 2 M MgCl?.

Tris-borate (TB) bu ffer 54 g of Tris base. 27.5 g of boric acid and 20 ml of 0.5 EDTA @H 8.0) were dissolved in ddH20, and adjusted to a volume of 1 L to form a 5X stock solution. A IX solution was prepared by a 15dilution of the 5X TBEstock solution with ddH20. APENDIX III

.Materials and solutions used in RAP-GST isolation

3X YTA ,Medium 16 g of tryptone. 10 g of yeast extract and 5 g of NaCl was added to 900 ml of ddHzO. The pH was adjusted to 7.0 with NaOH and the volume \vas adjusted to 1 L. The medium was sterlilze by autoclaving for 20 minutes. Once the medium had cooled. 1 ml of 100 mgml ampicillin stock solution (final concentration of 100 pg/ml) was added.

Glutathione Elution Buffer An entire bottle of the provided Dilution Buffer (50 ml) was poured into the provided bonle containing Reduced Glutathione. which was then dissolved by gently shaking. The solution was dispenced into 10 ml aliquots. and stored at -20°C. IPTG The total conents of a via1 containing lyophilized IPTG was dissolved in 20 ml of sterile ddH20 (100 mM). The solution was dispensed into 1 ml aliquots and stored at -îO°C.

Glutathione Solution 5 ml of sterile ddH2O were added to the bottle containing reduced glutathione. which was dissolved by gently shaking. The solution tvas aliquated into microfuge tubes and stored at -20°C. Materials and solutions used in Northern Blot analysis

Diethvlpyocarbonate (DEPC)-A Water 1 ml of diethylpprocarbonate (DEPC) was added to 999 ml of ddH20 and stirred ovemight. A 1 :10 dilution of DEPC water to dMzO was prepared and autoclaved for 40 minutes at 121 OC.

Sodium dodecvl sulphate (SDS)( 10%) 100 g of sodium dodecyl sulphate was dissolved in 900 ml of DEPC-A water and heated to assist with dissolution. Concentrated HCl was used to adjust the pH to 7.2. The final volume kvas adjusted to 1 L with DEPC-A water.

Sodium chloride/sodium citrate buffer (SCC) (20X stock) 175.3 g of sodium chloride and 88.2 g of sodium citrate were dissolved in 800 ml of DEPC-A water. The pH was adjusted to 7.0 with NaOH and the volume adjusted to 1 L with DEPC-A water.

Sodium chloride/sodium phosphate buffer (SSPE)(tOX stock) 175.3 g of sodium chloride. 27.6 g of sodium hydrogen phosphate and 7.4 g of EDTA were dissolved in 800 ml of DEPC-A water. The pH was adjusted to 7.4 with NaOH and the volume adjusted to 1 L with DEPC-A water.

3 -W-Morpholino) Propane-Sul fonic acid (MOPS)buffer ( 10X stock) 41.2 g of MOPS. 10.89 g of sodium acetate and 3.72 g of EDTA were dissolved in 800 ml of DEPC-A water. The pH ivas adjusted to 7.0 with NaOH and the volume adjusred to 1 L with DEPC-A water.

Denhadt's solution (5Xstock) 5 g of Ficoll 400. 5 g of polyvinylpyrolidone and 5 g of bovine serum albumin were added to DEPC-A water to a final volume of 500 ml. The solution was sterilized by filtration and aliquoted into 50 ml and stored at 20°C.

TEN buffer 584 mg of sodium chloride. 158 mg of Tris HCI and 200 pl of 0.5 EDTA (pH 8.0) were added to 80 ml of DEPC-A water. The pH was adjusted to 8.0 and the final walume was adjusted to 100 ml with DEPC-A water.

Sephadex G-50 3 g of Sehadex G-50 (medium grade) was allowed to swell in DEPC-A water. The mixture was washed a single time in DEPC-A water and equilibrated with TEN buffer (pH 8.0). The sephadex preparation was sterilized by autoclaving and stored at 4°C. Induced alteration in PEA sensitivity of MH-3T3 fibroblast-like cells by transforming growth factor-P

IDs0values for NIH-3T3 fibroblast-like cells treated with TGF-P.

Fold Change in Tirne Iba(ng/ml) Ceii Type Treatment 1 1 1 PEA Sensitivity ! Effect of TGF-P on NIH-3T3 fibroblast-like cellular PE.4 sensitivity. NIH-3T5 cells were pretreated with TGF-P for (A) 24 hours at various concentrations or at (B)7nglml for various rimes. Crlls were then challenged with 100 ngml of PEA for 2 hours.

Following toxin cxposure. cells were incubated with medium containing [3~]leucinefor

2 1 hours. Each data point represents the mean 2 standard error of either two (A) or threr

(B) separate experiments. O 3 6 12 18 24 Time (hours) Sources of supplies and materials

Material Source

BNL CL2 celis Amencan Type Culture Collection Rockville. MD

BNL 1ME A7R. 1 cells Amencan Type Culture Collection Roc kville. MD

[~~'PI~CTP Amersham Life Sciences. Oakville. ON

1251 Amersham Life Sciences, Oakville. ON

Nylon membrane (Hybond N) Arnersham Life Sciences, Oakville, ON

RediprimeOG mixture Amersham Life Sciences. Oakville. ON

Biofbge 13R Baxter Scientific. Osterode. Germany

BamH I Boehringer-Mannheim. Laval PQ

EcoR1 Boehringer-Mannheim. Laval PQ

Ethidium bromide Boehringer-Mannheim. Laval PQ

Rnase A Boehringer-Mannheim. Laval PQ

Bio-RadTMprotein assay Bio-Rad Laboratones. Richmond, CA

GS250 Molecula. Imager Bio-Rad Laboratories. Richmond. CA

Molecular Analyst Version 2.1 sofnirare Bio-Rad Laboratories, Richmond. CA

Molecular biology agarose Bio-Rad Laboratories. Richmond, CA

Quick Spin Column Bio-Rad Laboratories. Richmond, CA

.brlamar B lueTPA Biosource International, Montreal, PQ

Sprague-Dawley rats Charles River, St. Constant, PQ

2-0 silk ligature Clinical Sciences, Universis. of Guelph Stenle Gauze Clinical Sciences, University of Guelph

