4084 DOI 10.1002/pmic.200600028 Proteomics 2006, 6, 4084–4099
REVIEW
Cystic fibrosis enters the proteomics scene: New
answers to old questions
Mario Ollero1, 2, Franck Brouillard1, 3 and Aleksander Edelman1, 2
1
Inserm, U806, Paris, France
2
Université Paris-Descartes, Faculté de Médecine René Descartes, Paris, France
3
Université Paris-Descartes, Faculté de Médecine René Descartes, Plateau Protéomes IFR94, Paris, France
The discovery in 1989 of the gene encoding for the cystic fibrosis transmembrane conductance Received: January 12, 2006
regulator (CFTR) and its mutation as the primary cause of cystic fibrosis (CF), generated an Accepted: March 6, 2006
optimistic reaction with respect to the development of potential therapies. This extraordinary
milestone, however, represented only the initial key step in a long path. Many of the mechanisms
that govern the pathogenesis of CF, the most commonly inherited lethal pulmonary disorder in
Caucasians, remain even today unknown. As a continuation to genomic research, proteomics
now offers the unique advantage to examine global alterations in the protein expression patterns
of CF cells and tissues. The systematic use of this approach will probably provide new insights
into the cellular mechanisms involved in CF dysfunctions, and should ultimately result in the
finding of new prognostic markers, and in the generation of new therapies. In this article we
review the current status of proteomic research applied to the study of CF, including CFTR-
related interactomics, and evaluate the potential of these technologies for future investigations.
Keywords:
Biomarkers / CFTR chloride channel / Disease / Epithelium / Interactomics
1 Introduction problem. CF is a frequent autosomal recessive disorder
associated with multisystemic complications that compro-
The great expectations aroused upon the discovery of the mise functions of several epithelia. This includes meconium
CFTR gene and its mutation as the primary cause of cystic ileus, malabsorption, distal obstruction at the intestinal level,
fibrosis (CF), soon gave way to the certainty that not only was pancreatic insufficiency, and airway obstruction accom-
a genetic disease being faced, but also a much more complex panied by recurrent infections, inflammation and respiratory
insufficiency.
The underlying cause of CF is the presence of mutations
Correspondence: Dr. Aleksander Edelman, INSERM U806, in the CFTR gene that lead to defective function of the cor-
Faculté de Médecine, site Necker, 156 rue de Vaugirard, 75015
responding CF transmembrane conductance regulator
Paris, France
E-mail: edelman@necker.fr (CFTR), a 1480-amino acid transmembrane protein princi-
Fax: 133-1-40615591 pally located at the apical membrane of epithelial cells lining
digestive and respiratory epithelia, as well as exocrine glands,
Abbreviations: BALF, bronchoalveolar lavage fluid; CAL, CFTR- including lung, sinus, pancreas, intestine, sweat ducts, bile
associated ligand protein; CAP70, CFTR-associated 70-kDa pro- ducts, and vas deferens. The function of the CFTR protein
tein; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane con-
was first identified as a chloride channel activated by cAMP
ductance regulator; ClCA, Ca21-activated Cl channel; EBP50,
ezrin-radixin-moesin binding phosphoprotein of 50 kDa; FEV1,
and protein kinase A (PKA) [1]. However, additional func-
forced expiration volume; NHE-RF, Na1/H1 exchanger regulatory tions have been discovered, such as regulation of other
factor; PKA, protein kinase A; PDZ, conserved sequence elements channels [2], transport of molecules [3], among others (for
within PSD-95/Disc-large/ZO-1; QS, quorum-sensing review see [4]). Over one thousand different mutations of this
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
,Proteomics 2006, 6, 4084–4099 Clinical Proteomics 4085
gene have been identified to date. Among them, the deletion
of phenylalanine at the 508 position (delF508) is the most
abundant in the Caucasian population. Although the nature
of the mutation determines to some extent the localization
and function of the defective CFTR protein, the relationship
between genotype and phenotype is obscure.
