Inherent Flaw of Cholesterol Processing in Cell

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INHERENT FLAW OF CHOLESTEROL PROCESSING IN CELL

CULTURE AND IN VIVO MODELS OF CYSTIC FIBROSIS

NICOLE MARIE WHITE

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Thomas J Kelley

Department of Pharmacology

Case Western Reserve University

January 2007

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

______

candidate for the Ph.D. degree *.

(signed)______(chair of the committee)

______

(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein.

DEDICATION

To my parents who exemplify the contentment and fulfillment in life

that I aspire to, and to

Chris

whose continued support and belief of me served

as a buoy when I faltered.

TABLE OF CONTENTS

LIST OF FIGURES ...... iii

LIST OF ABBREVIATIONS...... iv

ACKNOWLEDGMENTS...... vi

ABSTRACT...... ix

CHAPTER 1: BACKGROUND...... 1

The inflammatory response in cystic fibrosis...... 1 Altered cell signaling events in cystic fibrosis ...... 5 Role of the isoprenoid/cholesterol pathway in cystic fibrosis disease pathology ...... 8 Evidence of altered metabolism in cystic fibrosis ...... 12 Niemann-Pick disease type C as a mechanistic model of the cystic fibrosis inflammatory response ...... 16 Statement of Purpose...... 18

CHAPTER 2: MECHANISTIC SIMILARITIES BETWEEN CULTURED CELL MODELS OF CYSTIC FIBROSIS AND NIEMANN-PICK TYPE C...... 27

Abstract...... 27 Introduction...... 28 Methods...... 30 Results ...... 33 CF-Like Cell Signaling Alterations in NPC Fibroblasts ...... 33 An Examination of NPC-Like Phenotypes in CF Model Cells ...... 34 Discussion...... 37

CHAPTER 3: ALTERED CHOLESTEROL HOMEOSTASIS IN CULTURED AND IN VIVO MODELS OF CYSTIC FIBROSIS ...... 58

Abstract...... 58 Introduction...... 59 Methods...... 61 Results ...... 69 Discussion...... 75

CHAPTER 4: ACUTE AND CHRONIC INHIBITION OF CFTR FUNCTION CAUSES CHOLESTEROL ACCUMULATION...... 98

Abstract...... 98 Introduction...... 99 Methods...... 102 Results ...... 107 Acute inhibition of CFTR function initiates altered cholesterol processing...... 107 Link of CFTR function in mouse models to disrupted cholesterol processing...... 110 Discussion...... 113

CHAPTER 5: SUMMARY, FUTURE DIRECTIONS, CONCLUSIONS...... 136

Summary...... 136 Implications for similar pathways important in disease pathology of CF and NPC...... 136 Deficient cholesterol transport elicits a CF-like cytokine production...... 139 Isoprenoid/cholesterol synthesis as a mechanism for cell signaling alterations in CF...... 140 Cholesterol relationship to CF fatty acid deficiency ...... 142 CF intestinal phenotype...... 146 Global CF metabolic disorders ...... 148 Preliminary Results and Future Directions...... 150 Indirect cholesterol accumulation caused by ceramide accumulation...... 152 Altered endosomal pH as a cellular trigger for cholesterol accumulation ...... 154 Altered endosomal calcium as a cellular trigger for cholesterol accumulation...... 155

REFERENCE LIST...... 168

LIST OF FIGURES Figure 1-1. De novo isoprenoid/cholesterol synthesis pathway...... 21 Figure 1-2. Sterol regulatory element binding protein cholesterol regulation...... 23 Figure 1-3. Major routes of cholesterol and CFTR trafficking...... 25 Figure 2-1. Reduced induction of (NOS2) in NPC compared with wt fibroblasts...... 42 Figure 2-2. Increased STAT1 protein expression in NPC compared with wt fibroblasts...... 44 Figure 2-3. Western blot of increased RhoA expression in NPC and CF model systems...... 46 Figure 2-4. Decreased Smad3 expression in NPC and CF model systems...... 48 Figure 2-5. Increased filipin staining in CF epithelial cells compared to controls...... 50 Figure 2-6. RhoA and STAT1 protein expression in IB3 (CF) and S9 (CFTR-corrected) cells. .52 Figure 2-7. ASM activity in pCEPR (CF) and pCEP (wt) 9/HTEo cells...... 54 Figure 2-8. Increased NPC1 mRNA levels in CF model cells compared with controls...... 56 Figure 3-1. Unesterified cholesterol accumulation in CF tissue...... 82 Figure 3-2. NBD-cholesterol accumulation in two CF cell culture systems...... 84 Figure 3-3. Lysosomal storage of NBD-cholesterol in CF cells...... 86 Figure 3-4. Microelectrode determination of membrane cholesterol content...... 88 Figure 3-5. Increased sterol response element (SRE) response in CF cells...... 90 Figure 3-6. Increased de novo cholesterol synthesis in Cftr-/- mouse tissue...... 92 Figure 3-7. Impaired cholesterol transport causes CF related cytokine release...... 94 Figure 3-8. The role of DHA in cholesterol transport...... 96 Figure 4-1. NBD-cholesterol accumulation in INH-172 treated control epithelial cells...... 122 Figure 4-2. Increased SRE response in INH-172 treated control epithelial cells...... 124 Figure 4-3. Microelectrode determination of INH-172 treated cell membrane cholesterol...... 126 Figure 4-4. Microelectrode determination of mouse tissue membrane cholesterol...... 128 Figure 4-5. De novo cholesterol synthesis in CF mouse tissue compared to matched controls. .130 Figure 4-6. MicroPET scan of 11C-acetate incorporation in CF mice...... 132 Figure 4-7. Quantification of 11C-acetate tissue count...... 134 Figure 5-1. Innate pro-inflammatory signaling events of CF...... 160 Figure 5-2. Altered pH affects cholesterol trafficking...... 162 Figure 5-3. Altered calcium reverses CF-like unesterified cholesterol accumulation...... 164 Figure 5-4. Altered calcium reverses CF-like NBD-cholesterol accumulation...... 166

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LIST OF ABBREVIATIONS

ΔF/ΔF: CFTRF508/F508 mice

ABCA1: ATP binding cassette protein A1

ASM: acid sphingomyelinase

BAL: Brochioalveolar Lavage

Ca2+ : calcium

CF: cystic fibrosis

CFTR: cystic fibrosis transmembrane conductance regulator

Cftr-/-: CFTRtmu/cftr mice

ER: endoplasmic reticulum

GC/MS: Gas Chromatography/ Mass Spectrometry

GGPP: geranylgeranyl pyrophosphate

GPN: glycy-L-phenylalanine-beta-naphthylamide

HMG-CoA: 3-hydroxy-3-methylglutaryl-Coenzyme A

IL-6: interleukin 6

IL-8: interleukin 8

IPP: isopentenyl pyrophosphate

FPP: farnesyl pyrophosphate

NBD-cholesterol: 25-[N-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)-methyl]amino]-27- norcholesterol

NFκB: nuclear (transfection) factor-κB

NPC: Niemann Pick disease type C

NO: nitric oxide

NOS2: nitric oxide synthase 2

PA: Pseudomonas aeruginosa

PBS: phosphate-buffered saline

R/R: CFTRR117H/R117H mice

RT-PCR: reverse transcriptase/polymerase chain reaction

SCAP: SRE binding protein cleavage activation protein

SRE: sterol regulatory element

SREBP: SRE binding protein

STAT1: signal transducer and activator of transcription-1

ACKNOWLEDGMENTS

My success is not my own, and sharing it with others makes it that much sweeter. Throughout the last five years, I have had many positive influences guiding me. One such instance was my fluke meeting with Mitch Drumm, who helped determine my research direction at Case. I conducted a brief research rotation in Mitch’s lab, but this experience opened up another door. Not only did I first learn about cystic fibrosis, the people in the field at Case, but I met Tom Kelley, my future advisor. It was Mitch who thought Tom’s research would interest me. He continued to be helpful in my development into a scientist. I’m thankful for the opportunity to have worked with him.

I was Tom’s first graduate student; we took a big risk agreeing to work together for five years. I was uncertain about joining such a young and small lab, but joining the Kelley lab is one of my better decisions in grad school. Tom is a great mentor, who was willing to bend and learn in the student/mentor relationship process. Tom is a great scientist who is well respected in the Case and CF communities. He has involved me in cutting-edge research that has been both rewarding and exciting. I am grateful for all I’ve learned from Tom. Beyond scientific pursuits, Tom has earned by respect. Occasionally, the frustration of graduate school had proven very difficult for me and many times I’ve called upon Tom’s understanding or guidance. In his way, he was compassionate and helpful. Having this support was incredibly helpful to me. Tom has also taught me to be more positive about situations. I’ve applied this outlook scientifically, but also in other aspects of my life. One of

Tom’s greatest attributes is the healthy balance between career and family he has learned.

Both Mitch and Tom enjoy their work but are also very involved in their families. I’ve watched them both manage this lifestyle, and I’ve taken notes for my future choices.

I would like to thank my other committee members: Dr. Maguire, Dr. Davis, and Dr.

Siegal. This group helped me to critically view my work and determine areas necessary of

improvement. I would also like to thank Dr. Amy Wilson-Delfosse who sat in on my oral

defense. I would also like to thank the members and my friends from the 8th Floor BRB.

Their support, both academically and socially, has helped me.

The above mentioned people helped me succeed while at Case, but there are many others who were involved in my struggles and successes prior to this point. My parents are wonderfully supportive of all my endeavors. They have provided an environment with space

for growing and learning, but they are involved enough to offer advice and encouragement

when its asked for. The look of pride in their eyes during/after my oral defense cemented this

huge accomplishment for me. I will always fondly remember joining my Mom in her lab and

“curing” Down’s syndrome on my days off from school. Although I didn’t know it at the

time, it was these experiences that shaped my future quests. It was there that I first learned

the magnificent and important applications of scientific research.

It is my father’s creative and curious nature that is present in both my brother and me.

At any time, surveying his coffee table reveals books on religion, ancient societies, and travel

guides from recent adventures. I’m continually amazed at the breadth of this interests and

ability to ingest it all. I tackle projects and problems as my father taught me approaching

things cautiously and nervously, and then when I’m ready, getting completely involved and

committed to the task. I’m thankful for my father’s encouragement and involvement in my

interests. It is evident how proud he is of his family. Inevitably, I need him to cycle through

the same basic pep talk every few years, but it helps to know how genuinely and

unconditional proud someone can be of me. The excitement and interest my parents have in

vii

my activities is infectious. All though I have tried to create my own life and style, I am

realizing the similarities I share with them both. I am proud of the women I am becoming

because them.

My parents provided a positive environment allowing me to grow, but it was my

brother, Brian, who set the path. He set the standards early and high for the White kids. It

was his determination of continual education, both formally and informally, that served as

my guide. I watched him reach for, and accomplish, these goals. My brother is one of my

greatest role models. I like to tell my friends that I have a brother who is getting a PhD in

Computer Science and a minor in Physics, because that interests him. I don’t know many

people with this vigor in knowledge and achievement. By attempting to follow is example, he

has taught me to challenge myself and be proud of what I accomplish.

An equally driven and supportive individual is my boyfriend, Chris. I admire his

devotion in all of his pursuits. There is grace and ease in which he fantastically accomplishes

each tack put before him, from his athletic prowess to his academic success. He has also set a

high standard of achievement to follow as there is no idea too outrageous or unattainable not

to at least be attempted. But he has shown me, by continual optimism and encouragement

that he will stand by me and help to accomplish any goal. He is always there, at the end of a

running race, to direct me to the finish just as he was there at my thesis defense. We did it! I

look forward to the future experiences and adventures we will share together.

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Inherent Flaw of Cholesterol Processing in Cultured and In Vivo Models of Cystic Fibrosis

ABSTRACT

Nicole M White

Cystic fibrosis (CF) is caused by a loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leading to lung infection and inflammation. Mechanisms that cause severe inflammatory responses in CF are not well established. It is hypothesized that

CF systems possess a flaw in intracellular cholesterol transport initiating increased isoprenoid/cholesterol synthesis and leading to pro-inflammatory events. To verify the role of isoprenoid/cholesterol synthesis in cell signaling regulation a model system was explored.

Niemann-Pick type C (NPC) disease is a lysosomal cholesterol storage disease due to improper intracellular cholesterol transport and increased isoprenoid/cholesterol synthesis.

Evidence of signaling similarities with CF models make NPC a functionally relevant model.

Additionally, models of CF exhibit increased unesterified cholesterol content as determined in NPC. CF epithelium of human airways further illustrates an accumulation of cholesterol compared to controls. Altered cholesterol homeostasis is indicated by an increase in membrane cholesterol content in CF cell culture and excised nasal epithelium. GC/MS analysis of deuterium incorporation of cholesterol in mouse tissue revealed that Cftr-/- mouse lung have an increase of newly made cholesterol compared to matched controls.

Confirmation of these results was obtained utilizing microPET technology to image live mice with 11C-acetate labeling indicating greater acetate incorporation in CF mice, specifically the

lung, compared to matched controls. These data establish an accumulation of intracellular cholesterol and an increase of isoprenoid/cholesterol synthesis.

Evidence of CFTR mediation of cholesterol processing was determined by examining different mouse models of CF disease severity. Comparison of CFTR mutations determined different phenotypic outcomes as measured by cholesterol parameters. A severe and mild mutation of CFTR, Cftr ΔF508/ΔF508, (ΔF/ΔF) and Cftr R117H/R117H (R/R) respectively, were compared for rates of de novo cholesterol synthesis. ΔF/ΔF mice exhibited nearly two fold increases in newly made synthesis compared to R/R mouse lung. Greater acetate incorporation and excised nasal epithelium membrane cholesterol were exhibited in ΔF/ΔF mice compared to R/R mice. All parameters measured in CF were significantly above levels in matched littermates. These data demonstrate by multiple approaches an inherent lesion in cholesterol homeostasis in CF that correlates with CFTR genotype severity.

CHAPTER 1: BACKGROUND

The inflammatory response in cystic fibrosis

Cystic fibrosis (CF) is the most common lethal autosomal recessive disease,

affecting approximately one in 25,000 Caucasian live births (1). The primary

physiological defect causing CF was identified in 1989 as mutations in a single gene

termed cystic fibrosis transmembrane conductance regulator (CFTR), a gene found to code for a cAMP-dependent chloride channel (2-4). A lack of CFTR at the apical membrane of epithelial cells causes decreased chloride secretion and increased sodium reabsorption (5, 6). CF is a multisystem disease, with CFTR-expressing tissues including sweat ducts, nasal epithelium, bile ducts, pancreas, lung, and intestinal epithelium (7). CF is characterized by thick mucus that lines and obstructs the bile ducts and airways.

A major manifestation of CF pathology is severe lung disease, causing 90% of the morbidity and mortality (8). Early in life CF patients show bacterial colonization of

Pseudomonas aeruginosa (PA) and continual chronic infection throughout life (9, 10).

CF is characterized as an excessive and prolonged pro-inflammatory response.

Characteristic markers of CF inflammation are increased pro-inflammatory cytokine production of interleukin (IL) 6 and 8, leukotriene B4, and neutrophil infiltration with decreased production of the anti-inflammatory IL-10 cytokine in the bronchoalveolar lavage (BAL) fluid (11-14). CF inflammation persists as an initial neutrophilic response that does not progress to a macrophage- and lymphocyte-mediated inflammatory response, as is normal for inflammatory events (11, 15).

Controversy persists over the primary cause of inflammation and lung disease in

CF patients. One hypothesis speculates severe lung disease is caused by an impaired

innate defense mechanism in the lung initiated by an electrolyte imbalance due to a lack

of CFTR at the epithelium surface (16). Altered sodium and chloride transport passively

affects the water concentration to decrease the airway surface liquid (ASL) above the epithelium. This affects ciliary beating and mucosal clearing, thus stopping or slowing

bacterial clearance, and contact of the ASL mucus layer with the cell surface.

Continuation of mucin secretion by goblet cells leads to subsequent formation of a thick

mucus and eventual plugging of the airways [reviewed in (17, 18)]. PA bacteria can

infiltrate this environment, adhere to cell surface mucins, and form biofilms resistant to

neutrophil penetration. These events of PA colonization set the stage for chronic infection.

