Cellular and animal models of cystic fibrosis, tools for drug discovery

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    Recent developments include novel therapeutics, ran-

    pancreatic insufficiency, intestinal lipid and bile salt malab-

    sorption are common inCF patients. The incidence of CF is 1/

    patients express a mutant form of CFTR that is partially or

    Pathology of CF-consensus and controversy

    CF has been aptly described as a generalized deficiency of

    secretory epithelia, its most characteristic clinical features are

    Drug Discovery Today: Disease Models Vol. 3, No. 3 2006

    San2500 newborn in Caucasians, more than 70,000 patients are

    registered worldwide. Over 1000 different CF mutations have

    been described. However, the most common mutation

    (DF508 or F508del) accounts for at least 70% of all CF alleles.

    This mutation causes amaturation defect of the CFTR protein

    resulting in enhanced turnover and severely reduced activity

    at the apical membrane. The realization that almost all CF

    summarized in Table 1. Although the general pattern of

    pathology in the CF population is nowwell established, there

    is a large variation in severity and progression of symptoms

    between individual patients. The major function attributed

    to the CFTR gene an apical epithelial chloride channel

    required for osmotic water transport across epithelia

    explains most aspects of the complexmulti-organ phenotype

    fairly well. This includes plugging of ducts in the pancreas

    and of the vas deferens, with subsequent tissue destruction,*Corresponding author: B.J. Scholte (b.scholte@erasmusmc.nl)

    1740-6757/$ 2006 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmod.2006.09.003 251ging from gene therapy to candidate drug treatments.


    Cystic fibrosis (CF) is caused by mutations of the CFTR gene,

    which encodes a hormonally regulated chloride-ion channel

    that is expressed at the apical membrane of epithelial cells. It

    is a vital component of osmotic water transport across secre-

    tory epithelia. The disease is characterized by recurrent lung

    infections with opportunistic pathogens, which result in

    progressive and irreversible loss of lung function. Further,

    It has been recognized for centuries that when a childs sweat

    tasted salty it would not thrive and die early. Only recently

    this has been explained as a deficiency of chloride resorption

    in the sweat gland duct, which is still a diagnostic hallmark of

    CF. It is clear nowwhy this abnormality, caused bymutations

    in the CFTR gene, is usually associated with pancreas fibrosis

    and lethal chronic lung disease (mucoviscidosis). Despite

    significant improvements in the past decades, the low life

    expectancy (35 years) and high morbidity of CF patients call

    for further development of therapeutic options.2Physiological Laboratory Department of Physiology, Development and Neuro3Erasmus University Medical Centre, Biochemistry Department, PO Box 2040

    Cystic fibrosis (CF) is a recessive inherited disease with

    a high incidence in the Caucasian population causing

    high morbidity and clinical costs. The disease is char-

    acterized by complex pathology of secretory epithelia,

    including chronic lung infections, pancreatic deficiency

    and intestinal disease. CF has been the subject of

    intense study both in the large and well-characterized

    patient population, and in different model systems.potentially active has resulted in an intensive search for

    pharmaceutical agents that improve CFTR trafficking and

    gating, and reduce CFTR turnover.1Erasmus University Medical Centre, Cell Biology Department, Ee1034, PO BoDRUG DISCOVERY



    Cellular and animalfibrosis, tools for drBob J. Scholte1,*, William H. Colledge2,


    Jan Tornell AstraZeneca, Sweden

    Andrew McCulloch University of California,

    Respiratory diseasesodels of cysticg discoveryartina Wilke3, Hugo de Jonge3

    040, 3000 CA Rotterdam, The Netherlands

    nce, University of Cambridge, Cambridge, UK CB2 3EG

    00 CA Rotterdam, The Netherlands

    Section Editor:Nelly Frossard Faculte de Pharmacie, Universite LouisPasteur, Illkirch, France

    Diego, USA

  • Drug Discovery Today: Disease Models | Respiratory diseases Vol. 3, No. 3 2006Glossary

    Corrector: pharmaceuticals that enhance the amount of mutant CFTR

    at the apical membrane, by improving folding and trafficking, or

    preventing breakdown.

    Immortalized cell: primary differentiated cells are isolated from

    donor tissue and transduced in culture with proto-oncogenes, usually

    derived from viruses, which overrule the apoptotic and senescence

    pathways that normally restrict cell growth. Alternatively, cells are

    isolated from a spontaneous primary tumor. Further selection of

    (sub)populations for growth under specified conditions leads to

    accumulation of secondary mutations, and a more or less stable

    phenotype. In general, expansion of cell lines with a differentiated

    phenotype is limited. See the Virtual Repository website (Links) for

    further information.