The effects of CFTR mutations on CFTR protein intra-
cellular trafficking and activity either as a chloride channel or
as a channel and/or transporter regulator in epithelia are
very well documented at present [5]. Since it has been estab-
lished that CFTR activity is that of an ionic channel, many
studies have emphasized its central role in the ionic equilib-
rium of epithelial cells. Accordingly, it has been proposed
that its deficiency results in defective anion secretion and
excessive Na1 reuptake across epithelia, leading to insuffi-
cient intraluminal hydration. This causes mucus accumula-
tion and impaired mucociliary clearance in airways, as well
as a defective impaction status in the intestine (Fig. 1A) [6].
Nevertheless, this paradigm of CF pathogenesis, based on
the principle of CFTR-controlled fluid homeostasis, is not
fully satisfactory since it does not explain how the CFTR
defect gives rise to the multiplicity in phenotypes of CF
patients bearing the same CFTR mutation, a fact that is
underlined by multiple phenotypic changes in different CF
models. Indeed, CFTR mutations have been associated with
a number of abnormalities in epithelia, including modifica-
tions of airway surface liquid volume/composition [6–9], in
antimicrobial activity [10], glutathione efflux [11], mucus Figure 1. Functions of CFTR. (A) Epithelial model for fluid secre-
sulfation [12], and cytokine release [13] (Figs. 1B, 2B). On the tion. The limiting factor for fluid secretion/absorption is CFTR, an
other hand, it is widely believed that other modifier genes cAMP-stimulated Cl2 channel. This channel regulates other
that are somehow related to CFTR functions or dysfunctions channels and/or transporters, such as (represented here) the
outwardly regulated Cl2 channel (ORCC), the Cl2/HCO32 anion
influence the variability of clinical CF phenotypes, even in
exchanger, and the Na1 channel (ENaC). In normal tissues Cl2
patients homozygous for the delF508 mutation [14, 15] ions are taken up into the cells by an NaK2Cl cotransporter. Its
(Fig. 2A). The diversity of defects suggests that CFTR is more intracellular concentration is above electrochemical equilibrium,
than a chloride channel that directly or indirectly controls which creates the driving force for Cl2 exit through apical Cl2
each of the above processes, and that it is somehow con- channels. This is followed by Na1 ions through the paracellular
nected to a plethora of functions by different cellular path- pathway (not shown), as well as by water, which also moves
ways. In addition, other Cl2 channels (Ca21-dependent Cl2 through aquaporin channels (not shown). Na1 is also absorbed
by the Na1 channel, and extruded from the cell by NaK2 ATPase
channel [16], outwardly regulated Cl2 channel [17] and/or
at the basolateral membrane. K1 ions are recycled by the K1
the voltage gated Cl2 channel ClC-2 [18, 19]) might alter- channel at the basolateral membrane. When CFTR is mutated Cl2
natively participate in fluid secretion. secretion is strongly diminished, and the epithelium becomes,
All together these observations point to the necessity of according to this model, hyperabsorptive for Na1. ENaC activity
global analyses to determine the level of expression of genes is increased, and Cl2 ions flow through the paracellular pathways
and/or their products, as well as their PTMs in CF tissues followed by water. The water content of the extracellular layer is
diminished, provoking impaired mucociliary clearance and
and cells, to unmask new important functions of CFTR.
favoring colonization by opportunistic pathogens. (B) Involve-
Such analyses, considered here as functional proteomics, ment of CFTR in different cellular processes.
will ultimately lead to a better understanding of CF physio-
pathology, and to the identification of severity biomarkers,
which will help develop new and better-adapted treatments.
In this review we present the interest and the problematics of 2 Global expression analyses in CF models
large-scale analytical methods in the context of CF, summa-
rize the strategies and results corresponding to the prote- 2.1 Interest of large-scale methods
omic studies performed to date, as well as the perspectives
for a more comprehensive and effective future screening of The recently developed “omic” methods are supposed to
the protein expression patterns that might lead to a more allow not only the gain in knowledge that is acquired from
rational conception of therapy. each set of experiments, but also to provide additional infor-
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
, 4086 M. Ollero et al. Proteomics 2006, 6, 4084–4099
mation, as compared to the more conventional approaches,
by yielding insights into the intricate interactions and net-
works among different genes or proteins [20]. This kind of
data, difficult to be obtained by reductionist methods, should
improve our understanding of the complex and multi-
factorial CF pathology, which involves multiple interacting
components of innate immunity, cell signaling pathways,
cellular trafficking and ion transport, among others. It must
be remembered that the diverse alteration patterns observed
in CF epithelia may represent either the primary con-
sequences of CFTR mutations or the end result of a series of
secondary downstream events (or both). One of the main
problems encountered by global analysis of gene expression
will be to discriminate between these two possibilities.