However, there is also substantial evidence to support the idea of a “primed” lung

with an intrinsic imbalance of inflammatory mediators due to lost CFTR function. This is

supported by the evidence in patients of an excessive inflammatory response in the

absence of bacterial colonization. Comparison of CF and healthy 12 month old infant

BAL fluid revealed increased neutrophil count and IL-8 production, common

inflammatory markers to CF disease, in CF infants. A plausible conclusion for the

presence of inflammation is bacterial infection. However, stringent determination of

bacterial and fungal levels indicated approximately half of the CF patients were negative

for any pathogens with none of the patients colonized with the most common CF bacteria,

P. aeruginosa (PA). Separation of the CF patients based on the presence of bacterial

cultures determined a greater inflammatory response from patients with pathogens;

nevertheless, an elevated response remained in the CF negative group compared to

controls (12). Muhlebach, et al. verified that levels of inflammatory markers are noticeably increased in CF patients compared to controls independent of bacterial infection. BAL fluid from 50 CF patients, approximately half were infected with PA, showed significant elevations of IL-8 and neutrophil count after adjusting for bacterial burden compared to healthy and infected control groups (13). Another study of older patients, mean age of 19, showed similar conclusions. Although CF BAL fluid cytokine levels were significant above that of healthy controls, direct comparison of cytokines IL-

1, IL-8, and TNF-α found in BAL fluid of PA-positive and PA-negative CF groups were not significantly different from each other (15). These studies of BAL fluid indicate a response in the absence of bacterial infection in CF patients significantly different from both healthy control and other respiratory disease groups early in life and continuous throughout it. These studies highlight the intricacies of CF disease pathology and illuminate a pivotal role of CFTR function in the intracellular immune response not fully explained by an impaired innate defense mechanism of the lung.

Cell culture models of CF also support the idea of a direct connection of CFTR to the CF inflammatory response. Human airway epithelial cells overexpressing the regulatory (R) domain of CFTR, 9/HTEo-pCEPR, behave similarly to CF epithelium with the characterization of less chloride conductance than 9/HTEo cells transfected with the pCEP empty vector (19). Phosphorylation of the R domain allows channel opening and activation. Investigating these cells for inflammatory markers relevant in CF revealed increased PA binding corresponding to increased production of IL-8 compared to control cells (20). This cell line is a sufficient tool to study CF related inflammation as it recapitulates alterations previously studied in ΔF508 CFTR epithelium while delineating

the importance of CFTR function to regulate bacterial response in the absence of a

trafficking defect.

CF animal models also support the hypothesis of CFTR function tied directly to

severe lung disease. Mouse models of CF inflammation indicate an inflammatory

response prior to bacterial infection. Human fetal tracheal grafts implanted in immunodeficient mice produce a model of naïve CF and non-CF airways. Prior to bacterial infection, naïve CF grafts exhibited increased IL-8 content and neutrophil/macrophage localization compared to non-CF graphs. Upon PA challenge, naïve CF grafts displayed leukocyte infiltration, epithelial shedding, and bacterial binding

within 3 hours of infection. Conversely, non-CF airway grafts initiated these events six

hours post infection (21). This model supports the theory of CF airways with an imbalanced and hyperactive inflammatory environment present early in development

prior to bacterial exposure. Mice lacking Cftr (Cftr-/-) provide another model for an

excessive inflammatory response inherent to defective CFTR. Pseudomonas-laden

agarose beads were employed as a model of infection to investigate a difference in Cftr-/-

and wild-type littermate bacterial response. No difference in bacterial colonization was

observed between CF and control mice; however, increased inflammatory mediators and

leukocyte concentrations in the BAL fluid and significant mortality in the CF mice

compared to littermates was determined three days post infection (22). It can be

concluded from the bead model studies that altered bacterial colonization is not sufficient

to cause the excessive inflammatory response in CF, adding additional evidence for the

importance of CFTR function mediating the inflammatory response.

Altered cell signaling events in cystic fibrosis

There is no established view linking CF pathology to its etiology of disrupted

CFTR function. Therefore, inflammation is studied extensively to functionally connect

CF-related alterations in cell signaling pathways to CFTR. Many of these alterations are nuclear (transcription) factor kappa (NFκB) mediated as noted by increased activation in

CF model systems (23-26). NFκB is a known regulator of cytokines and pro- inflammatory molecules, established to be important in CF disease pathology, such as inducible nitric oxide synthase, IL-6, and IL-8 (27). Cellular stress associated with mistrafficked CFTR accumulated in the endoplasmic reticulum (28) and binding of PA to asialoGM1 glycosphingolipid receptor (29, 30) are partial explanations for increased

NFκB activity in CF. Additionally, there is evidence for a dysregulated inflammatory response in CF independent of cellular stress. Blackwell’s group investigated bronchial epithelial cell models of CF and determined decreased inhibition of NFκB due to hypophosphorylated IκK-β (24). More recently, another group discovered a failure of

CFTR null mice to produce IκK-α once it was degraded to explain activation of NFκB and prolonged inflammation in these mice compared to controls (26). Other groups have confirmed an intrinsic dysregulation of the inflammatory response in CF. Comparison of different CFTR mutations in cell lines for NFκB activation determined that independent of a CFTR trafficking defect, there was increased NFκB activation and IL-8 expression in

CF cells compared to controls (20, 31).

Other inflammatory aberrations of CF are not driven by NFκB activation.

Increased exhaled nitric oxide (NO) is a hallmark of various inflammatory lung diseases, such as asthma and bronchiectasis (32). NO, an antibacterial agent normally simulated by

bacterial lipopolysaccharide of PA in the epithelium of the airway, is an important first

defense against bacterial challenge (33). Although CF patients have chronic infection of

PA there are lower levels of exhaled NO compared to healthy controls (34-37). This alteration in primary response to bacterial challenge may help explain the excessive inflammation present in CF. A mechanism to understand attenuated levels of NO in CF is to investigate cell signaling molecules known to regulate NO levels. It was established that human, mouse, and cell culture models of CF epithelium exhibit decreased levels of

inducible nitric oxide synthase (NOS2) (38, 39). NOS2 is necessary to cleave L-arginine

to form NO.

Studies of decreased NOS2 levels support the hypothesis of a defective inflammatory response directly produced by altered CFTR function. Cftr-/- mice

expressing human CFTR in the intestine using the fatty acid binding protein (FABP) promoter (FABP-hcftr) were used to examine the effect of tissue specific effect of CFTR

correction. Confirmation of CFTR function in FABP-hcftr mice was revealed with

corrected electrophysiological properties in the ileum of these mice comparable to wild-

type mice. However, these mice exhibited a potential difference in the nasal epithelium similar to Cftr-/- mice (40). Evaluation of NOS2 expression levels in FABP-hcftr mice

showed increased expression compared to Cftr-/- mouse ileum, but exhibited reduced expression of NOS2 levels similar to Cftr-/- nasal epithelium. Analogous results were shown in other CFTR corrected models. Receptor-mediated CFTR gene delivery to the

epithelium of Cftr-/- mice showed a dose- and time-dependent correction of nasal

potential differences and NOS2 levels and no change in control mice (41). Genetic

models and gene therapy techniques of targeted CFTR insertion and correction both

corrected electrical potential differences and NOS2 expression levels. These mouse models indicate the importance of CFTR function in signaling alterations.

Exploration of upstream mediators revealed NOS2 regulation via signal transducer and activator of transcription-1 (STAT1). Although protein levels of STAT1 are increased in nasal epithelium of Cftr-/- mice (42), activity of this protein is downregulated due to protein inhibitor of activated STAT1 (PIAS1). A mechanism predicted to be controlling these cellular events was the small signaling GTPase, RhoA. It was demonstrated in vascular smooth muscle cells that decreased RhoA increased NOS2 expression (43). These small GTPases are known to regulate such functions as cell migration, protein trafficking, and inflammation (44). A cell line overexpressing RhoA revealed CF-like protein patterns of reduced STAT1 activity, increased expression of

PIAS1, and reduced NOS2 expression (45), supporting the hypothesis of increased RhoA in CF model systems. Increased RhoA expression and activity was determined in CF mouse nasal epithelium and CF-like airway epithelial cells (45). It was also demonstrated that inhibition of RhoA leads to increased NOS2 expression in airway epithelial cells via regulation of STAT1.

Other unrelated signaling pathways were determined to be altered in CF model systems. Transforming growth factor (TGF)-β1 has several roles in modulating cytokine production, such as reduced levels of IL-8 (46); moreover, increased expression of TGF-

β1 was implicated in more severe pulmonary disease (47). Closer investigation of this pathway revealed that Smad3 is reduced in cell culture and Cftr-/- nasal epithelium (48).

Howe et al. reported the regulation by TGF-β1 signaling to control the levels of CFTR function of CFTR. This group showed an inverse relationship between chloride transport

of CFTR and TGF-β1 signaling (49). The premise behind studying cell signaling events

in CF is that all are attributed to lost CFTR function and diverge from a common

upstream signaling intermediate. Modulating these pathways revealed a commonality of

proper modification and activation by the isoprenoid/cholesterol pathway.

Role of the isoprenoid/cholesterol pathway in cystic fibrosis disease pathology

Data suggest the link between increased RhoA GTPase activation and decreased

TGF-β1/SMAD3 signaling in CF models is isoprenylation-dependent activation of each pathway. Isoprenylation is a protein modification system adding 15 or 20 carbon isoprenoid units to proteins therein properly localizing them to the membrane for activation. GTPases require prenylation, farnesylation or geranylgeranylation, to be properly localized and activated (50-52). It was also determined that isoprenoids directly interact with SMAD3 promoter regions for transcriptional regulation of the pathway (53).

Cholesterol and isoprenoids are both produced from acetyl coenzyme A (acetyl-

CoA) through an addition of a 5-carbon isoprenoid compound. Acetate and Coenzyme A produce acetyl-CoA. Acetyl-CoA is first converted to 3-hydroxy-3-methyglutarly-CoA

(HMG-CoA) through the actions of HMG-CoA synthase. In the committed step in the pathway, HMG-CoA conversion to mevalonate is catalyzed by HMG-CoA reductase.

The conversion of mevalonate to the inorganic phosphate, isopentenyl pyrophosphate

(IPP), and further to farnesyl pyrophosphate (FPP), serves as an important branch point in

this pathway. This 15-carbon isoprenoid, FPP, produces squalene and ultimately

cholesterol. Conversely, FPP can also synthesize isoprenoids to activate and influence

other cell functions by conversion to geranylgeranyl pyrophosphate (GGPP). FPP and the

more stable GGPP modify and activate various Rho and Rac GTPase proteins. The

pathway of isoprenoid/cholesterol synthesis is diagramed in Figure 1-1.

One mechanism to control intracellular cholesterol levels is the sterol regulatory

element binding protein (SREBP) located in the membrane of the endoplasmic reticulum

(ER). It is SREBP-cleavage-activating protein (SCAP) that senses the lack of cholesterol

at the ER via its five membrane sterol-sensing domain. Next SCAP interacts with SREBP and moves from the ER to the Golgi. SREBP protein is cleaved by proteases and consequently the N-terminus translocates into the nucleus to increase transcription of genes regulated by the sterol regulatory element (SRE) as diagrammed in Figure 1-2

[reviewed in (54)]. Genes containing SRE regions include important enzymes within the

isoprenoid/cholesterol pathway, such as HMG-CoA synthase, HMG-CoA reductase,

farnesyl-diphosphate synthase, and squalene synthase. The function of these enzymes

within the isoprenoid/cholesterol pathway is depicted in Figure 1-1. Elevated activation

of these genes lead to increased isoprenoid/cholesterol synthesis and activation of

isoprenoid-dependent pathways such as GTPases.

Indirect evidence for increased activation of the isoprenoid/cholesterol pathway is

illustrated by increased protein expression and activation of RhoA in CF models (45).

Confirmation and support for the importance of isoprenoid regulation in CF was shown by other groups. Direct evidence of increased isoprenoid synthesis was obtained from microarray analysis of messenger RNA and matched control small intestine of mouse models. Multiple enzymes important for metabolic processes were increased in CF mice small intestine compared to controls. Specifically, isoprenoid enzymes farnesyl disphosphate synthetase and squalene synthase/farnesyl diphosphate farnesyl transferase

were increased in the CF mice compared to matched controls (55). Additional support for increased isoprenoid/cholesterol synthesis in CF models was shown by manipulating the pathway with pharmaceutical compounds. Inhibition of cholesterol synthesis with mevastatin, an HMG-CoA reductase inhibitor, influenced NOS2, RhoA, and STAT1 signaling by reversing protein expression patterns normally present in CF models (45, 56).

Isoprenoid mediated activation of NOS2 was shown to influence NOS2 directly and through RhoA activation in CF models. Inhibiting cholesterol synthesis with mevastatin increased RNA levels in epithelial cells transfected with NOS2-luciferase. Addition of isoprenoid intermediates farnesyl pyrophosphate (FFP) or geranylgeranyl pyrophosphate

(GGPP) reversed mevastatin induced NOS2 levels (56). It is established that NOS2 is negatively regulated by RhoA protein activated by geranylgeranyl isoprenylation (57, 58).

Additionally, inhibited RhoA with a Rho-associated kinase (ROCK) inhibitor in CF cells led to increased activation of NOS2 protein (56). These studies were confirmed in vivo with the restoration of NOS2 protein in nasal epithelium in mice treated with mevastatin via osmotic pumps for 72 hours (45).

Intervention in the isoprenoid/cholesterol pathway also corrected CF signaling changes in the TGF-β1 pathway. Two different CF models of human epithelium exhibited an increase in TGF-β1 Smad3 protein expression by inhibiting isoprenoid production with farnesyl or geranylgeranyl transferase inhibitors (53). This regulation appears to be at the transcriptional level via activation of Sp1/Sp3 sites at the SMAD3 promoter. As predicted all of the previously studied cell signaling pathways in the TGF-

β1 pathway and RhoA/NOS2 appear to be mediated by isoprenoids.

Various groups have implied aspects of CFTR regulation as isoprenoid dependent.

These groups speculate that maintenance of intracellular homeostasis requires an intricate

regulation between isoprenoid synthesis and CFTR trafficking. Initial experiments

identified this connection by utilizing an HMG-CoA reductase inhibitor, lovastatin, to

inhibit cholesterol synthesis. The result showed a decrease in chloride current caused by a lack of CFTR trafficking to the plasma membrane (59). Inhibiting cholesterol synthesis consequently inhibited isoprenoid production. Inhibition caused by lovastatin was rescued, in two different cell lines, with the addition of the isoprenoids, FPP or GGPP; however, there was no change in CFTR function with the addition of cholesterol. The

distinction of intermediate products necessary for CFTR function highlight the

significance of isoprenoid production within the cholesterol pathway for protein

trafficking. More direct evidence of isoprenoid action on CFTR function was recently

delineated. It is known that various isoprenoid dependent proteins such as small GTPases

are fundamental chaperones (44). Cheng et al. determined that a GTPase in the Rho

family, TC10, is required for selective trafficking, as well as functioning of CFTR at the

plasma membrane (60).

Inhibition of CFTR function through inhibition of isoprenoid/cholesterol synthesis and trafficking of CFTR by an isoprenoid activated Rho protein reveal an intimate connection between CFTR function and isoprenoid synthesis. In chronic conditions of altered CFTR function at the plasma membrane there is presumably a constant cellular response of increased isoprenoid synthesis and activation of small GTPases. Hence, in CF it is hypothesized that due to a loss of CFTR function there is a persistent increase of isoprenoid/cholesterol synthesis. The increase of isoprenoids and activation of Rho

proteins initiates pro-inflammatory cell signaling events previously determined in CF.

Past studies do not address the origin of an intimate cellular correlation between

isoprenoids and CFTR or what cellular response the cell is sensing to initiate increased synthesis to correct CFTR function. Moreover, the consequences of chronic compensation for a lack of CFTR function are not presented. These questions propelled the current body of work.

Evidence of altered metabolism in cystic fibrosis

Although implicating isoprenoid/cholesterol synthesis in CF pathophysiology is novel, indications of other metabolic changes are prevalent in CF disease. Pathology in the intestine of CF patients led to the hypothesis of malabsorption and malnutrition, and more recently a primary defect in lipid metabolism due to CFTR dysfunction. Fatty acid and cholesterol synthesis share a common precursor molecule of acetate and common regulatory mechanisms through the activation of SREBP proteins [reviewed in (61)]. The commonalities of these pathways provide interesting potential interconnections and implications in CF disease pathology. The preliminarily results of this are presented in

Chapter 3.

Eighty five percent of CF patients present with an essential fatty acid deficiency

(EFAD) [reviewed in (62)]. EFAD in CF is well established as a decrease in linoleic acid and docosahexaenoic acid (DHA) and an increase in arachidonic acid (AA) (63-65).

These essential fatty acids are not synthesized by humans but obtained through diet (66).

Linoleic acid and AA are the beginning and the end (respectively) of the omega-6 pathway. AA is important for mediating inflammatory processes as it is the precursor for

the generation of eicosanoids such as prostaglandins, thromboxanes, and leukotrienes

(66). Prostaglandins and leukotrienes have many pro-inflammatory actions such as

production of IL-6. Increased IL-6 production is a hallmark of CF lung disease. Therefore,

because their altered levels are thought to have profound effects in CF disease pathology,

fatty acids are studied extensively.

Numerous factors may contribute to an altered fatty acid profile in CF.

Historically, it was speculated that malabsorption and malnutrition due to pancreatic

insufficiency resulted in EFAD (67, 68); but, similar fatty acid profiles are present in well

nourished patients without pancreatic insufficiency (64, 69). However, current research

has shown an inherent altered fatty acid metabolism primary to defective CFTR function

(70-72). Investigation of four groups—normal, chronic bronchitis, PA infections (non-

CF), and CF—revealed different profiles of fatty acids present in bronchial secretions.