    Mouse model: we can distinguish recombinant mutant mouse strains,

    generated by homologous recombination of an endogenous target gene

    in mouse embryo stem cells. Further, transgenic mouse strains, which

    are made by injection of a gene expression cassette in mouse oocytes,

    resulting in random integration of the cassette in the mouse genome. Aninflammation and fibrosis. However, despite intense research

    in the past three decades there are still several major issues

    unresolved (Outstanding issues). In particular, the events

    leading from CFTR dysfunction to chronic lung disease are

    not completely understood. One line of thought is that

    abnormal and viscous luminal secretions in CF lungs pro-

    mote bacterial colonization, followed by chronic inflamma-

    tion and irreversible tissue remodeling. An alternative view is

    that CF epithelia have inherent pro-inflammatory properties,

    owing to abnormal cytokine secretion and signaling. This

    suggests that inflammation and tissue remodeling may actu-

    ally precede bacterial infection (Outstanding issues). Another

    contested issue is whether CF lung disease is causedmainly by

    fluid hyper-resorption through sodium channels of the sur-

    face epithelium, or by a deficiency of CFTR dependent secre-

    tion from submucosal glands. Clearly, improvement of CF

    therapy requires an answer to these questions. The limited

    annotated list of available CF mutant models can be found at the Virtual

    Repository website (Links).

    Patch clamp analysis: allows ion current detection of a single ion

    channel molecule in a membrane patch attached to a micropipette [45].

    The use of this approach in the different model systems is still very

    important in the field of CF drug discovery. Like planar lipid technology it

    allows a detailed study of an ion channel while it interacts with other

    molecules, including inhibitors and potentiators. An introduction to this

    and related techniques can be found at the Virtual Repository website


    Potentiator: pharmaceuticals that enhance intrinsic activity of mutant

    CFTR, usually by increasing the open probability (Po) of the ion channel.

    Ussing chamber: a device in which a layer of epithelial cells can be

    mounted between two buffer compartments. This allows the

    measurement of electrical resistance, capacitance and net electrical

    currents generated by the epithelium. In open circuit mode electrical

    voltage (PD) can be measured. In short circuit mode the current

    required to clamp the voltage to zero (Isc) is determined. With specific

    inhibitors and by creating transepithelial ion gradients the contribution

    of different of transport systems can be identified. An introduction to

    this and related techniques can be found at the Virtual Repository

    website (Links).

    252 www.drugdiscoverytoday.comavailability of patient tissue and the constraints on clinical

    research make the use of model systems inevitable in this

    pursuit, despite their disadvantages and caveats. This review

    points the reader toward recent developments in this field

    and their potential for drug discovery.

    Properties of CFTR and its mutant forms

    CFTR is amember of the ABC transporter family characterized

    by two series of six membrane-spanning helices interspaced

    by a large intracellular domain (R-domain), and two ATP

    binding domains neighboring the R-domain and C-terminal

    domain, respectively (Fig. 1) [1,2]. In contrast to other mem-

    bers of this family, which act as ATP driven transporters, CFTR

    acts as a gated anion channel. The activation of its conduc-

    tance requires both binding of ATP and hormone induced

    phosphorylation of the R-domain.

    The protein is synthesized on the endoplasmic reticulum,

    glycosylated in the Golgi system and routed to the apical

    membrane of epithelial cells [3,2]. At the apical membrane it

    anchors to the subapical cytoskeleton and recycles to a sub-

    apical vesicular pool [4]. This complex process involves inter-

    actions with various chaperones and routing complexes

    (Fig. 1). Each of these interactions is a possible target for

    therapeutic intervention [5]. In the CF populationmore than

    a thousand different mutations have been described, which

    are found in all functional domains of the proteins [6] (CF

    Mutation Database). The three base pair in-frame deletion of

    a phenylalanine at codon 508, is the major CF mutation

    which occurs on about 70% of all CF-chromosomes and in

    >90% of all CF patients worldwide. In most publications the

    indication DF508 is still used, although F508del is actually

    correct in current genetics. The mutant DF508 CFTR protein

    lacks a phenylalanine in the intracellular domain, which

    causes inefficient folding at the endoplasmic reticulum,

    and subsequent breakdown of the nascent protein [2,3]. Only

    a further ten mutations occur with the frequency of more

    than 1%. CF mutations are classified in six categories [7]. In

    Class I mutations, no CFTR protein is synthesized, predomi-

    nantly because the CFTR mRNA is subjected to cryptic stop-

    codon induced decay. Class II mutants are defective in post-

    translationalmaturation and trafficking. Category III consists

    of regulatory mutants of the CFTR protein, Class IV mutants

    have altered channel properties. Class V mutations lead to

    significantly reduced amounts of wild-type CFTR protein, in

    most cases resulting from aberrant splicing, and the class VI

    mutants are characterized by increased CFTR protein turn-

    over at the apical membrane (Fig. 1).