2.2 Learning from microarrays
Powerful techniques, such as DNA microarrays, which cur-
rently make it possible to undertake the analysis of global
gene expression at the transcription level have been intro-
duced into the field of CF research [7, 21–27]. This approach
has already given a new glimpse on the consequences of the
CFTR defect at the molecular level, as well as some good
lessons about the relevance of models, the reliability of data
for further research, and the use of alternate global gene
expression procedures, such as proteomics.
For example, the analysis of mRNA expression in whole
lung tissue from cftr2/2 mice by Xu and collaborators [26]
revealed a significant increase in 29 and decrease in 25 out of
the approximately 12 500 different genes represented on the
array. Those genes consistently modulated by the absence of
CFTR were sorted in different groups according to the func-
tion of the proteins they encode, including signal reception
and transduction (PEG-3, Grin 2d, Npr-3, ADRB-3), gene
Figure 2. CF phenotype and CFTR regulatory functions. (A) On transcription (CEBPd), inflammation (IL-1b, IL-4, cal-
the left, two chest X-ray images from two siblings homozygous granulin-S100 family), intracellular trafficking and degrada-
for the delF508 mutation, showing a diffuse bronchiectasis tion (proteosome 26S subunits, AP-2 a1, kinesin3a), and ion
change for sibling 1 with severe CF phenotype (top) and normal transport (solute carrier 38, Kir 4.2). These data led to an
X-ray chest for sibling 2 with mild disease (bottom). On the right,
interesting discussion about how those genes might partici-
the functional assay based on halide-sensitive fluorescent dye,
SPQ [95], was used on nasal ciliated cells from the same two
pate either in the improvement of lung function or in CF
siblings. This method allows the rate of Cl2 transport as the rate pathogenesis. The authors developed a model of cascade
of fluorescent changes to be measured in response to the effect compensating for the absence of functional CFTR
exchange of extracellular Cl2 with NO32, an anion that passes protein. However, it cannot be excluded that the gene
through CFTR but, unlike Cl2, does not quench indicator fluores- expression changes detected arise from nonepithelial lung
cence. After addition of cAMP agonists, activation of CFTR results cells, and that they are only indirectly related to the lack of
in Cl2 efflux and NO3 influx, producing an increase in fluores-
CFTR function in epithelia.
cence. In the experimental design shown here, Cl2 was replaced
by iodide (I), as CFTR is permeable to I, and SPQ fluorescence is Cellular heterogeneity of samples, in particular in epi-
more strongly quenched by I2 than by Cl2. As shown, there is no thelial lung diseases such as CF, appears therefore as an
change after addition of a cAMP agonist to the nasal cells of sib- important parameter that may bias the interpretation of data
ling 1, as expected for non-functional CFTR, whereas SPQ fluo- obtained by large-scale analyses. Using microarray hybridi-
rescence varied after addition of a cAMP agonist to the nasal cells zation to compare the mRNA expression profiles in well-dif-
of sibling 2, suggesting the CFTR/chloride channel and/or an
ferentiated human airway epithelial primary cultures from
alternative Cl2 pathway are functional in the cells of this patient.
(B) CFTR regulatory functions. It is admitted today that CFTR is a
CF and non-CF donors, Zabner et al. [7] have overcome the
multifunctional protein displaying functions different from the problem of lung cell heterogeneity. By studying this model,
first demonstrated cAMP-regulated Cl2 channel activity. These they avoided some of the difficulties associated with the use
potentially important functions are listed. of in vivo specimens, such as the secondary effects of CF,
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