Groups with infection and inflammation, chronic bronchitis, PA infections, and CF

showed a trend toward unsaturation of fatty acids, but increased AA was singular to CF

(70). Freedman et al. reported that CFTR-expressing organs, ileum, pancreas, and lung

specifically show decreased DHA and increased AA in Cftr-/- mice absent of overt lung

disease (72). These papers indicate that altered fatty acid metabolism is not secondary to

malabsorption or inflammation, but a primary defect in CF disease pathology. Plasma,

nasal and rectal biopsy specimens of pancreatic-sufficient and -insufficient CF patient

subjects revealed similar alterations of fatty acid levels compared to control subjects (73).

Additional breakdown of the control group and analysis of epithelial nasal scrapings support a CFTR functional role in modulating AA and DHA levels. Five groups of control subjects included normal, heterozygote for ΔF508 CFTR, inflammatory

bowel disease (IBD), upper respiratory tract infection (URI), and asthma patients. CF

patients exhibited a ratio of AA to DHA significantly above all groups. Interestingly, the

ratio of AA to DHA in nasal mucus in the heterozygotes was intermediate between CF

and normal group. This trend was also present in the URI and asthma group. There was

no difference in the mean ratio of AA to DHA in the IBD group compared to controls.

Altered AA to DHA ratios are present due to infection and inflammation, as seen in

groups with CF, URI, and asthma; however, the greatest difference from control subjects

was present in CF subjects. A comprehensive investigation of inflammation independent

of CFTR function indicated an ablated AA to DHA ratio compared to controls although

not as compromised as shown in CF patients. Moreover, the intermediate change of fatty

levels present in heterozygote CFTR implies the necessity for CFTR function in the

absence of disease inflammation. This study supports the hypothesis of a fatty acid

deficiency primary to CF.

The ability to correct fatty acid levels also indicates a basic defect inherent in CF.

Oral administration of DHA to Cftr-/- mice exposed to Pseudomonas lipopolysaccharide

(LPS) for three days exhibited a suppression of neutrophil infiltration and a decrease in

eicosanoids; control mice exposed to LPS exhibited no change (74). Karp et al. also

acknowledge the importance of lipid mediators in disease pathology. The initial

hypothesis and confirmation of a suppressed lipoxin/nuetrophil ratio validated the administration of a lipoxin analogy, an ecosianoid, to PA challenged mice. BAL fluid collected 5 days after challenge revealed a shift of the inflammatory response with suppression of neutrophil count and a decrease in bacterial count (75).

Other key mediators of lipid regulation and inflammation are peroxisome proliferator-activated receptors (PPAR). The pro-inflammatory response of PPAR ligands

and the comparable manifestations in CF indicate decreased PPARγ activity and validate

exploration into the orphan nuclear receptor family (76). PPARs are a family of

transcription factors that regulate lipid homeostasis. PPARγ is highly expressed in

adipocytes and macrophages to promote adipocyte differentiation, lipid storage, and

glucose metabolism [reviewed in (77)]. Ollero et al. confirmed that PPARγ expression and activity is significantly diminished in Cftr-/- mice (78). Specifically there was

decreased PPARγ in CFTR-regulated tissue such as ileum and lung with no change in fat

or liver. This study establishes a potential mechanism for altered fatty acid synthesis

inherent in CF that is found in CFTR regulated tissue. Regulation of PPARγ suggests

crosstalk with cholesterol pathways as revealed with inhibition studies of cholesterol synthesis. Treatment of atorvastatin, directly inhibiting cholesterol synthesis, activated

PPARγ activity in human monocytes (79).

Other groups present evidence that lipids play a role in the inflammatory response in CF. Grassme et al. have reported the importance of ceramide rich signaling platforms to internalize Pseudomonas aeruginosa, the common bacteria colonized in CF patients

(80). This group and others have also shown that CFTR is present and necessary for

internalization of PA in these platforms (80, 81). Clustering of CFTR in lipid platforms is

a first defense against bacterial infection. Additionally, direct binding of PA to CFTR is a

proposed mechanism for bacterial clearance (82). Therefore, defective CFTR would lead

to less internalization and to improper immune response and thus to the inflammatory

response seen in CF. Ceramide importance in the immune response is highlighted in the

Grassme studies by the use of a model system of impaired ceramide production, acid sphingomyelinase knockout (ASM-/-) mice. This enzyme, ASM, is necessary to break down sphingomyelin to ceramide. These mice are a model for Niemann-Pick disease type

A and B, two diseases from a group of lipid disorders.

Niemann-Pick disease type C as a mechanistic model of the cystic fibrosis inflammatory response

Niemann-Pick disease encompasses a group of lipid disorders. Niemann-Pick type

C (NPC) is similar to type A and B as there is ceramide accumulation; however, this is secondary to cholesterol accumulation. NPC disease is a well characterized lipid disorder of accumulated unesterified cholesterol, and therefore, model some aspects of what we predict is occurring in CF epithelium. It is hypothesized in this study that altered cholesterol transport is caused by a loss of CFTR function, thus altering cholesterol transport, and initiating CF-related cell signaling events. This is a novel approach to understanding the inflammatory response in CF, therefore, a well-defined model of cholesterol accumulation was employed to determine potential mechanisms in CF.

Niemann-Pick is primarily a neurological disease characterized by endosomal and lysosomal accumulation of unesterified sphingomyelin and cholesterol [reviewed in

(83)]. Niemann-Pick disease is a progressive neurological disease likened to childhood

Alzheimer’s disease. Various classes of Niemann-Pick disease have been determined according to disease etiology. Niemann-Pick type A (NPA) and Niemann-Pick type B

(NPB) are caused by mutations in the gene for acid sphingomyelinase (ASM) (84). NPA is characterized as a near complete loss of ASM function resulting in severe neurological

disorder while NPB is a non-neuropathic form with 90% loss of ASM function and adult

onset (84). Loss of ASM activity results in the accumulation of sphingomyelin and

indirectly influences the trafficking of other lipids such as cholesterol. The third class of

disorders manifests as an accumulation of unesterified cholesterol in the endosomes and

lysosomes termed Niemann-Pick type C (NPC) (83, 85). NPC is caused by mutations in

the gene for Npc1 or Npc2 (HE1) (86, 87). NPC1/NPC2 proteins reside in the endosomes

and serve as cholesterol chaperones to shuttle cholesterol from the endocytic pathway.

It is at the ER that cholesterol is esterified by acyl-coenzyme A: cholesterol

acetyltransferase (ACAT) and then stored or transported out of the cell as cholesterol

esters. The ER serves as a site for regulation of cholesterol homeostasis via the

SRE/SREBP pathway described above and diagrammed in Figure 1-3. A consequence of

delayed or lack of cholesterol trafficking to the ER is increased activity of SREBP protein

and expression of SRE-containing genes, important for increasing cholesterol by

increasing synthesis or exogenous cholesterol uptake. Reports indicate significant

increased HMG-CoA reductase activity and endogenous cholesterol synthesis in

fibroblasts isolated from NPC patients compared to controls (88, 89). Altered cholesterol sensing at the ER causing upregulation of isoprenoid/cholesterol synthesis is predicted to occur in CF cells, ultimately leading to previously studied inflammatory cell-signaling changes.

Interestingly, NPC and CF have shared pathophysiology. Hepatosplenomegaly is common in NPC, absent in only 10 –15% of patients, and also present in CF due to portal occlusion (85). NPC is also characterized as a lung disease with inflammation and fibrosis of unknown cause (90). Significant presence of lung disease is seen in NPB with

only ten percent prevalence in NPC patients with the Npc1 mutation (91). Six out of

seven NPC patients with the rare mutation in Npc2/HE1 died of respiratory failure due to

airway disease prior to developing neurological symptoms (92).

Other cholesterol diseases also show altered lung physiology. Tangier’s disease is

caused by a defect of ABCA1 function, and therefore, efflux of cholesterol to HDL, and

accumulation of cholesterol. A model of Tangier’s disease, ABCA-/- mice, exhibited

enrichment of total cholesterol in the lung, as well as respiratory distress caused by

altered lung function and structure (93). Moreover, there are reports of increased

expression of GTPases, specifically RhoA in Tangier’s disease (94). Lung disease present

in lipid disorders indicates the importance of cholesterol in regulating lung structure and

function.

The reported phenotypic outcome of lung disease in NPC makes it an interesting

model for the study of CF lung disease. NPC provides a well characterized model of cholesterol accumulation independent of CFTR function. Exploring this model will help

determine if similar alterations of cholesterol processing are occurring in CF to cause

altered cell signaling events. Further definition of NPC as a model of cell signaling

events in CF is detailed in Chapter 2. This study determined that CF models have

accumulation of unesterified cholesterol as a mechanism for increased cholesterol

synthesis. Investigation into perturbed NPC cholesterol processes served as a guide for

potential mechanisms explaining CF cell signaling alterations.

Statement of Purpose

Mechanisms that cause severe inflammatory responses in CF are not well

established. The present study was conducted to establish a novel pathway as an

important upstream mediator of inflammatory cell-signaling events. It is hypothesized

that CF systems possess a flaw in intracellular cholesterol transport resulting in increased

isoprenoid/cholesterol de novo synthesis, thus leading to pro-inflammatory signaling

events. Upregulation of the isoprenoid/cholesterol synthesis pathway, caused by a lack of

cholesterol reaching the ER, is a key event in initiating cell-signaling events in CF. To

demonstrate the important role of isoprenoid/cholesterol synthesis in cell signaling events,

an established model system of accumulated unesterified cholesterol leading to increased

isoprenoid/cholesterol synthesis was explored. Niemann Pick type C disease (NPC) was

assessed for cell-signaling alterations hypothesized to be isoprenoid dependent. These

studies revealed, within a CFTR independent model, similarities with cell signaling

events exhibited in CF models. Comparisons of these models indicate the importance of

cholesterol processing in mediating cell signaling events. Parallel to NPC fibroblasts, CF

cell culture models exhibit accumulation of cholesterol in the endosomal/lysosomal

vesicles (Chapter 2). Additional characterization of altered cholesterol processing in CF

models revealed unesterified cholesterol accumulation in human airways, increased

membrane cholesterol in cell culture and excised nasal epithelium of Cftr-/- mice, and increased de novo cholesterol synthesis in Cftr-/- mouse lung and liver (Chapter 3).

Examination of cholesterol processing in CF revealed an intimate connection of CFTR

function in cholesterol processes. Inverstigations of different models of CFTR inhibition

illustrated that acute inhibition of CFTR function is sufficient to elicit altered cholesterol

processing. Two other murine models of CF, with varying quantity of CFTR function,

also exhibit increased membrane cholesterol in the nasal epithelium and increased rate of cholesterol synthesis in mouse lung. The parameters of altered cholesterol were more

dramatic in the severe model of CF compared to the mild model of CF (Chapter 4). These

studies establish deficient cholesterol processing in CF models as a direct consequence of

CFTR function and determine that altered cholesterol is sufficient in causing pro-

inflammatory signaling events in CF. Implementing the novel pathway of isoprenoid/cholesterol synthesis in the CF inflammatory response facilitates the understanding of CF disease pathology for enhanced therapeutic interventions.

Figure 1-1. De novo isoprenoid/cholesterol synthesis pathway.

Initiation of cholesterol synthesis begins with acetyl-CoA enzyme synthesized from acetate and Coenzyme A. Acetyl-CoA is converted to 3-hydroxy-3-methyglutarly-CoA

(HMG-CoA). HMG-CoA is converted to mevalonate, through the action of HMG-CoA reductase, in the committed step of cholesterol synthesis. An important branch point occurs at isoprenoid pyrophosphate (IPP), the beginning of isoprenoid production.

Farnesyl pyrophosphate (FPP) can produce squalene or geranylgeranyl pyrophosphate

(GPPP). FPP and GGPP are important carbon compounds that modify and activate small

GTPases such as RhoA. Isoprenoid-mediated activation of SMAD3 and NOS2 was also previously determined.

Statins are a selective pharmacological inhibitor of HMG-CoA reductase. This compound inhibits cholesterol synthesis and indirectly isoprenoid synthesis.

Important enzymes are shown in italics.

statins Acetyl-CoA HMG synthase HMG-CoA HMG-CoA reductase mevalonate

IPP Rho GTPase

FPP GGPP SMAD3 Squalene synthase NOS2 squalene

lanosterol

cholesterol

oxysterols

Figure 1-2. Sterol regulatory element binding protein cholesterol regulation.

Low intracellular sterol levels are sensed by proteins residing in the endoplasmic

reticulum (ER). Sterol regulatory element (SRE) binding protein (SREBP) and its

chaperone, SREBP chaperone activating protein (SCAP), interact and move to the Golgi.

Here two proteases, S1P (site-1 protease) and S2P (site-2 protease), cleave the N- terminus of SREBP to activate it. The active protein translocates to the nucleus to bind to

SRE sites of promoter regions of genes involved in cholesterol synthesis and uptake. One such gene is HMG-CoA reductase, the enzyme involved in the committed step of cholesterol synthesis.

Sterols

SCAP SREBP

SRE

Nucleus

Golgi

S2P

SREBP S1P

Adapted from Horton JD et al. Cold Spring Harb Symp Quant Biol. (2002) 67: 491-8.

Figure 1-3. Major routes of cholesterol and CFTR trafficking.

Cholesterol is endocytosed in clatherin coated pits through the low-density lipoprotein receptor (LDLR) pathway at the plasma membrane. Cholesterol is released into the endosomal/lysosomal system while LDLR is recycled to the plasma membrane. Proteins

in the late endosomes, Niemann-Pick C 1 (NPC1) and NPC2, are thought to mediate

cholesterol exit from the endosomal system to the plasma membrane. Mutations in these

genes cause accumulation of lysosomal cholesterol and the disease known as Niemann

Pick type C disease. The majority of intracellular cholesterol is stored at the plasma

membrane. Salvaged cholesterol is transported through the endocytic pathway to the

plasma membrane via the Golgi. De novo cholesterol synthesized in the ER is trafficked

to the plasma membrane via Golgi independent pathways. Excess cholesterol is

transported from the plasma membrane to the ER where is it esterified by acyl-coenzyme

A:cholesterol acetyltransferase (ACAT) for storage in lipid droplets in the cytosol. Efflux

of cholesterol to high-density lipoprotein (HDL) particles occurs at the plasma membrane

through an ABC type transporter, ATP-binding cassette transporter (ABCA1). Mutations

in ABCA1 cause accumulation of cholesterol esters and decreased HDL resulting in

Tangier’s disease.

CF transmembrane conductance regulator (CFTR) is also trafficked through similar

pathways. CFTR is produced in the ER, trafficked to the Golgi for maturation, and lastly

the plasma membrane through the endosomal pathway. CFTR is also constitutively

recycled back to the plasma membrane through endosomes. At each of these stages of

transport Rho and Rab GTPases are important for proper CFTR localization.

Cholesterol trafficking is represented by black lines and CFTR trafficking by dotted lines.

HDL LDL

LDL Receptor plasma membrane ABCA1 CFTR

endosome

Golgi late endosome Npc1/Npc2 lysosome

lipid droplets

ACAT

ER • cholesterol sensing • de novo cholesterol synthesis Nucleus

Adapted from: Ioannou YA Nat Rev. Cell. Biol. (2001) 2: 657-668.

CHAPTER 2: MECHANISTIC SIMILARITIES BETWEEN CULTURED CELL MODELS OF ∗ CYSTIC FIBROSIS AND NIEMANN-PICK TYPE C

Nicole M. White+*, Deborah A. Corey+, and Thomas J. Kelley+* Departments of Pediatrics+ and Pharmacology* Case Western Reserve University and Rainbow Babies and Children’s Hospital, Cleveland, OH

Abstract

Recent data demonstrate that inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase

restores normal signal transducer and activator of transcription-1 and inducible nitric

oxide synthase expression regulation in cystic fibrosis (CF) cells through the modulation

of RhoA function. These findings lead to the hypothesis that alterations in the cholesterol

synthesis pathway may be an initiating factor in CF-related cell signaling regulation. A

disease with a known lesion in the cholesterol synthesis pathway is Niemann-Pick type C

(NPC). The hypothesis of this study is that CF cells and NPC fibroblasts share a common

mechanistic lesion and should exhibit similar cell signaling alterations. NPC fibroblasts

exhibit similar alterations in signal transducer and activator of transcription-1, RhoA,

SMAD3, and nitric oxide synthase protein expression that characterize CF. Further

comparison reveals NPC-like accumulation of free cholesterol in two cultured models of

CF epithelial cells. These data identify novel signaling changes in NPC, demonstrate the

cholesterol-synthesis pathway is a likely source of CF-related cell signaling changes, and

that cultured CF cells exhibit impaired cholesterol processing.

∗ The data presented in the chapter has been published in 112.