    Once at the apical membrane, CFTR acts as an element in

    a functional molecular network. To perform its main func-

    tion in osmotic fluid transport, CFTR shows direct and

    indirect interactions with several other transport systems

    and cytoskeletal elements (Fig. 1). Fluid homeostasis acrossepithelia requires simultaneous transport of chloride,

  • Vol. 3, No. 3 2006 Drug Discovery Today: Disease Models | Respiratory diseases

    Table 1. Comparative pathology of CF in human and mutant micea

    Human pathology Tissues affected

    in CFTR mutant

    Mouse pathology

    Recurrent bacterial lung

    infections, chronic inflammation,

    airway remodeling, irreversible

    loss of function

    Lung No CF type lung disease; hyper-inflammation,

    reduced clearance upon challenge; incidental

    inflammation and tissue remodeling

    High amiloride response, low chloride

    conductance, frequent polyps

    Nasal epithelium


    Increased amiloride response,

    goblet cell hyperplasia

    Hyposecretion, viscous mucus, plugging Submucosal gland

    (nasal, bronchial)

    Nasal and tracheal glands present,

    no published reports on (dys)function

    Bacterial biofilm, viscous/purulent

    mucus, defective mucociliary clearance,

    goblet cell hyperplasia, inflammation,

    tissue remodeling

    Trachea, bronchial

    epithelium (ciliated/goblet)

    Reduced mucociliary clearance


    Not well characterized in early disease,

    CFTR expression in CLARA cells

    (strain and age dependent)

    Bronchiole (CLARA cells) Spontaneous remodeling

    and inflammation

    No known pathology, CFTR

    expression in cultured type II cells

    Alveoli (type I/II) No known pathology

    Meconium ileus (perinatal intestinal

    obstruction), distal intestinal obstruction

    syndrome (DIOS), chronic malabsorption,

    reduced body weight, failure to thrive

    Intestine Mucus plugging crypt and lumen, lipid and bile salt

    resorption defect, reduced body weight, failure to

    thrive. Goblet cell hyperplasia, Paneth cell

    dysfunction, chronic inflammation

    Frequent biliary disease,

    progressive cirrhosis

    Liver No morbidity, progressive liver

    disease in aging CF KO mice

    CFTR expression, altered function

    suspected but not shown directly

    Biliary duct CFTR expression, reduced fluid output.

    Duct damage upon induction of

    colitis in KO mice, plugging

    in aging mice

    Increased frequency of bile stones Gall bladder No cAMP induced Cl or water secretion

    Defective bicarbonate and pancreatic

    enzyme secretion in most CF

    patients, resulting in lipid maldigestion,

    reduced body weight, failure to thrive

    Pancreas Ranging from normal to partially affected

    No pathology Tooth Abnormal enamel (white teeth)

    No pathology, reduced

    flow rate

    Salivary gland Adrenergic fluid secretion defective

    High NaCl in sweat

    (defective resorption

    in duct)

    Sweat gland Footpad glands only, no report on deficiency


    (male infertility 100%)

    Vas deferens Frequent plugging or absent (male infertility)

    Frequent obstruction Oviduct Frequent obstruction (female infertility)

    No pathology reported Cornea Deficient chloride permeability, enhanced amiloride response,

    P. aeruginosa uptake deficient

    No pathology reported Smooth muscle CFTR expression, altered function and innervation

    No pathology reported Mast cells, dendritic cells CFTR expression, no pathology reported

    No known pathology Kidney Functional CFTR in tubular system, abnormalities in

    Na+ and K+ transport, no overt pathology

    a This table summarizes highlights of comparative human and mouse CF pathology. For general reviews on human pathology see [6,7] for a review on CF mouse pathology [23,24]. A fully

    annotated version of this table can be found at the Virtual Repository and EuroCareCF websites (Links). The main affected organs are indicated in the middle column.

    www.drugdiscoverytoday.com 253

  • Drug Discovery Today: Disease Models | Respiratory diseases Vol. 3, No. 3 2006sodium, potassium and bicarbonate ions. Combined trans-

    cellular current and paracellular flow through epithelial tight

    junctions determines the net flux and equilibrium para-

    meters. CFTR is not the only chloride channel expressed in

    epithelial cells. Notably, the epithelial calcium activated

    chloride channel (CaCC), which has not been cloned as

    yet, may function as an important compensatory chloride

    channel in CF, in particular in CF airways.

    Considering the complex nature of these interactions, it is

    not surprising that CF pathology is highly variable in the CF

    population. This depends not only on the type of the CFTR

    mutation and on environmental factors, but also on the

    genetic makeup of the patient. The identification of modifier

    genes, genes that play a prominent role in CF pathology,

    could identify important new clues toward therapeutic inter-

    vention [8,9]. Clearly, the most important and most relevant

    Figure 1....