Matrigelm Collaborative Research, Bedford, MA

Mauispersew ColIaborative Research . Bedford. MA

Vitrogen Collagen Corporation. Pa10 Alto. CA

Anhydrous ethy 1 alcohol Commercial Alcohols Inc.. Brampton. ON

Bacto-tryptone Difco Laboratones, Oakville. ON

Bacto-yeast extract Difco Laboratones, Oakville, ON

50 ml tubes Fisher, Scientific. Nepean. ON

Bovine serum albumin Fisher, Scientific. Nepean. ON

C hloro form Fisher, Scientific, Nepean. ON

EDTA Fisher, Scientific, Nepean, ON

Formaldehyde Fisher, Scientific, Nepean. ON

Formamide Fisher, Scientific, Nepean. ON

Isopropy 1 alcohol Fisher. Scientific. Nepean. ON

Mrthylamine HCl Fisher. Scientific. Nepean. ON

MOPS Fisher, Scientific. Nepean. ON

Sodium acetate Fisher, Scientific, Nepean. ON

Sodium chloride Fisher. Scientific. Nepean, ON

Sodium citrate Fisher. Scientific, Nepean. ON

Sodium hydrogen phosphate Fisher, Scientific, Nepean. ON

Sodium hydroxide Fisher. Scientific. Nepean, ON

Potassium acetate Fisher, Scientific, Nepean, ON

Potassium chloride Fisher. Scientific, Nepean, ON UV Crosslinker Fisher, Scientific, Nepean, ON

Micro plate reader Flow Laboratones, Mississauga ON

Bovine insdin Gibco/BRL, Burlington, ON

Collagenase Gibco/BRL. Burlington. ON

Dulbecco's modified Eagle's medium Gibco/BRL, Burlington ON

EBSS Gibco/BRL. Burlington. ON

Glucose GibcolBRL, Burlington. ON

Hank's Balance Salt Solution GibcoBRL. Burlington, ON

HEPES GibcolBRL. Burlington, ON

Magnesium chloride Gibco/BRL. Burlington. ON

Penicillin/streptomycin Gibco/BRL. Burlington. ON

RPMI medium Gibco/BRL. Burlington. ON

Salmon sperm DNA Gibco/BRL. Burlinpon. ON

TrizoITS' reagent Gibco/BRL. Burlington. ON

Trypan blue GibcoBRL. Burlington, ON

Williams' E medium Gibco/BRL. Burlington. ON

leucine ci ne ICN. Montrea1,PQ

Boric acid ICN. MontredPQ

Fetal bovine serum ICN. MontrealPQ

Liquid scintillation cocktail ICN, Montreai.PQ

SDS ICN. Montreal,PQ

Tris base ICN, MontredPQ

Triton X-I O0 ICN. Montreal-PQ Filter mats LKB Wallac, Turku, Finland

Plate hwester LKB Wailac, Turku, Finland

Influxm pinocytic cell-loading reagent Molecular Probes. Eugene. OR

Sodium pentobarbital MTC Pharmaceuticals, Cambridge. ON

Diaphot phase contrast microscope Nikon Instruments, Canada

F301 camera Nikon Instruments, Canada

PBS Pathobiology. University of Guelph. ON

Trypsin Pathobiology, University of Guelph. ON

CDNB Pharmacia Biotech. Baie d'lirfe. PQ

Ficol1400 Pharmacia Biotech. Baie d'Urfe. PQ

Flexi-prep kit Pharmacia Biotech, Baie d'Urfe. PQ

Glutathione Pharmacia Biotech, Baie d'Urfe. PQ

Glutathione Elution Buffer Pharmacia Biotech. Baie dwUrfe.PQ

Glutathione Sepharose JB Column Pharmacia Biotech. Baie d0Urfe. PQ

GST Detection Module Pharmacia Biotech. Baie doUrfie. PQ

GST purification module Pharmacia Biotech. Baie d'Urfe. PQ

IPTG Pharmacia Biotech. Baie d'Urfe. PQ

Sephades (3-50 Pharmacia Biotech. Baie d0Urfe. PQ

W-Spectrophotometer Pharmacia Biotech. Baie d'Urfe. PQ

Iodobeads Pierce Chernicals Company. RocMord IL

Rotating Hybridization Incubator Robbins Scientific, Sunnyvale, CA

24-well plates Sarstedt hc.. St. Leonard, PQ

60 mm culture dishes Sarstedt Inc.. St Leonard. PQ 96-well plates Sarstedt Inc., St. Leonard, PQ

T-75 flasks Sarstedt Inc., St. Leonard, PQ

AmpiciIIin Sigma, Oakville, ON

Diethylpyrocarbonate Sigma, Oakville. ON

L-glutamine Sigma. Oakville. ON

L-proline Sigma. Oakville. ON

LPS Escherichia coli 0 1PB8 Sigma. Oakville, ON

Percoll Sigma, Oakville. ON

Dexamethasone Vetoquinol Canada Inc.. Joliette. PQ