Introduction

Several studies indicate that inherent inflammatory cell signaling pathways exhibit altered regulation in cystic fibrosis (CF) epithelial cells. Our laboratory has identified altered expression of individual proteins mostly related to inflammatory signaling in CF epithelium, including reduced expression of the inducible form of nitric oxide synthase

(NOS2) (39, 40, 95), reduced expression of SMAD3 (a transforming growth factor-β1 signaling protein) (48), altered regulation of signal transducer and activator of

transcription-1 (STAT1) activity (42, 96), and elevated expression of the small GTPase

RhoA (45). Our overall hypothesis has been that each of these CF-related alterations is

due to changes in a single process regulated by CF transmembrane conductance regulator

(CFTR) function that is capable of influencing a broad range of signaling interactions.

We have recently demonstrated that STAT1 activation and NOS2 expression can be

normalized in CF epithelial cells by interfering with isoprenoid/cholesterol synthesis by

influencing RhoA signaling through 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)

reductase inhibition with mevastatin (45). These data strongly implicate

isoprenoid/cholesterol-dependent pathways as the triggering event in mediating altered

CF cellular responses.

Niemann-Pick is primarily a neurological disease characterized by endosomal and

lysosomal accumulation of unesterified cholesterol and sphingomyelin [reviewed in (83)].

There are multiple variations of Niemann-Pick disease that exhibit similar phenotypes.

Niemann-Pick Types A (NPA) and B (NPB) are the result of mutations in the gene for

acid sphingomyelinase (ASM). NPA is characterized by an almost complete loss of ASM

activity, whereas NPB exhibits approximately a 90% reduction in ASM function.

Reduced ASM activity results in an accumulation of sphingomyelin and other lipids,

including unesterified cholesterol in endosomes and lysosomes. Niemann-Pick type C

(NPC) is the direct result of impaired cholesterol transport due to gene mutations in

NPC1 (86). The hypothesis of this study is that CF cells and NPC fibroblasts share a

common mechanistic lesion and should exhibit similar cell signaling alterations.

Circumstantial evidence based on disease phenotypes suggests the presence of

some shared mechanisms between CF and NPC. In addition to neurological symptoms,

one of the more pronounced phenotypes in NPC is excessive lipid accumulation in the

liver and spleen leading to hepatosplenomegaly and progressive liver failure. Interestingly,

patients with CF are also prone to developing hepatosplenomegaly, although this is

typically attributed to ductal occlusion and only affects ~20% of patients (97). However,

at least one report describes CF liver disease as being characterized by excessive lysosomal accumulation of lipids (98). In addition to hepatosplenomegaly, nonpulmonary radiographic features of Niemann-Pick disease include metacarpal widening and osteoporosis (99), both secondary features of CF. CFTR is not expressed in the brain to

any great extent, accounting for a lack of CF-related neurological symptoms in CF.

However, Niemann-Pick disease is characterized in part by severe pulmonary

manifestations. A recent study examining the more rare form of NPC, NPC2, reported

that six of seven patients followed in the study died of respiratory failure due to severe

airway disease (92). Pulmonary involvement has been reported in each type of Niemann-

Pick disease, but airway disease appears to be more common in NPB and NPC2.

Data in this manuscript provide evidence of shared cell signaling regulatory

mechanisms in cell models of NPC and CF. These data show that NPC and CF cell

models share a number of cell-regulatory alterations, including reduced NOS2 and

SMAD3 expression, and elevated STAT1 and RhoA expression. Data also indicate that

secondary effects of lost CFTR function influences normal cholesterol processing

pathways.

Methods

Cell Culture. NPC fibroblasts (GM03123A) containing two missense mutations in

the NPC1 gene were obtained from Coriell Cell Repository (Camden, NJ). Control

human fibroblasts (CRL-2076) were obtained from American Type Culture Collection

(ATCC, Manassas, VA). The cells were grown at 37°C in 95% O2–5% CO2 on Falcon 10 cm diameter tissue culture dishes (Biosource International, Camarillo, CA) in modified

Eagle's medium (MEM) with 2 mM L-gluatmine containing 15% fetal bovine serum and

1 µ/ml penicillin/streptomycin. IB3–1 cells (ΔF508/W1282X) and S9 cells (IB3–1 cell

stably transfected with the full-length wild-type CFTR as controls) were developed by

Pamela L. Zeitlin (Johns Hopkins University, Baltimore, MD). These cells were grown at

37°C in 95% O2–5% CO2 on Falcon 10 cm diameter tissue culture dishes in LHC-8 Basal

Medium (Biofluids, Biosource International) with 5% fetal bovine serum. Human

epithelial 9/HTEo- cells overexpressing the CFTR regulatory (R) domain (pCEPR; CF-

phenotype) and mock-transfected 9/HTEo- cells (pCEP, wild-type phenotype) were a

generous gift from the lab of Dr. Pamela B. Davis (Case Western Reserve University,

Cleveland, OH). Cells were cared for as previously described (19).

Mice. Mice lacking CFTR expression (Cftrtm1Unc) were obtained from Jackson

Laboratories (Bar Harbor, ME). CFTR wild-type mice were siblings of Cftr–/– mice. All

mice were used between 6 and 8 wk of age. CF mice were fed a liquid diet as described

by Eckman and colleagues (100). Mice were cared for in accordance with the Case

Western Reserve University IACUC guidelines by the CF Animal Core Facility. Excised

mouse nasal epithelium was obtained from both wild-type and CF animals and was

processed as previously described (42).

Western Immunoblotting. Antibodies against RhoA (mouse), Smad3 (rabbit),

NOS2 (mouse), and STAT1 (mouse) were obtained from Santa Cruz Biotechnology

(Santa Cruz, CA). Antibody against actin (rabbit) was obtained from Sigma-Aldrich (St

Louis, MO). Protein samples were prepared by homogenizing excised nasal epithelium or

60-mm dishes of cultured cells in ice-cold lysis buffer (50 mM Tris, pH 7.5, 1% Triton X-

100, 100 mM NaCl, 50 mM NaF, 200uM Na2VO4, and 10 µg/ml pepstatin and leupeptin) for 30 min at 4°C while shaking. Cell lysates were microcentrifuged at 4°C at 14,000 rpm

for 10 min. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel

electrophoresis containing 20–40 µg of protein of 6–12% acrylamide gel. The samples

were transferred to an Immobilon-P membrane (Millipore, Bedford, MA) at 15 V for 30

min. The blots were blocked overnight in phosphate-buffered saline (PBS: 138 mM NaCl,

15 mM Na2HPO4, 1.5 mM KCl, and 2.5 mM KH2PO4) containing 5% nonfat dehydrated

milk and 0.1% Tween-20 at 4°C. Blots were incubated overnight for Smad3 or NOS2 or

1–2 h for other primary antibodies (all at 1:1,000 dilution) in PBS containing 5% nonfat

dehydrated milk and 0.1% Tween-20. Blots were washed three times in PBS and

incubated in secondary antibody conjugated to horseradish peroxidase for 1 h at room

temperature (dilution 1: 4,000; Sigma). Blots were washed again in PBS before visualizing using Super Signal chemiluminescent substrate (Pierce, Rockford, IL) and

exposing the membrane to Kodak scientific imaging film (Kodak, Rochester, NY).

Quantification of protein expression was accomplished by densitometry software on the

VersaDoc (Quality One; BioRad, Hercules, CA).

Filipin Staining. Cells were treated as previously described by Kruth and coworkers (101). Briefly, cells were grown to 75–90% confluence on Fisherbrand

coverslips. Cells were rinsed three times with PBS and then fixed with 2%

paraformaldehyde for ~30 min. Cells were rinsed once more with PBS and then incubated

with 0.05 mg/ml filipin (Sigma-Aldrich) in PBS for 1 h on a shaker in the dark. Filipin

was dissolved fresh in dimethylformamide before each experiment. Cells were rinsed in

PBS before mounting using SlowFade Light antifade (Molecular Probes, Eugene, OR) on

slides. A Leica DMIRE2 confocal microscope (Leica Imaging Systems, Mannheim,

Germany) using the HCX PL AP x63 1.4 oil objective was used to view fixed samples of

cells at room temperature using Leica light software.

Acid Sphingomyelinase Assay. ASM assay was obtained from Molecular Probes and the protocol was followed accordingly on 9HTEo- cells using the two-step ASM

assay with 45 µg protein. Fluorescence was measured using a Molecular Devices Fmax

fluorescent plate reader.

Real-Time Reverse Transcriptase/Polymerase Chain Reaction Analysis of NPC1

mRNA Expression. NPC1 mRNA levels were determined on a LightCycler quantitative

polymerase chain reaction (PCR) machine (F. Hoffmann-La Roche Ltd, Basel,

Switzerland). Primers used to amplify a portion of the human NPC1 gene were 5'-

ACACCTTCTCTCTCTTTGCGGG-3' and 5'-GCTTGTTCCATCTTCAGCACCTC-3'.

Total RNA was obtained from cells by the TRIzol method and cDNA was produced using

Moloney murine leukemia virus with random primers according to the manufacturer's

instructions (GIBCO-BRL, Gaithersburg, MD).

Results

CF-Like Cell Signaling Alterations in NPC Fibroblasts

NOS2 induction. The observation of reduced NOS2 expression in the presence of

apparently aggressive inflammatory signaling provides an intriguing insight into cell

signaling alterations in CF cells (39, 40, 95). We have previously demonstrated that

altered NOS2 regulation was dependent on CFTR function (40), but how CFTR

influences cell signaling is still unknown. To determine if impaired cholesterol regulation

contributes to altered NOS2 expression, we examined NPC as a model system. As seen in

CF cells, human NPC fibroblasts fail to induce NOS2 protein expression in response to cytomix (1 ng/ml tumor necrosis factor-α, 0.5 mg/ml interleukin-1β, 100 U/ml interferon-

γ). However, robust NOS2 expression is seen in control wt fibroblasts in response to

identical stimulation (3.4 + 0.5 fold) (Figure 2-1). These results demonstrate that NPC

cells exhibit the same impaired ability to induce NOS2 expression characteristic of CF epithelial cells.

STAT1 expression. Previous studies indicated that alterations in STAT1 regulation

contribute to impaired NOS2 induction in CF cells. Despite a reduction in STAT1-

mediated signaling, STAT1 protein levels were elevated in multiple models of CF

epithelium (42). To further determine if CF-related cell signaling changes are conserved

in NPC fibroblasts, STAT1 protein levels were evaluated. When evaluating STAT1 levels

in NPC fibroblasts, an increase in protein expression (1.9 + 0.3 fold) above control cells

was found (Figure 2-2). STAT1 activity in NPC cells is yet to be determined. These

results further indicate that similar pathways are altered in NPC cells as seen in CF model

cells.

RhoA expression. We have recently reported that CF cells exhibit increased protein

expression of RhoA and that RhoA inhibition with the HMG-CoA reductase inhibitor

mevastatin normalized STAT1 and NOS2 regulation (45). Similar to findings in CF

model systems, investigation of RhoA protein levels in NPC cells showed increased

expression (4.3 + 1.2 fold) compared with controls (Figure 2-3). RhoA is modified by

prenylation and an increase in protein expression may possibly be the result of increased

isoprenoid/cholesterol synthesis reported in NPC (102, 103). The effectiveness of

mevastatin in normalizing CF-related signaling alterations and the known lesion in this

system in NPC models suggest that increased isoprenoid/cholesterol synthesis maybe an upstream mediator of the signaling alterations reported in CF model systems.

SMAD3 expression. Although not directly related to NOS2 regulation, another CF-

related cell signaling alteration we have observed is decreased expression of the

transforming growth factor-β1 signaling protein SMAD3 (48). An examination of NPC

fibroblasts revealed that SMAD3 protein expression is reduced 64% relative to wild-type

controls (Figure 2-4). These results indicate that multiple inflammatory pathways affected in NPC cells parallel the signaling changes seen in CF model systems.

An Examination of NPC-Like Phenotypes in CF Model Cells

The above data demonstrate that the protein expression of four proteins, NOS2, STAT1,

RhoA, and SMAD3, are identically altered in NPC fibroblasts as they are in CF model

systems. These results show that NPC fibroblasts are similar to CF cells from the

standpoint of cell signaling regulation and support a role for cholesterol-mediated

regulation of signaling in CF. The following experiments focus on determining if CF

model cells exhibit NPC-like phenotypes.

Filipin staining. Because NPC fibroblasts exhibit CF-like cell signaling changes, we

examined whether CF model cells shared any phenotypes characteristic of NPC. The

defining characteristic of NPC cells is elevated content of free cholesterol as determined by filipin staining (104, 105). Filipin from Streptomcyces filipinensis is a bacterial protein

that binds to unesterified cholesterol and fluoresces in the ultraviolet range. A

representative staining pattern of control and NPC fibroblasts is shown in the top panel of

2- 5. The 9/HTEo-pCEPR (CF-phenotype) cells exhibit a nearly identical filipin staining

pattern as observed in NPC controls. Similar results were obtained with a second CF

model cell line, IB3 cells, compared with S9 controls. There is some degree of staining in

S9 cells that may indicate incomplete rescue of this phenotype by transgene expression of

CFTR. However, there is a clear difference in staining intensity and pattern between S9 and IB3 cells. The elevated levels of unesterified cholesterol and the punctuated staining pattern point to an inherent flaw in intracellular cholesterol processing in two models of

CF epithelial cells.

The possible incomplete rescue of cholesterol processing in S9 cells as indicated

by filipin staining calls into question whether signaling differences are preserved in this

model system. We have shown above signaling differences in CF mouse nasal epithelium

as a comparison to NPC model cells. We have also previously reported the NOS2, RhoA,

STAT1, and SMAD3 signaling changes in mice and in the pCEP/pCEPR cells (39, 40, 42,

45, 48, 95, 96). However, no direct examinations of these pathways have been performed

in the S9/IB3 cell model. We examined RhoA and STAT1 protein expression in these cell

lines since these changes appear to be upstream in the signaling consequences related to

cholesterol processing. As shown in Figure 2-6, STAT1 exhibits elevated expression in

IB3 cells compared with CFTR-corrected S9 controls, which is consistent with our other

CF models. However, RhoA expression is robust in both IB3 and S9 cells, suggesting that

the partial correction of cholesterol esterification in S9 cells is insufficient to completely

modulate RhoA regulation. These data do not address whether RhoA is constitutively

active in S9 and IB3 cells as we have previously reported in mouse models of CF (45).

Further characterization of the ability of ectopic CFTR expression to influence cholesterol

processing and related signaling cascades in CF cells is needed.

Potential Mechanisms of Increased Filipin Staining in CF Model Cells

ASM activity. There are various forms of Niemann Pick disease, two of which are characterized as ASM deficiencies that cause a secondary accumulation of lipids.

Sphingomyelinase converts sphingomyelin to ceramide at the plasma membrane. To

narrow the focus of a true model system and illuminate a possible mechanism of why and how de novo synthesis of cholesterol is upregulated in CF cell models, ASM activity was

investigated using 9/HTEo-pCEP (control) and pCEPR (CF-phenotype) cells because

filipin staining differences were more distinct in this model system. Two different

experiments with a total of eight samples for each cell type were averaged and reported in

Figure 2-7. There appears to be no significant difference in ASM activity between

9/HTEo- pCEPR (CF-phenotype) and pCEP2 (control) cells. Two positive controls were

used; one to measure sphingomyelinase from Staphyloccus sp. and hydrogen peroxide to

ensure the assay was functional. A negative control of reaction buffer was also measured

and used to subtract background from each sample. This in vitro study implies that the

ASM is functional in CF cells and not the cause of the cholesterol accumulation seen with

filipin staining.

NPC1 mRNA expression. NPC is caused by a direct lipid transport problem resulting

from a mutation in the NPC1 gene. A potential cause of elevated filipin staining in CF

model cells is an indirect decrease of NPC1 expression leading to the development of

NPC-like phenotypes. Therefore, NPC1 mRNA content was examined using quantitative

real-time reverse transcriptase (RT)-PCR. Baseline expression levels of NPC1 show a 2.7

+ 0.4 fold increase in pCEPR 9/HTEo (CF-phenotype) cells compared with controls (n =

5, Figure 2-8). Similarly, IB3 cells exhibit a 1.7 + 0.2 fold increase in NPC1 mRNA

content compared with control S9 cells. These data suggest that reduced NPC1 expression is not a direct cause of elevated filipin staining and possibly indicate that CF-phenotype

cells are responding to an increase in free cholesterol by upregulating the expression of a

cholesterol transport protein.

Discussion

Understanding the relationship between CFTR function and the regulation of cell

signaling cascades is an ongoing goal. Alterations in the regulation of NOS2, STAT1,

SMAD3, and RhoA protein expression in CF models have previously been identified (39,

40, 42, 45, 48, 95, 96). The observation that inhibition of HMG-CoA reductase activity

with mevastatin normalized STAT1 and NOS2 regulation in CF cells through RhoA-

dependent mechanisms lead to the hypothesis that alterations within the

isoprenoid/cholesterol synthesis pathway represent a potential source of cell signaling

changes in CF cells. To address this hypothesis, we turned to a model system that has a well-defined increase in endogenous isoprenoid/cholesterol synthesis, NPC. Our data demonstrate that NPC fibroblasts exhibit identical alterations in NOS2, STAT1, RhoA,

and SMAD3 that we have reported in CF epithelial cells. The reproducibility of these

protein expression profiles between CF and NPC model systems support our hypothesis

that an alteration in cholesterol-related pathways is a potential source of improper cellular

responses in CF.

The above studies reveal that NPC fibroblasts resemble CF cells from the

standpoint of altered expression of various signaling proteins. We next examined two cultured models of CF epithelial cells for similarities to typical NPC phenotypes.

Increased staining of free, unesterified cholesterol by filipin is a defining characteristic of

NPC cells. Filipin staining of free cholesterol revealed increased staining in CF-

phenotype pCEPR 9/HTEo- cells and in immortalized IB3 CF cells compared with

respective controls. The staining patterns obtained in CF model cells were very similar

from those obtained from NPC fibroblasts. These data indicate that inherent cholesterol

processing mechanisms are altered in CF cells in a manner similar to what is observed in

NPC.

In an effort to identify a cause of impaired cholesterol processing in CF model

cells, two Niemann-Pick–related mechanisms were examined. An examination of ASM

activity revealed no significant change between 9/HTEo- pCEP (control) and pCEPR

(CF-phenotype) model cells. These findings are consistent with those reported, but not

shown, by Grassme and colleagues (80). However, our results are obtained in an in vitro

assay and do not speak to in vivo substrate availability or ceramide transport to the plasma

membrane. Another mechanistic possibility is that an indirect influence of CFTR function

on NPC1 expression leads to NPC characteristics in CF cells. Expression of NPC1

message was examined in two models of CF epithelia and a significant increase in NPC1 expression compared with wt controls was observed. These data do not address function,

but do suggest that CF cells are responding to a lesion in normal cholesterol processing by

increasing expression of a cholesterol transport protein. A caveat to these studies is that

both the CF cell line models used are immortalized, clonal cell lines and global

conclusions about CF pathogenesis have to be made with caution. However, signaling

changes in these cell lines have been consistent with data from CF mouse models and

human samples making them useful models to begin examining these novel processes.

An inherent flaw in cholesterol processing in CF has multiple potential

consequences. In addition to cell signaling regulation, the impairment of cholesterol

processing may also influence CF susceptibility to bacterial infection. Recent results by

Kowalski and Pier demonstrate that CFTR clusters in lipid rafts in response to bacterial

challenge, but the expression of ΔF508 CFTR impedes this process (81). Although

inherent problems in ΔF508 CFTR localization may be responsible for this result, a

Niemann-Pick–like impairment in intracellular lipid transport may also contribute to this

observation. The findings by Kowlaski and Pier are similar to those by Grassme and

coworkers, who recently reported that ceramide-rich lipid rafts were necessary for innate

defense against Pseudomonas aeruginosa (80). Evidence continues to mount in support

of the hypothesis that alterations in lipid-related processes contribute to poorly explained

aspects of CF pathogenesis.

How a loss of CFTR function would lead to the development of NPC-like

phenotypes is not obvious, and alterations is ASM activity or NPC1 expression do not

seem to be involved. One possibility is the direct regulation of cholesterol or other lipid

transport by CFTR. Circumstantial support for this possibility is the considerable structural similarity shared between CFTR and the ATP binding cassette protein A1

(ABCA1) (106). ABCA1 is primarily a cholesterol transport protein important in regulating cholesterol efflux across the plasma membrane, and the protein whose

dysfunction is associated with Tangier's disease (94). In an interesting parallel with our

CF-related data, one report demonstrates that RhoA protein expression is elevated in cells

from patients with Tangier's disease (94), suggesting a further link between cholesterol

transport and Rho GTPase regulation. More direct evidence of lipid transport mediated by

CFTR has been published showing CFTR-mediated transport of sphingosine-1-phosphate,

although no cholesterol transport through CFTR was observed (107). Another possibility

is an alteration in the intracellular environment caused by CFTR dysfunction. Picciano

and colleagues demonstrate that CFTR undergoes endosomal recycling that is dependent on the C-terminal tail of CFTR (108). Schweibert and colleagues have demonstrated that

CF-related cell signaling alterations can also be influenced by interactions at the CFTR C-

terminus (109). Related to CFTR presence in endosomes, Poschet and coworkers have shown that endocytic vesicles are hyperacidified in CF lung epithelial cells due to the loss of CFTR function (110). Altered endosomal environment due to lost CFTR function may

cause an indirect impairment of cholesterol processing mechanisms. Finally, impaired

fatty acid processing in CF has been demonstrated in multiple reports (72, 73, 111).

Increased filipin staining in our CF model cells suggests that cholesterol is not being re- esterified. Possible explanations for this observation are that cholesterol is not properly

reaching the endoplasmic reticulum (ER), as is the case in NPC, or that there is a lack of

fatty acid substrate to be used for cholesterol esterification.

Understanding how CFTR function influences cholesterol processing will require

considerable investigation. Data presented in this manuscript demonstrate that NPC and

CF are very similar at the cellular level. A comparison of CF and NPC may be useful in

increasing our understanding of pathogenic mechanisms in both diseases.

Acknowledgements

This work is supported by NIH/NHLBI grant HL64899. The authors thank Dr. P. Davis for providing materials necessary for the completion of this study, and P. Bead for

technical assistance.

Figure 2-1. Reduced induction of (NOS2) in NPC compared with wt fibroblasts.

(A) NOS2 protein expression in Cftr+/+ (wt) and Cftr-/- (CF) mouse nasal epithelium. (B)

Western blot of NOS2 and actin expression in NPC and wild-type (wt) control fibroblasts

in the presence (+) or absence (-) of cytomix (CM). (C) Densitometry analysis of NOS2

expression relative to actin protein content (NOS2/actin) in NPC and wt fibroblasts. Data

represent averages of three separate experiments. Number (n) of samples is shown in

parentheses above each bar. Significance determined by t test; *P = 0.002. Error bars

represent SEM.

A. CF wt

NOS2

B. NPC wt CM --+ + NOS2

actin

(6) C. 4.0 * 3.0

2.0 (NOS2/actin)

Relative density (5) 1.0 (5) (6)

0 + + CM -- wt NPC

Figure 2-2. Increased STAT1 protein expression in NPC compared with wt fibroblasts.

(A) STAT1 protein expression in Cftr+/+ (wt) and Cftr–/– (CF) mouse nasal epithelium.

(B) Western blot of STAT1 and actin expression in NPC and wt fibroblasts. (C)

Densitometry analysis of STAT1 expression relative to actin protein content

(STAT1/actin) in NPC and wt fibroblasts. Data represent averages of three separate experiments. Number (n) of samples is shown in parentheses above each bar.

Significance determined by t test; *P = 0.03. Error bars represent SEM.

A. CF wt

STAT1

B. NPC wt

STAT1

actin

C. 2.5 (7)

2.0 *

1.5 (8) 1.0 (STAT1/actin) Relative density 0.5

0 wt NPC

Figure 2-3. Western blot of increased RhoA expression in NPC and CF model

systems.

(A) RhoA and actin expression in NPC and non-NPC cells. RhoA protein expression in

Cftr+/+ (wt) and Cftr–/– (CF) mouse nasal epithelium. (B) Western blot of RhoA and actin expression in NPC and wt fibroblasts. (C) Densitometry analysis of RhoA expression relative to actin protein content (RhoA/actin) in NPC and wt fibroblasts. Data represent averages of three separate experiments. Number of replicates is shown in parentheses above each bar. Significance determined by t test; *P = 0.02. Error bars represent SEM.

A. CF wt

RhoA

B. NPC wt

RhoA

actin

C. 6 (9)

5 * 4

3 (RhoA/actin)

Relative densityRelative 2 (8) 1

0 wt NPC

Figure 2-4. Decreased Smad3 expression in NPC and CF model systems.

(A) SMAD3 protein expression in Cftr+/+ (wt) and Cftr–/– (CF) mouse nasal epithelium.

(B) Western blot of SMAD3 and actin expression in NPC and wt fibroblasts. (C)

Densitometry analysis of SMAD3 expression relative to actin protein content

(SMAD3/actin) in NPC and wt fibroblasts. Data represent averages of three separate experiments. Number of samples is shown in parentheses above each bar. Significance determined by t test; *P < 0.001. Error bars represent SEM.

A. CF wt

Smad3

B. NPC wt

Smad3

actin

C. (11) 1.0

0.8

0.6 (11) * (RhoA/actin) 0.4 Relative density

0.2

0 wt NPC

Figure 2-5. Increased filipin staining in CF epithelial cells compared to controls.

Increased filipin staining in NPC fibroblasts compared with wt fibroblasts are used for staining control and for comparison. 9/HTEo- pCEP (wt) and pCEPR (CF-phenotype) images are representative of results observed with 16 coverslips/cell type over four separate experiments. S9 (CFTR corrected) and IB3 (CF) images are representative of results obtained with 14 coverslips/cell type over three separate experiments.

NPC non-NPC

pCEPR pCEP2

IB3 S9

Figure 2-6. RhoA and STAT1 protein expression in IB3 (CF) and S9 (CFTR- corrected) cells.

(A) Western blot of RhoA, STAT1, and actin protein expression in IB3 (hatched bars) and S9 cells (closed bars). (B) Densitometry analysis of RhoA and STAT1 expression relative to actin protein content in IB3 and S9 cells. Data represent averages of three separate experiments. Significance determined by t test and error bars represent SEM.

A. S9 IB3 B. * p = 0.002 S9 RhoA IB3 16 14 12 10 STAT1 8

(protein/actin) 6 4 Relative protein expression protein Relative 2 actin 0 RhoA STAT1

Figure 2-7. ASM activity in pCEPR (CF) and pCEP (wt) 9/HTEo cells.

Negative (neg cont) and positve controls (ASM and H2O2) were performed as described

in MATERIALS AND METHODS. The bars are averages of two separate experiments. Number of

replicates is shown in parentheses above each bar. Significance determined by t test and no significant difference was found. Error bars represent SEM.

8000

6000

4000 Fluorescence (arbitrary units) 2000 (8) (8)

0 pCEP pCEPR neg cont ASM H2O2

Figure 2-8. Increased NPC1 mRNA levels in CF model cells compared with controls.

(A) Representative melting curve real-time RT-PCR traces showing 106 copies/µl standard, samples from pCEP and pCEPR cells, and a water control. (B) Quantified real- time RT-PCR determination of NPC1 mRNA expression in pCEP/pCEPR and S9/IB3 cell models were examined. Results are given as a ratio of NPC1 mRNA and GAPDH mRNA (NOS2/GAPDH) normalized to wt phenotype cells (pCEP and S9, respectively).

Number of replicates is given in parentheses. Significance determined by t test with *P =

0.005 and **P = 0.04.

A. standard

pCEPR

pCEP water

(5) B. 3.0 * 2.5 (5) 2.0 * 1.5 (6) (5) (NPC1/GAPDH) 1.0 Relativecontent mRNA 0.5

0 pCEP pCEPRS9 IB3

CHAPTER 3: ALTERED CHOLESTEROL HOMEOSTASIS IN CULTURED AND IN VIVO ∗ MODELS OF CYSTIC FIBROSIS

Nicole M. White*, Dechen Jiang†, James D. Burgess†, Ilya R. Bederman‡, Stephen F. Previs‡§, and Thomas J. Kelley*+ Departments of Pediatrics and Pharmacology*, Department of Chemistry†, Department of Nutrition‡, Department of Medicine§, Case Western Reserve University and Rainbow Babies and Children’s Hospital, Cleveland, OH

Abstract

Determining how the regulation of cellular processes is impacted in cystic fibrosis

(CF) is fundamental to understanding disease pathology and to identifying new

therapeutic targets. In this study, unesterified cholesterol accumulation is observed in

lung and trachea sections obtained from CF patients compared to non-CF tissues

suggesting an inherent flaw in cholesterol processing. An alternate staining method

utilizing a fluorescent cholesterol probe also indicates improper lysosomal storage of

cholesterol in CF cells. Excess cholesterol is also manifested by a significant increase in

plasma membrane cholesterol content in both cultured CF cells and in nasal tissue

excised from Cftr-/- mice. Impaired intracellular cholesterol movement is predicted to

stimulate cholesterol synthesis, a hypothesis supported by the observation of increased de

novo cholesterol synthesis in lung and liver of Cftr-/- mice compared to controls.

Furthermore, pharmacological inhibition of cholesterol transport is sufficient to cause

CF-like elevation in cytokine production in wt cells in response to bacterial challenge, but

has no effect in CF cells. These data demonstrate via multiple methods in both cultured

and in vivo models that cellular cholesterol homeostasis is inherently altered in CF. This

∗ The data presented in the chapter has been, in part, published in 136.

perturbation of cholesterol homeostasis represents a potentially important process in CF pathogenesis.

Introduction

Cystic fibrosis (CF) is caused by the lost function of the cAMP-dependent chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). However, it is not clearly understood what mechanisms lead to aggressive inflammatory signaling in CF. Previous work demonstrated that cultured models of CF epithelial cells exhibited intracellular accumulation of unesterified cholesterol in a manner similar to what is observed in cells from patients with Niemann-Pick type C (NPC) disease (112). NPC is a disease of impaired intracellular cholesterol transport resulting in free cholesterol accumulation in late endosomes and lysosomes (104, 113). It was determined that NPC fibroblasts share a number of cell signaling alterations previously identified in CF cells including reduced inducible nitric oxide synthase (NOS2) expression, increased RhoA and signal transducer and activator of transcription-1 (STAT1) protein expression, and reduced SMAD3 protein expression (112). These data suggest that improper cholesterol processing is a trigger for altered inflammatory signaling responses in CF cells. Further support for the importance of cholesterol-related pathways in CF cell-signaling regulation in CF cells has been demonstrated previously by the impact of isoprenoid/cholesterol synthesis on these signaling cascades. Treatment with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, mevastatin, resulted in the correction of

STAT1 and NOS2 signaling in both cultured cell models and mouse models of CF (48)

(45, 56). Similarly, inhibition of isoprenoid transferase activity resulted in correction of

SMAD3 expression and TGF-β1 signaling in CF cells (53).

The above findings suggest a relationship between cholesterol accumulation and

the isoprenoid/cholesterol synthesis pathway in CF. NPC cells have been shown to

exhibit increased cholesterol synthesis despite increased intracellular storage, likely due

to a lack of cholesterol transport to the endoplasmic reticulum (ER) (114). The goal of

this manuscript is to further investigate cholesterol processing in CF models as a potential

mechanism for alterations in CF inflammatory pathways. The hypothesis of this study is

that a loss of CFTR function leads to altered cholesterol trafficking resulting in increased

cholesterol synthesis. This perturbation in cholesterol regulation is proposed to contribute

to the inflammatory response present in CF.

The importance of lipid regulation in CF inflammatory responses has been

explored previously. Kowalski and Pier have reported that plasma membrane cholesterol is essential for CFTR localization and for proper responses to Pseudomonas aeruginosa

(PA) (81). Similarly, Grassme et al. have demonstrated the importance of ceramide-rich signaling platforms to internalize PA using a mouse model of Niemann-Pick type A disease (80). Another component of an anti-inflammatory pathway related to cholesterol homeostasis reported to be deficient in CF models is the peroxisome proliferation activated receptor-γ (PPARγ) (78). PPARγ has been demonstrated to be deficient in NPC cells as well, suggesting a similar regulatory relationship in CF cells (115). Each of these studies supports the hypothesis that alterations in cholesterol processing and internal trafficking would have important consequences on inflammation and on bacterial response at the plasma membrane in CF.

The cholesterol pathway has also been implicated in the regulation of CFTR

trafficking to the apical membrane. Shen et al. demonstrated that treatment with the

HMG-CoA reductase inhibitor, lovastatin, reduced CFTR-mediated chloride transport and CFTR trafficking to the apical membrane (59). Cheng et al. have also recently shown that lovastatin inhibits CFTR trafficking by inhibition of the Rho family small GTPase

TC10 (60). These studies indirectly raise the possibility that observed alterations in cholesterol processing in CF cells may be an adaptive response by cells to increase CFTR content at the plasma membrane.

The current study details multiple anomalies in cholesterol related regulation in both cultured cell models and in primary tissue of CF origin including intracellular cholesterol accumulation, increased de novo cholesterol synthesis, and elevated plasma membrane cholesterol content. It is concluded that CF epithelial cells possess an inherent flaw in cholesterol regulation due to the loss of CFTR activity or expression. These data demonstrate in primary tissue and in multiple model systems that aberrations in cholesterol homeostasis is a CF-related phenotype that potentially influences a number of relevant cell-signaling events. The control of both cholesterol synthesis and processing represent new avenues for therapeutic development for CF.

Methods

Cell culture. IB3-1 cells, human epithelial with the ΔF508 mutation (CF-

phenotype), and S9 cells, IB3-1 cell stably transfected with the full-length wt CFTR

(control) were a generous gift from Pamela L. Zeitlin (Johns Hopkins University,

o Baltimore, MD). These cells were grown at 37 C in 95% O2 -5% CO2 on Falcon 10 cm

diameter tissue culture dishes in LHC-8 Basal Medium (Biofluids Camarillo, CA) with

5% FBS. The human alveolar type-II epithelial adenocarcinoma cell line (A549) were

grown under the above conditions in Ham’s F-12 Kaign’s Modification (Biofluids, Inc.,

Rockville, MD) with 10% fetal bovine serum. Human epithelium 9/HTEo- cells over

expressing the CFTR R domain (pCEPR) and mock-transfected 9/HTEo- cells (pECP2),

the wild-type phenotype, were a generous gift from the lab of Dr. Pamela B. Davis (Case

Western Reserve University). Cells were cared for as previously described (19).

Mice. Mice lacking CFTR expression (CFTRtm1Unc) were obtained from Jackson

Laboratories (Bar Harbor, MA). CFTR wild-type mice were siblings of Cftr-/- mice. All

mice were used between six and eight weeks of age and are back-crossed over ten

generations onto a C57Bl/6 background. CF mice were fed a liquid diet as described by

Eckman and colleagues (100). Mice were cared for in accordance with the Case Western

Reserve University IACUC guidelines by the CF Animal Core Facility. Nasal scrapings

of mouse epithelium were obtained from both wild-type and CF animals.

NBD-cholesterol staining. Cells were seeded at a density of 150 -200,000

cells/well on Fisherbrand coverslips. Fifty µg/mL of NBD-cholesterol or 25-[N-[(7-

nitrobenz-2-oxa-1, 3-diazol-4-yl)-methyl]amino]-27-norcholesterol (Molecular Probes,

Eugene, OR) was added for approximately 24 h. Cells were then incubated in fresh media

for another 4 h. Cells were fixed in 2% paraformaldehyde for 30 minutes and then rinsed

in phosphate buffered saline (PBS) three times before being mounted using SlowFade

Light antifade (Molecular Probes). Confocal images were taken using a Leica DMIRE2

confocal microscope (Leica Imaging Systems, Manneheim, Germany) using the HCX PL

AP x 63 1.4 oil objective. Images are representatives of average pictures taken of the z

stacks. For experiments with gly-phe-B-naphthylamide, GPN, and docosahexaenoic acid,

DHA, (Sigma-Aldrich, St. Louis, MO) cells were incubated the full time at a final concentration of 50µM in DMSO (GPN) or 10 µM in methanol (DHA).

Flow cytometry. Approximately 300,000 cells were plated in 6 well dishes and treated as described with NBD-cholesterol; however, the cells were allowed to sit in fresh media overnight. Cells were trypsinized, rinsed once in PBS, and placed in fresh PBS for analysis. The EPICS-XL-MCL (Beckman Coulter, Miami, FL) has a 488 air-cooled argon ion laser at 15mW. A 525nm band pass filter was used to collect 200,000 events per sample.

Filipin staining. Cells were treated as previously described by Kruth et al. (101).

Briefly, cells were grown 75 -90% confluency on Fisherbrand coverslips. Cells were rinsed three times with PBS and then fixed with 2% paraformaldehyde for approximately

30 minutes. Cells were rinsed once more with PBS and then incubated with 0.05mg/mL filipin (Sigma-Aldrich, St Louis, MO) in PBS for 1 h on a shaker in the dark. Filipin dissolved fresh in dimethylformamide before each experiment. Cells were rinsed in PBS before mounting using SlowFade Light antifade (Molecular Probes) on slides. Cells were visualized in the ultraviolet range using wide field microscope on a Zeiss Axiovert 200 and Metamorph software. A 63X objective was used for all images. Cells were treated for

1 h with GPN at a final concentration of 50 µM or DHA for 24 h at a final concentration of 10 µM.

Tissues were fixed in 2% Paraformaldehyde and placed in paraffin blocks. Tissue was sectioned at 5um. These sections were stained with filipin as described above. However, confocal images were taken using a Leica DMIRE2 confocal microscope (Leica Imaging

Systems) using the HCX PL AP x 100 oil objective. Staining was done with one CF

trachea and lung and one control tissue.

Transfections. The human sterol response element (SRE)-luciferase reporter

construct (SRE-luc) was provided by Dr. Timothy Osborne (the University of California

at Irvine). The SRE-luc construct consists of the SRE region of the HMG-CoA synthase

promoter. Cells were seeded at a density of 50,000 cells/well in 24 well tissue culture

dishes 24 h prior to transfection. For each transfection 0.6µL of FuGene 6 (Roche,

Indianapolis, IN) was incubated for 5 minutes in 100µL of OptiMEM (Gibco BRL,

Gaithersburg, MD). Then 0.03 µg of DNA and 0.008 µg of pRL-TK were added to the

FuGene/OptiMEM mix and incubated for another 15 minutes. One hundred µl of diluted

o transfection mix was added to each well and cells incubated at 37 C in 95% O2/5% CO2

for 24 h. To address the role of de novo cholesterol synthesis, cells were also examined

under serum free conditions. Cells were cultured without serum for 24 h to eliminate

exogenous cholesterol influences on SRE regulation. Cell were also treated with the

HMG-CoA reductase inhibitor, mevastatin (Calbiochem San Diego, CA) (50 µM) and/ or

50 mg/mL cholesterol (Sigma-Aldrich) for 24 h. For other experiments cells were also

treated with the 10µM DHA (Calbiochem) for 24 h. Cells were then lysed in 1X Passive

Lysis Buffer (Promega, Madison, WI) at room temperature for 15 min, and assayed for

luciferase activity according to manufacturer instructions (Promega, Madison, WI).

Results are expressed in Relative Light Units (RLU) and normalized to Renilla luciferase activity.

Measuring cholesterol synthesis in vivo. CFTRtm1Unc mice and the matched

controls were given an intraperitoneal injection (i.p.) (~ 24 µL per g body weight) of

2 deuterated saline (9 g NaCl in 1000 mL of 99% H2O, Sigma-Aldrich, St. Louis, MO).

After 8 h, mice were sacrificed using carbon dioxide. Blood was taken from the heart and plasma collected. Whole lungs and approximately 1.0 g of liver and small intestine were collected. Tissue samples were hydrolyzed in 1N KOH/70% ethanol (v/v) for 2 at 70oC vortexing occasionally. Samples were then evaporated to dryness, redissolved in 2 ml of water and acidified using 12N HCl. Cholesterol was extracted twice by addition of ethyl ether (3 ml). The pooled ether extracts were evaporated to dryness under nitrogen and then converted to the trimethylsilyl cholesterol derivatives by reacting with 60 µl of bis(trimethylsilyl) trifluoroacetamide + 1% trimethylchlorosilane (Regis, Morton Grove,

IL) (TMS) at 60° C for 20 min. The 2H-labeling of cholesterol was determined using an

Agilent 5973N-MSD equipped with an Agilent 6890 GC system. The cholesterol was run

on a DB17-MS capillary column (30 m x 0.25 mm x 0.25 µm). The oven temperature

was initially held for 1 min at 150° C, then increased by 20° C per min to 310° C and

maintained for 8 min. The split ratio was 20:1 with helium flow 1 ml per min. The inlet

temperature was set at 270° C and MS transfer line was set at 310° C. Under these conditions, cholesterol elutes at ~11.1 min. Electron impact ionization was used in all analyses with selected ion monitoring of m/z 368-372 (M0-M4, cholesterol), dwell time of 10 ms per ion.

The 2H-labeling of mice plasma water was determined by exchange with acetone

as described by McCabe et al. (116). Briefly, plasma was diluted 2-fold with distilled

water and reacted with 2 μl of 10 N NaOH and 4 μl of a 5% (v/v) solution of acetone in

acetonitrile for 24 h. Acetone was extracted by addition of 600 μl of chloroform followed

by addition of 0.5 g Na2SO4. Samples were vigorously mixed and a small aliquot of the

chloroform was transferred to a GC-MS vial. Acetone was analyzed using Agilent equipment described above. The oven temperature program was: 60º C initial, increase by 20º C per min to 100º C, increase by 50º C per min to 220º C and maintain for 1 min.

The split ratio was 40:1 with a helium flow of 1 ml per min. The inlet temperature was set at 230º C and the mass spectrometer transfer line was set at 245º C. Acetone eluted at

~1.5 min. The mass spectrometer was operated in the electron impact mode (70 eV).

Selective ion monitoring of m/z 58 and 59 was performed using a dwell time of 10 ms

per ion.

Calculation of cholesterol synthesis. Following correction for natural enrichment

(117), rates of de novo cholesterol synthesis were calculated using the formula:

2 Total labeling ([(M1 x 1) + (M2 x 2) + (M3 x 3) + (M4 x 4)]) / n / H-labeling of plasma

water x time

where Mi represents isotope labeled isomeric species of cholesterol (M1 being singly-

labeled, M2 doubly labeled and etc (118) and “n” represents the number of exchangeable

hydrogens, assumed to be 25 for cholesterol (119).

Electrochemical measurements of cholesterol. Platinum microelectrodes were

fabricated in house (11.5 µm and 100 µm diameter wire, Goodfellow Corp.) as described

(120). Platinum wire was inserted into glass capillaries (Kimax-51, Kimble products) and

placed inside a heated platinum coil. The glass was pulled to create a thin insulating

layer on the platinum wire. The capillary microelectrodes were polished using a beveling

machine (WPI, Inc.) to produce a disk electrode. The microelectrodes were immediately

immersed in a 5 mM hexane solution of 11-mercaptoundecanoic acid (95%, Aldrich

Chem. Co) for 2 h to form a carboxylic acid terminated monolayer on the electrode

surface. Then, the microelectrodes were treated 2 mM 1-ethyl-3 -(3-dimethylaminopropyl) carbodiimide (EDC) (Sigma Chem. Co.) in 100 mM PBS solution (pH 7.4) for 30 min. to activate the carboxyl groups to an acylisourea intermediate. The modified electrode was immersed in 1 mg/ml recombinant cholesterol oxidase (Oriental Yeast Co. Ltd., 42.0 units/mg) solution for 3 hrs allowing this intermediate to react with amine immobilizing the enzyme on the electrode surface.

Data Acquisitions: Amperometric measurements were conducted using a two-electrode cell and a voltammeter-amperometer (Chem-Clamp, Dagan corp.). The three-pole bessel filter in the voltammeter-amperometer was set to 100 Hz. The output was further processed using a noise-rejecting voltmeter (model 7310 DSP, Signal Recovery Inc.) to digitally filter 60-Hz noise. An Ag/AgCl (1 molar KCl) reference electrode was used for all experiments, and the applied potential is 780mV versus NHE for all experiments. All experiments were performed in 100mM phosphate buffer (pH 7.4) at 36°C.

Single cells and excised tissue were captured by a capillary prepared in house using an IM-6 microinjector (Narishige International USA, Inc.). The electrode was initially positioned about 50 mm from the cell surface or tissue inner edge for acquisition of baseline data. The electrode was repositioned for contacting the biological sample and acquisition of electrode response.

Cytokine Measurements. 9/HTEo-pCEP and pCEPR were plated in 24 well plates at a density of 500,000 cells per well. These cells were treated for 24 h with 5µg/mL

U18666a, a cholesterol transport inhibitor. After being placed in serum free media overnight, some of the cells were challenged with 1 x 109 CFU of Pseudomonas aeruginosa in the presence and absence of U18666a. After one h cells were sterilized

with 50µg/mL gentamicin. The supernatants were collected 18 h later and assayed for IL-

6 and IL-8 production. Cytokine levels are assayed by the Cell Mediator core facility

using immuno reagents obtained from R&D Systems.

Western Immunoblotting. Antibodies against NOS2 (mouse) were obtained from

BD Transduction Labs (Billerica, MA). Antibody against actin (rabbit) was obtained from

Sigma-Aldrich (St Louis, MO). Protein samples were prepared by 60-mm dishes of

cultured cells in ice-cold lysis buffer (50 mM Tris, pH 7.5, 1% Triton X-100, 100 mM

NaCl, 50 mM NaF, 200uM Na2VO4, and 10 µg/ml pepstatin and leupeptin) for 30 min at

4°C while shaking. Cell lysates were microcentrifuged at 4°C at 14,000 rpm for 10 min.

Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis

containing 20–40 µg of protein of 6–12% acrylamide gel. The samples were transferred to

an Immobilon-P membrane (Millipore, Bedford, MA) at 15 V for 30 min. The blots were

blocked overnight in phosphate-buffered saline (PBS: 138 mM NaCl, 15 mM Na2HPO4,

1.5 mM KCl, and 2.5 mM KH2PO4) containing 5% nonfat dehydrated milk and 0.1%

Tween-20 at 4°C. Blots were incubated overnight for NOS2 ( 1:500 dilution) in PBS

containing 5% nonfat dehydrated milk and 0.1% Tween-20. Blots were washed three times in PBS and incubated in secondary antibody conjugated to horseradish peroxidase

for 1 h at room temperature (dilution 1: 4,000; Sigma). Blots were washed again in PBS

before visualizing using Super Signal chemiluminescent substrate (Pierce, Rockford, IL)

and exposing the membrane to Kodak scientific imaging film (Kodak, Rochester, NY).

Quantification of protein expression was accomplished by densitometry software on the

VersaDoc (Quality One; BioRad, Hercules, CA).

Results

In vivo accumulation of unesterified cholesterol. Previous observations of unesterified

cholesterol accumulation in CF were limited to two cultured cell models (112). To determine if this phenotype is conserved in vivo and relevant to human disease, cholesterol content in sections from CF and non-CF trachea and upper lung airways was determined by filipin staining. Filipin is a protein from the bacteria Streptomcyes filipinensis that binds to unesterified cholesterol becoming detectable in the ultraviolet range. Compared to control tissue, CF trachea exhibited increased intracellular cholesterol content and more intense staining (Figure 3-1A). The same pattern of

accumulation observed in the CF trachea was also present in the epithelium of the upper

airway of the lung (Figure 3-1B). These data support previous findings of increased

intracellular unesterified cholesterol content in cultured CF models and demonstrate that

primary tissue of CF origin exhibits the aberrant cholesterol transport phenotype.

NBD-cholesterol accumulation in CF cell models. Filipin staining demonstrates

accumulation of unesterified cholesterol in cell and tissue models of CF. In order to

determine if these findings were due to a disruption of transport or simply due to deficient re-esterification, the transport of a fluorescently labeled cholesterol analog was examined. Utilizing a fluorescent cholesterol probe, 25-[N-[(7-nitrobenz-2-oxa-1, 3- diazol-4-yl)-methyl]amino]-27-norcholesterol (NBD-cholesterol) cholesterol trafficking in cultured cell models was measured. After approximately 24 h of incubation with NBD- cholesterol, cells were placed in fresh media for 4 h before being fixed. Confocal images demonstrate a clear accumulation of NBD-cholesterol in two different cultured CF cell models (9/HTEo- pCEPR and IB3) compared to respective controls (Figure 3-2A).

Quantification of NBD-cholesterol accumulation was accomplished using flow cytometry

analysis. Cells were treated similarly as before but allowed to process cholesterol in fresh

media for longer periods of time (approximately overnight). There is a significant

increase in mean florescence present in both CF-like cells; 9/HTEo- pCEPR had a 1.4 +

0.2 fold (p=0.003) increase compared to 9/HTEo- pCEP controls and IB3 cells had a 2.0

+ 0.2 fold (p=0.005) increase compared to S9 controls (Figure 3-2B). These data confirm

by separate technique the observation of cholesterol accumulation in CF cells initially determined by filipin staining and suggest that the observation is due to a flaw in lipid transport mechanisms.

Based on the increased content of free cholesterol in CF cells and tissues, it is postulated that cholesterol is accumulating in late endosomes and lysosomes, similar to

NPC. In order to determine if the cholesterol accumulation phenotype could be reversed,

9/HTEo- pCEPR (CF-phenotype) cells were treated with gly-phe-B-naphthylamide

(GPN), a cathepsin C substrate that causes lysosomal disruption (121). The pattern of

cholesterol accumulation was examined by visualization of NBD-cholesterol. 9/HTEo-

pCEPR cells were treated with 50µM GPN and 5µg/mL NBD-cholesterol for

approximately 24 h. GPN treatment reduces cholesterol accumulation in pCEPR to near control (pCEP) levels (Figure 3-3A). NBD-cholesterol content was objectively determined by flow cytometry analysis. Flow cytometry analysis reveals a significant decrease in NBD-cholesterol fluorescence in GPN treated cells compared to untreated

9/HTEo- pCEPR cells (Figure 3-3B). GPN treatment significantly reduces mean fluorescence in pCPER (CF-phenotype) cells approximately 34% to control (pCEP) levels. These data suggest that cholesterol is at least partially being accumulated in

lysosomal compartments. The use of NBD-cholesterol for trafficking studies must be

viewed with some caution as the hydrophobic NBD moiety can potentially interact with

membranes itself and interfere with processing. However, consistency and quantification

balanced these concerns for this study.

Increased cholesterol content in the plasma membrane of CF model systems.

Intracellular cholesterol accumulation in cultured and in in vivo CF models suggest a

direct flaw in cholesterol transport mechanisms. NPC cells have been reported to exhibit reduced plasma membrane cholesterol as a result of impaired NPC1 function (122).

Given the potential importance of membrane cholesterol content in immune responses to bacterial challenge (81) and in cell signaling through lipid raft formation, plasma membrane cholesterol content was measured in 9/HTEo- pCEP and pCEPR cells and in excised nasal epithelium from wt and Cftr -/- mice. Plasma membrane cholesterol content was measured utilizing a microelectrode containing cholesterol oxidase as described in

METHODS (120). In both cultured cell models and in nasal epithelial tissue, CF samples

exhibited an approximately 2-fold (p < 0.01) increase in detectable current indicating

increased membrane cholesterol content (Figure 3-4). These results are consistent with

earlier work demonstrating increased cholesterol content in plasma membranes of CF

lymphoblasts (123). Whether this increase in plasma membrane cholesterol content in CF

cells and tissues is due to elevated NPC1 expression (112) or to passive diffusion of cholesterol-rich lipid droplets is unclear. These data do demonstrate another manifestation of disrupted cholesterol homeostasis in both CF cells and tissue.

Increased sterol response element (SRE) activation in CF cell models. An increase in

unesterified cholesterol and impaired intracellular cholesterol trafficking suggest a

limited amount of cholesterol is reaching the ER in CF epithelial cells. A loss of

cholesterol sensing at the ER is predicted to lead to elevated expression of specific genes

containing sterol regulator elements (SRE) in their promoters to increase intracellular

cholesterol levels through exogenous uptake or endogenous synthesis (54). Utilizing an

SRE-luciferase construct containing the promoter regions of HMG-CoA synthase, an

enzyme involved in the cholesterol synthesis, gene responsiveness was measured in

9/HTEo- pCEP (wt-phenotype) and pCEPR (CF-phenotype) cells. In serum free media,

CF-phenotype cells exhibit a 2.7 + 0.4 fold (p < 0.001) increase in SRE-driven luciferase

expression above wt-phenotype controls (Figure 3-5). These data are consistent with the

hypothesis that impaired cholesterol processing in CF cells limits ER cholesterol delivery.

Disruption of extracellular cholesterol delivery to the ER due to

endosomal/lysosomal accumulation of cholesterol would predict that SRE regulation in

CF cells would be more dependent on de novo cholesterol synthesis relative to wt cells.

Inhibition of de novo cholesterol synthesis in CF cells would be hypothesized to lead to

significantly increased SRE activation in CF models cells compared to controls.

Treatment of CF-phenotype 9/HTEo- pCEPR cells and control pCEP cells with the

cholesterol synthesis inhibitor mevastatin leads to a 3.2 + 6.6 fold (p = 0.002) increase of

SRE-luc in pCEPR cells compared to only a 2.4 + 1.9 fold (p = 0.008) increase (3-5).

These data support the hypothesis that a disruption of cholesterol transport mechanisms

in CF cells results in alterations in cholesterol homeostasis.

In vivo new cholesterol synthesis in Cftr-/- mouse tissue. Lysosomal accumulation of cholesterol and activation of SRE-containing genes is predicted to stimulate de novo

cholesterol synthesis due to a lack of cholesterol transport to the ER. An increase in de

novo cholesterol synthesis is reported in NPC models (114) supporting this hypothesis.

To directly test this hypothesis in an in vivo model of CF, Cftr-/- mice were

intraperitoneally (i.p.) injected with deuterium labeled water. After eight hours, deuterium incorporation into newly made cholesterol in various tissues was determined using GC/MS analysis. Cftr-/- mouse lung had a 1.7 + 0.1 fold (p < 0.001) increase of

cholesterol synthesis compared to matched controls in an 8 h time period (Figure 3-6).

Cholesterol synthesis in the liver was also increased 1.8 + 0.2 fold (p < 0.001) in Cftr-/-

mice compared to control. No difference in cholesterol synthesis between wt and Cftr-/-

mouse small intestine was observed. These data demonstrate an inherent alteration in

cholesterol homeostasis leading to increased de novo cholesterol synthesis in an in vivo

model of CF compared to wt controls. D2O can potentially impact ion channel function

such as some proton channels in airway epithelium (124). The potential influence of D2O on ion transport must be considered when interpreting these data.

Impaired cholesterol transport is sufficient to cause CF-like cytokine release. Previous

work demonstrated identical alterations in the expression of a number of signaling proteins between cultured CF and NPC cell models (112). In order to determine if alterations in cholesterol homeostasis described in this manuscript are capable of influencing inflammatory signaling, 9/HTEo- pCEP and pCEPR cells were treated with the cholesterol transport inhibitor described above, U18666a (5 µg/mL). Cells were challenged with 1 x 109 CFU/well PA for 1 h and then assayed for IL-6 and IL-8

production after 24 h as previously described (125). Wild-type pCEP cells exhibited a

54.7 + 13.1 % (p = 0.002) increase in IL-6 and a 74.5 + 15.6 % (p < 0.001) increase in

IL-8 production in U18666a treated cells compared to cells challenged with bacteria

alone (Figure 3-7A, 7B). CF-phenotype pCEPR cells exhibited no difference in cytokine

production in response to U18666a treatment. The effect of U18666a exposure on

cholesterol transport in 9/HTEo- pCEP and pCEPR cells is demonstrated in Figure 3-7C

to confirm that cholesterol transport is being inhibited.

To further verify that U18666a treatment recapitulated CF-phenotypes, the impact

of U18666a on NOS2 expression was examined in Figure 3-7D. Both NPC and CF cells

fail to induce NOS2 expression (112), therefore, U18666a treatment is predicted to impair NOS2 induction as well. A549 cells were chosen for these studies due to their robust induction of NOS2 protein expression (56). Exposure to U18666a (5 µg/mL) for

72 hours results in an inhibition of NOS2 expression in response to 3 µL/mL cytomix

(CM). However, NOS2 expression is restored by the addition of 50 µM mevastatin for 24 hours prior to CM treatment, a finding consistent with previous results showing mevastatin-mediated correction of NOS2 expression in CF mouse models (45). These data in combination with previous work strongly supports the hypothesis that impaired cholesterol transport is an initiating factor in CF-related cell signaling.

The restoration of NOS2 expression with mevastatin directly implicates isoprenoid-dependent pathways in the effects mediated by U18666a. In addition to inhibiting cholesterol transport, U18666a has been shown to inhibit cholesterol synthesis by indirect interactions while considerably stimulating isoprenoid production (126).

The role of docosahexaenoic acid (DHA) in cholesterol transport regulation. Restoration

of DHA levels in CF models have been shown to effectively modulate inflammatory

responses (72, 74). In order to determine if DHA treatment was mediating its anti-

inflammatory influence in CF by intervening within the cholesterol transport pathway,

filipin staining and NBD-cholesterol trafficking was examined in 9/HTEo- pCEPR CF-

model cells in the presence of DHA. DHA (10 µM) has no effect on cholesterol

accumulation visualized by either filipin staining or NBD-cholesterol visualization

(Figures 3-8A, 8B). However, DHA is effective at reducing sterol response element (SRE)

activation as measured by SRE-luciferase reporter assay. Basal SRE activity is increased

2.6 + 0.3 fold (p < 0.001; n=10) in pCEPR CF model cells compared to control pCEP

9/HTEo- cells, consistent with increased cholesterol synthesis observed in Cftr-/- mice

(Figure 3-8C). DHA treatment reduces SRE-driven luciferase expression by approximately 50% in both pCEP and pCEPR cells. Whether reduced SRE activity in the presence of DHA is responsible for its anti-inflammatory properties needs further examination, but this interaction may represent a point of convergence between these pathways.

Discussion

Previous work demonstrated an increase in filipin staining in two cultured cell models of CF indicating an accumulation of free cholesterol compared to respective control cells (112). The pattern of free cholesterol accumulation was similar to that characteristic of NPC cells. In a comparative study, NPC cells were found to share a number of cell-signaling characteristics with CF epithelial cells including increased

RhoA expression and reduced NOS2 induction (112). These earlier results suggest that aberrations in cholesterol processing in CF epithelial cells could represent an important initiating step in aggressive inflammatory signaling. The goal of this manuscript is to further define cholesterol homeostasis in CF epithelial cells using both cultured models

and primary samples to determine if intracellular accumulation of free cholesterol is

potential source for other down-stream signaling events.

The current study demonstrates free cholesterol accumulation by filipin staining

in human CF bronchial airway and tracheal sections compared to non-CF tissues. These

data are consistent with results obtained in cultured models of CF epithelial cells and

suggest that free cholesterol accumulation is a CF-related phenotype relevant to the in

vivo condition. The cholesterol accumulation phenotype was also confirmed in cultured cell models using a second technique by visualizing the accumulation of a fluorescent cholesterol analog, NBD-cholesterol. Correction of the cholesterol transport phenotype by expressing full-length CFTR in S9 cells, coupled with the fact that pCEPR cells are a model of functional CFTR inhibition, suggest that CFTR activity is necessary for proper cholesterol movement.

Another aspect of cholesterol processing is the regulation of cholesterol transport to the plasma membrane. Given the potential importance of membrane cholesterol in regulating cell signaling and bacterial responses (81), membrane cholesterol content was examined in cultured cells and nasal epithelial tissue from CF mice by utilizing a

cholesterol-specific electrode. CF cells and tissue both exhibit an approximately two-fold

increase in membrane cholesterol content compared to respective controls. The existence

of this cholesterol phenotype in mice null for CFTR expression further indicates that a

loss of CFTR function, as opposed to a trafficking defect, is responsible for altered

cholesterol movement. Whether the increase in membrane cholesterol content in CF cells

is due to passive diffusion of lipid droplets to the membrane or to an increase in active

transport mechanisms is unclear. Passive transport is unlikely since NPC cells would

demonstrate a similar increase. Previous results demonstrated increased NPC1 mRNA

content in CF cells (112) suggesting the possibility of increased NPC1-driven membrane

cholesterol transport in CF. Specific mechanisms are currently being explored. Based on

comparison to NPC cells, it is improbable that membrane cholesterol content is

significantly affecting signaling regulation regarding RhoA and NOS2 expression as CF

and NPC cells have similar signaling but opposite membrane cholesterol content changes.

However, how bacterial interactions are influenced by increased membrane cholesterol

content is not addressed by these studies.

The functional consequences of aberrant cholesterol transport in CF cells also

need to be explored. Endosomal/lysosomal accumulation of free cholesterol is expected

to result in increased de novo cholesterol synthesis as described in NPC (114). Indirect

evidence of increased synthesis was shown with increased SRE-containing gene response

in CF cells compared to controls. Inhibition of cholesterol synthesis in CF cells with mevastatin caused a dramatic response in CF cells compared to controls revealing a reliance on cholesterol synthesis in regulating cholesterol homeostasis. To directly address increased de novo cholesterol synthesis, in vivo examination of synthesis was measured in lung, liver, and small intestine of wt and Cft -/- mice. Both lung and liver exhibit increased de novo cholesterol synthesis in Cftr-/- mice compared to controls, although cholesterol synthesis is unchanged in the small intestine. Why cholesterol synthesis is elevated in the liver is unclear. Previous reports suggest that CFTR is not expressed in hepatocytes, with most expression centered in bile duct epithelial cells (127).

These data imply a functional cross-talk between cell types within the liver or the

possibility that hepatocytes do have some low-level CFTR expression. Overall, these data

are consistent with a disruption of cholesterol homeostasis in CF.

The impact of disrupted cholesterol transport on epithelial inflammatory signaling

was also examined. Pharmacologically inducing endosomal/lysosomal accumulation of

free cholesterol with the compound U18666a in wt 9/HTEo- pCEP cells resulted in

elevated IL-8 and IL-6 production in response to PA challenge. However, U18666a

treatment had no influence on cytokine production in CF-phenotype 9/HTEo- pCEPR

cells. These data establish that disruption of cholesterol transport is sufficient to mimic

CF-like inflammatory signaling. The lack of effect of U18666a in pCEPR cells

demonstrates that U18666a is unlikely having a non-specific effect on cytokine

production and that impaired cholesterol movement is already contributing to responses

to PA challenge in these cells. U18666a also recapitulates the CF-phenotype of impaired

NOS2 induction in A549 cells, further verifying cholesterol transport inhibition as a

mechanistic source of cell signaling changes in CF cells. NOS2 expression inhibition by

U18666a is restored by mevastatin consistent with findings in CF mouse models (56).

The restoration of NOS2 expression with mevastatin directly implicates isoprenoid-

dependent pathways in the effects mediated by U18666a. In addition to inhibiting

cholesterol transport, U18666a has been shown to inhibit cholesterol synthesis by indirect interactions while considerably stimulating isoprenoid production (126). These data are consistent with previous work demonstrating the role of the isoprenoid-modified RhoA in

CF signaling cascades (45, 56).

Although not completely analogous, these data are consistent with a report by

Grassme et al. demonstrating aggressive inflammatory responses in a mouse model of

Niemann-Pick type A, an acid sphingomyelinase null mouse (80). In addition to a loss of

ceramide production, this mouse model also exhibits endosomal/lysosomal cholesterol

accumulation. Grassme et al. show that challenge with PA elicits excessive production of

IL-1 from these mice (80), suggesting that lipid transport is essential in modulating

inflammatory responses.

The mechanism by which cholesterol accumulation triggers increased cytokine

production is currently unclear. In addition to the associated cell signaling alterations

previously observed (112), several studies demonstrate a relationship between lipid

pathways and inflammation in CF. Oral treatment of docosahexaenoic acid (DHA) as a

means to correct the DHA and arachidonic acid (AA) imbalance in Cftr-/- mice corrected

intestine pathology and the inflammatory response in lipopoysaccharid (LPS) treated

mice (72, 74). Similarly, treatment with the AA metabolite lipoxin (LXA4) ameliorated

CF-like inflammation. Mice challenged with PA and treated with LXA4 to restore deficient endogenous levels exhibited a suppression of neutrophil inflammation and decreased bacterial burden (75). This manuscript demonstrates that DHA does not correct cholesterol transport, but its ability to inhibit SRE activation may be a source of its anti- inflammatory properties. This relationship, however, needs to be further explored.

In addition, the loss of the anti-inflammatory properties of PPARγ is likely directly related to the cholesterol transport aberration in CF cells. NPC cells have also been shown to be deficient in PPARγ activation (115) and RhoA is a known inhibitor of

PPARγ (128). Inhibition of RhoA function by preventing isoprenoid/cholesterol synthesis with statins restores NOS2 expression in Cftr-/- (45) mice and has been shown in other

systems to increase PPARγ signaling (79). A pilot study to determine the potential efficacy of statin therapy in reducing CF-related inflammation is underway.

Determining the relationship between CFTR and intracellular cholesterol trafficking is a clear next goal. CFTR is part of the super family of ABC transports and other family members such as ABCA1 are important for cholesterol trafficking. Direct cholesterol trafficking by CFTR has yet to be seen, however, there is some evidence that it can transport lipids such as sphingosine-1-phosphate (107). Is has also been determined that both channels, ABCA1 and CFTR, are inhibited by glibenclamide and inhibition of

ABCA1 causes cholesterol accumulation (129). These data potentially indicate a direct role of chloride function and cholesterol regulation. Another link between cholesterol and

CFTR function is proper pH of the endocytic pathway. The importance of pH has been shown in cholesterol trafficking and in CF model systems. Furuchi et al. has determined that an acidic pH is needed to properly facilitate cholesterol transport from the endosomal/lysosomal vesicles (130). CFTR is known to be recycled within the endosomal pathway (108, 131), and the regulation of organelle pH by CFTR has been examined with mixed conclusions (110, 132, 133). The relative ambiguity of the role of

CFTR in pH regulation makes conclusions regarding the role of these processes in cholesterol regulation difficult to assess. However, the building evidence regarding organelle pH and cell signaling is compelling. Recent work linking endocytic acidification and disrupted nitric oxide production directly draws these processes together

(134). This same group has also demonstrated that reversing the acidification with chloroquine corrects CF-related changes in TGF-β1 production (135). Based on these

relationships, strong consideration of organelle pH as a mechanistic source of aberrant cholesterol transport in CF must be given.

Data presented in this manuscript demonstrate an inherent defect in cholesterol homeostasis in multiple models of CF, including primary lung and trachea tissue.

Aberrations in cholesterol processing associated with CF cells and tissue include endosomal/lysosomal storage, elevated plasma membrane cholesterol content, and elevated rates of de novo cholesterol storage in lung tissue. Data also demonstrate that inhibition of cholesterol transport is sufficient to elevate cytokine production in a manner consistent with the CF-phenotype. In conclusion, these data demonstrate a novel cell biological process disrupted in CF that has profound implications on the understanding of the pathobiology of CF and represents important new therapeutic targets for the treatment of CF.

Acknowledgments: This work is supported by a grant from the Cystic Fibrosis

Foundation and by NIH/NHLBI grant HL080319. Technical support for this project was provided by the Flow Cytometry Core Facility of the Comprehensive Cancer Center of

Case Western Reserve University and University Hospitals of Cleveland (P30 CA43703) and the inflammatory mediator core of the cystic fibrosis center (P30 DK 27651), and

NIH RoadMap 1R33DK070291-01 and the Mt. Sinai Health Care Foundation (Cleveland,

OH). The authors thank Dr. Pam Davis (CWRU) for providing cell lines necessary for the completion of this study, Dr. Serpil Erzurum (Cleveland Clinic Foundation) for providing CF and control human lung sections, Dr. Laura Liscum (Tufts University) for helpful discussions of cholesterol transport mechanisms, and to P. Bead for technical assistance.

Figure 3-1. Unesterified cholesterol accumulation in CF tissue.

A) Filipin staining of non-CF and CF trachea epithelium tissue. B). Filipin staining of non-CF and CF of epithelium from upper airway tissue. To the right of each filipin stain is a transmitted image to indicate tissue structure. Images are representative of multiple sections of each sample. Trachea and lung tissue is from separate individuals. The bar on each image indicates 30 µm.

non-CF CF A) trachea

B) lung

Figure 3-2. NBD-cholesterol accumulation in two CF cell culture systems.

A) Cells were incubated for 24 hours with NBD-cholesterol, a fluorescent cholesterol probe, and then placed in fresh media for 4 hours before being fixed. 9/HTEo- pCEP and

S9 (wt) and pCEPR and IB3 (CF-phenotype) images are representative of average confocal images found over 5 experiments. B) NBD-cholesterol accumulation was quantified using flow cytometry analysis. Significance was determined by t test. Error bars represent SEM (n = 8 for each). *p = 0.002, **p = 0.005. The bar on each image indicates 8 µm.

pCEP pCEPR

S9 IB3

400 * 400 ** 300 300

200 200

100 100 mean fluorescence 0 mean fluorescence 0 pCEP pCEPR S9 IB3

Figure 3-3. Lysosomal storage of NBD-cholesterol in CF cells.

A) Cells were treated for 24 hours with 50 µM gly-phe-B-naphthylamide (GPN) and 5

µg/mL NBD-cholesterol. Images are average projections of z-stacks representative of results from 3 experiments. B) Quantification of decreased NBD-cholesterol accumulation in GPN treated cells was shown by flow cytometry analysis. Filled bars represent 9/HTEo-pCEPR (CF) cells and open bars represent 9/HTEo-pCEP (wt) cells.

Significance was determined by t test comparing non-treated (nt) pCEPR cells and GPN treated pCEPR. Error bars represent SEM (n = 3). * p = 0.008. The bar on each image indicates 45 µm.

A) pCEP pCEPR pCEPR + GPN

400 pCEP 300 * pCEPR

200

100 mean fluorescence mean

0 nt nt GPN

Figure 3-4. Microelectrode determination of membrane cholesterol content.

A) Representative traces of membrane cholesterol determination in 9/HTEo–pCEP (WT) and pCEPR (CF) cells. Control trace consists of an examination of pCEPR (CF) cells with a platinum electrode without incorporation of cholesterol oxidase to determine cholesterol specificity of current response. B) Representative traces of membrane cholesterol determination in excised nasal epithelium from Cftr-/- (CF) and sibling

Cftr+/+ (WT) mice. Control trace consists of an examination of Cftr-/- (CF) nasal tissue with a platinum electrode without incorporation of cholesterol oxidase to determine cholesterol specificity of current response. C) Quantification of responses between

9/HTEo–pCEPR (CF) and pCEP (WT) cells. Responses are reported relative to WT response (response ratio) to indicate the fold-increase in response. Error bars represent

SEM, n = 5 for each. Significance determined by t test. *p < 0.01. D) Quantification of responses between Cft -/- (CF) and sibling Cftr+/+ (WT) nasal tissue. Responses are reported relative to WT response (response ratio) to indicate the fold-increase in response.

Error bars represent SEM, n = 5 for each. Significance determined by t test. *p < 0.01.

A) 9/HTEo -cellsB) Mouse Nasal Epithelium

0.1pA CF 20 pA 2s 3s WT WT Control Control contact contact

C) 9/HTEo -cells D) Mouse Nasal Epithelium

3 3 * 2 2 *

1 1 Response ratio Response Response ratio Response

0 0 WT CF WT CF

Figure 3-5. Increased sterol response element (SRE) response in CF cells.

9/HTEo cells were incubated in serum, serum free (SF) conditions for 24 h with or without 50µg/mL cholesterol (chol), or 50µM mevastatin (mev) or both for an additional

24 h. Filled bars represent 9HTEo-pCEPR (CF) cells and open bars represent 9HTEo- pCEP (wt) cells. Data are normalized to serum control levels over 3 experiments.

Number (n) of samples is shown in parenthesis above each bar. Significance was determined by t test. Error bars represent SEM. * p = < 0.001, # p < 0.05.

50 (12) pCEP 45 pCEPR 40 * 35 (10) 30 * 25

(8)

(Fold difference) 15

RLU SRE-luc/renila * 10 (12) # 5 (12) 0 Serum + - - - - Serum Free - + + + + Chol - - + - - Mev - - - + - Chol/Mev - - - - +

Figure 3-6. Increased de novo cholesterol synthesis in Cftr-/- mouse tissue.

Deuterium incorporation into newly synthesized tissue cholesterol was measured by

GC/MS. Data is normalized to each tissue wt control and reported as fold increase of % newly synthesized cholesterol/8 h. Percent newly synthesized cholesterol in wt tissues

used as references were 4.2 + 0.5% lung, 11.3 + 1.7% liver, and 16.5 + 0.9 % small

intestine. Filled bars represent Cftr-/- mice (CF) and open bars represent their matched wt sibling controls. Significance was determined by t test; * p < 0.001. Error bars represent SEM. The number of replicates is shown in parenthesis above each bar and represents individual assays on multiple tissue samples obtained from five separate mice of each genotype over three experiments.

2.5 wt

(19) Cftr-/- 2 (21) * * * 1.5 (14) (21) (21) (16) 1 (fold increase) (fold

0.5 % new cholesterol synthesis/8h cholesterol % new 0 lung liver small intestine

Figure 3-7. Impaired cholesterol transport causes CF related cytokine release.

9/HTEo cells were treated with 5µg/mL U18666a (U18), cholesterol transport inhibitor,

for 24 h with and without Pseudomonas aeruginosa (PA) challenge for 1 h. The results

are shown as fold-increase normalized to untreated wt-phenotype pCEP 9/HTEo- ccells

from 3 experiemnts. A) 9/HTEo-pCEP cells treated with U18 and PA show increased IL-

6 release. B) 9/HTEo-pCEP cells treated with U18 and PA show increased IL-8 controls compared to PA treatment alone. Error bars represent SEM, The number of replicates is shown in parenthesis above each bar. Significance is determined by t test; * p = 0.002, # p = 0.0005. C) Representative image confirming U18 treatment (5 µg/mL) causes unesterified cholesterol accumulation visualized with filipin staining in 9/HTEo-pCEP and pCEPR cells.

* A) (8) pCEP 14 pCEPR

12 (5) 10 (8) (5) 8 6 4 Fold increase Fold (8) (8) (8) IL-6 (pg/mg protein) (pg/mg IL-6 2 (8) 0 U18 - + - + - + - + PA --+ + - - ++ B) 25 (5) 20 # (5) 15 10 (8)

Fold increase (8) IL-8 (pg/mg protein) 5 (8) (8) (8) (8) 0 U18 - + - + - + - + PA --+ +++- -

C) pCEP pCEPR

U18

Figure 3-8. The role of DHA in cholesterol transport.

A) Filipin staining of CF-phenotype pCEPR cells with and without exposure to DHA (10

µM) for 24 hours. Control 9/HTEo-pCEP cells are shown as a control. No effect of

DHA treatment is observed. Images are representative of duplicate experiments. B)

NBD-cholesterol visualization of CF-phenotype pCEPR cells with and without exposure

to DHA (10 µM) for 24 hours. Control 9/HTEo-pCEP cells are shown as a control. No effect of DHA treatment is observed. Images are representative of two coverslips each treatment viewing multiple fields. C) DHA reduces SRE activation in pCEP and pCEPR

cells. SRE activity is elevated in pCEPR cells compared to pCEP control cells (p <0.001).

DHA reduces SRE activity by approximately 50% in both cell types. Data presented as a

fold difference of the ratio of reporter construct per renilla luciferase control as relative

light units (RLU; firefly/renilla) normalized to untreated wt-phenotype pCEP 9/HTEo-

cells. Error bars represent SEM and significance determined by t-test comparing DHA-

treated to untreated cells for each respective cell line; * p < 0.01 (n = 8 to 12 for each

condition over two separate experiments).

pCEP (wt) pCEPR (CF) A)

10 µM DHA

B) pCEP (wt) pCEPR (CF)

10 µM DHA

C) 3.5 (12) pCEP 3 pCEPR 2.5 2 1.5 * (10) (8) 1 (8)* (Fold difference) 0.5 RLU (SRE-luc/renilla) 0 -DHA + DHA

CHAPTER 4: ACUTE AND CHRONIC INHIBITION OF CFTR FUNCTION CAUSES CHOLESTEROL ACCUMULATION

Nicole M. White*, Dechen Jiang†, James D. Burgess†, Stephen F. Previs‡^, Nicolas Salem§, Yu Kuang§, Fangjing Wang§, Chris Flask§, Zhenghong Lee§, and Thomas J. Kelley* Departments of Pediatrics and Pharmacology*, Department of Chemistry†, Department of Nutrition‡, Department of Medicine^, Department of Radiology§ Case Western Reserve University and Rainbow Babies and Children’s Hospital, Cleveland, OH

Abstract

Cystic fibrosis (CF) is caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR) ultimately resulting in severe lung disease.

The precise role of CFTR function in regulating pathophysiology is not well established.

There are conflicting ideologies explaining CF disease pathology and the role of CFTR function in this process. New evidence has established the importance of cholesterol homeostasis in CF pathology. In this study, we provide evidence for the loss of CFTR function causing an alteration in intracellular cholesterol transport. We found direct inhibition of CFTR function, by inhibitor 172 (INH-172), caused the accumulation of a fluorescent cholesterol probe in control epithelial cells, 9/HTEo-pCEP, similar to untreated 9/HTEo-pCEPR (CF-phenotype) cells. Altered cholesterol transport led to increased sterol regulatory element (SRE)-luciferase responses in INH-172 treated cells.

SRE elements are binding sites within promoter regions of genes involved in cholesterol regulation. The importance of CFTR function was also demonstrated through the disruption of cholesterol homeostasis in chronic mouse models of CF, ΔF508/ΔF508

(ΔF/ΔF) and R117H/R117H (R/R). Using live animal imaging, we demonstrated that both mouse models exhibited increased membrane cholesterol in excised nasal epithelium,

increased newly made cholesterol, and 11C-acetate label incorporation as compared to

matched controls. These mouse models represent a range of CFTR function. These results

support arguments for the necessity of CFTR function in maintaining proper intracellular

cholesterol regulation independent of a CFTR trafficking defect.

Introduction

The primary defect causing cystic fibrosis (CF) is the loss of a cAMP regulated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR). Most morbidity and mortality is associated with infection and inflammation resulting in severe lung disease (8). While an excessive inflammatory response is believed to be innate to CF,

and fundamental to a lack of CFTR function, the mechanisms that cause severe

inflammatory responses in CF are not well established (12, 13, 15). The goal of this work

is to support a role of CFTR functionality in CF pathophysiology by examining the previously determined disruption of cholesterol processes in CF model systems.

A lack of cholesterol sensing at the ER caused by altered trafficking of cholesterol

was discovered as the key event causing upregulation of de novo cholesterol synthesis in

CF models (136). Niemann Pick type C disease (NPC), a model of cholesterol

accumulation and increased endogenous cholesterol synthesis, provided clues of the

consequences from altered cholesterol transport (83, 88 ). In CF, indirect evidence of

upregulated isoprenoid/cholesterol synthesis was determined from the reversal of CF cell

signaling events by the addition of an HMG-CoA reductase inhibitor, mevastatin.

Mevastatin treated CF models were found to exhibit decreased activation of RhoA

GTPase and increased inducible nitric oxide synthase (NOS2) and TGF-β1/Smad3

pathway signaling (45, 53, 56). These data demonstrate that the cholesterol synthesis pathway is an important upstream mediator of pro-inflammatory cell signaling events. In addition, isoprenoid/cholesterol synthesis was discovered as the upstream mediator connecting cell signaling events. Overall, all of these pathways are hypothesized to be a consequence of innate signaling changes due to dysfunctional CFTR.

Two predominate theories exist regarding the role of CFTR in initiating inflammatory signaling. The first theory asserted by many researchers is that unexplained pathology in CF relates to altered intracellular trafficking of mutant CFTR (137).

Substantial evidence supports a second theory of CFTR function as pivotal for proper cellular function. Established cell lines overexpressing the regulatory (R) domain of

CFTR, 9/HTEo-pCEPR, provide a beneficial model of CF that exhibit a lack of chloride conductance and an inherent excessive inflammatory response (19, 20). Increased inflammatory responses in these cells indicated a clear need for CFTR function. The mouse model null for Cftr (Cftr-/-) did not exhibit overt lung disease; however, utilizing the Pseudomonas-agarose bead model for controlled challenge of bacteria indicated increased inflammatory mediators with increased mortality in CF treated mice comparison to treated control littermates (22).

Direct evidence for CFTR-mediated regulation of inflammation was revealed by modulating previously established decreased levels of NOS2 in CF models (95). CFTR gene transfer using serpin-enzyme complex receptor corrected NOS2 levels and nasal potential differences in Cftr-/- compared to controls (41). Additionally, mice with human

CFTR introduced to the intestinal epithelium using the fatty acid binding protein (FABP) promoter (FABP-hcftr) possess identical transepithelial potential differences in nasal

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epithelium compared to Cftr-/-. However, in the ileum the electropotential difference is corrected, correlating with normalized levels of NOS2 in the FABP-Cftr mice similar to control animals (40). Two different techniques correcting CFTR expression rescued the electrical potential differences inherent in CF and NOS2 protein expression supporting the hypothesis of CFTR function directly mediating alterations in inflammatory signaling.

The most common CFTR mutation, a deletion in phenylalanine residue (ΔF508) of the nucleotide binding domain, results in incomplete CFTR processing and accumulation in ER, and correlates with severe disease in CF patients (138 , 139). Less common CFTR gene mutations, such as the missense mutation, R117H, associate with milder disease (140). R117H, a mutation in the membrane spanning domain of the CFTR protein results in proper protein trafficking with modified chloride function at the membrane (141). Utilizing different mouse models containing different extents of CFTR function will help establish a link between CFTR function and cholesterol processing inherent in CF models.

We believe CFTR function to be central to changes in cholesterol processing in

CF models. The goal of this study is to further determine disruptions in cholesterol homeostasis primary to a CFTR defect. In the present study, acute inhibition of CFTR in epithelial cell culture models indicated a trend toward altered NBD-cholesterol trafficking and homeostasis. Further investigations of murine models containing physiologically relevant CFTR mutations also indicate an accumulation of cholesterol at the membrane of excised nasal epithelium, increased rate of newly made cholesterol synthesis, and 11C-acetate label accumulation in the lung compared to matched controls.

These cholesterol processes were previously determined to be markers for altered

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cholesterol processing and important in the CF inflammatory response (136). The ΔF/ΔF mouse, a model of lacking CFTR function, and the R/R mouse, a model of diminished

CFTR function, both exhibited disruptions in cholesterol processes. These data

demonstrate the importance of CFTR function for proper cholesterol processing in CF

models.

Methods

Cell culture. Human epithelium 9/HTEo- cells overexpressing the CFTR R

domain (pCEPR) and mock-transfected 9/HTEo- cells (pECP), the wild-type phenotype, were a generous gift from the lab of Dr. Pamela B. Davis (Case Western Reserve

University). Cells were cared for as previously described (19).

Mice. Mice lacking CFTR expression (CFTRtm1Unc) were obtained from Jackson

Laboratories (Bar Harbor, MA). CFTR wild-type mice were siblings of Cftr-/- mice.

Mice with the ΔF508 CFTR (CFTRtm1Kth) and R117H CFTR (CFTRtm2Uth) mutations, on

mixed backgrounds, were a gift from Dr. Kirk Thomas and Dr. Mario Capecchi from the

University of Utah. These mice were backcrossed into the C57BL6/J background for at

least 10 generations, and were between seven to nine weeks of age before being used for

experimentation. Cftr-/- and ΔF508/ΔF508 mice were fed a high-fat diet rodent chow,

Harlan Teklad 9F Sterilizable Rodent Diet (W) 7960, Harlan Teklad, Madison, WI).

Colyte (Schwarz Pharma, Wilwaukee, WI) was supplemented in autoclaved water to

prevent gastrointestinal obstruction. Wild-type and R1117/R117H mice were fed a

standard rodent chow of irradiated Prolab RMH 3000, Agway, Inc (Syracuse, NY) after

weaning. Mice were cared for in accordance with the Case Western Reserve University

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IACUC guidelines by the CF Animal Core Facility. Nasal scrapings of mouse epithelium were obtained from both wild-type and CF animals.

NBD-cholesterol staining. Cells were seeded at a density of 150-200,000