Department of Molecular Genetics, Leiden Institute of Chemistry,
P. O. Box 9502, 2300 RA Leiden, The Netherlands, the
Department of Pathology, Leiden University Medical
Center, P. O. Box 9600, 2300 RC Leiden, The Netherlands, and the
§ Center for Applied Molecular Biology, School of Pharmacy,
University of London, 29/39 Brunswick Square, London WC1N 1AX, United
Kingdom
Received for publication, January 16, 2001, and in revised form, March 14, 2001
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ABSTRACT |
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The protective barrier provided by stratified
squamous epithelia relies on the cornified cell envelope (CE), a
structure synthesized at late stages of keratinocyte differentiation.
It is composed of structural proteins, including involucrin, loricrin,
and the small proline-rich (SPRR)
proteins, all encoded by genes localized at human chromosome 1q21. The
genetic characterization of the SPRR locus reveals that the various
members of this multigene family can be classified into two distinct
groups with separate evolutionary histories. Whereas group 1 genes have
diverged in protein structure and are composed of three different
classes (SPRR1 (2×), SPRR3, and SPRR4), an active process of gene
conversion has counteracted diversification of the protein sequences of
group 2 genes (SPRR2 class, seven genes). Contrasting with this
homogenization process, all individual members of the SPRR gene family
show specific in vivo and in vitro expression
patterns and react selectively to UV irradiation. Apparently,
creation of regulatory rather than structural diversity has been the
driving force behind the evolution of the SPRR gene family.
Differential regulation of highly homologous genes underlines the
importance of SPRR protein dosage in providing optimal barrier function
to different epithelia, while allowing adaptation to diverse external insults.
An essential function of stratified squamous epithelia is to
provide a protective barrier for the organism against extracellular and
environmental factors. The cornified cell envelope
(CE),1 a specialized
structure formed beneath the plasma membrane of differentiated cells,
is a major component responsible for this protective function (1-3).
The CE is an insoluble ~15-nm-thick layer, which is the result of
extensive cross-linking of several proteins by both disulfide and
N Human SPRRs consist of a multigene family (16-18), clustered in a
170-kilobase region within the epidermal differentiation complex (EDC)
on human chromosome 1q21 (16, 19-22). Orthologs of these genes have
also been described in other mammalian species (reviewed in Ref. 23).
The SPRR proteins have an identical structure consisting of head
(amino-) and tail (carboxyl-terminal) domains, comprising several
glutamine and lysine residues, and a proline-rich central repetitive
domain. Whereas the head and tail domains show a high degree of
homology with other CE precursors (e.g. involucrin and
loricrin; Ref. 24), the internal repeats, which vary in both number and
consensus sequence, distinguish the various members of this gene
family, allowing their classification into several SPRR classes. Based
on the specific sequences of these internal domains, secondary
structure algorithms have predicted various degrees of flexibility for
different classes (SPRR2 < SPRR1 < SPRR3) (25).
The SPRR classes are differentially regulated in various types of
epithelia, and their expression is modulated in response to
environmental insult (UV irradiation), aging, diseased states, and
following carcinogenic transformation (18, 23, 26-37). Among all
cornified envelope precursor proteins identified to date, SPRRs are the
only ones that are encoded by a multigene family. Important questions
concerning the reason for this complexity remain to be addressed. To
provide novel insights to these questions, we have characterized the
whole human SPRR locus, including the identification of several new
members, the refinement of the physical and transcriptional maps, the
comparison of gene and deduced protein structures, and the
establishment of in vivo and in vitro expression patterns at the single gene level and after UV irradiation.
Cosmid Library Screening and SPRR Contig Assembly--
The
chromosome 1 cosmid library ICRFc112 (38) was screened with SPRR class
specific probes (16). A total of 27 cosmids covering the SPRR locus
were identified and used for preliminary contig assembly (21). A
minimal tiling path comprising seven cosmids was chosen for further
analysis, and overlap was verified by cosmid walking (39). Cosmids,
linearized with NruI or CpoI (both are unique
restriction sites in the vector), were partially digested with either
BamHI, BglII, EcoRI,
HindIII, KpnI, NcoI, PstI,
or XbaI and analyzed by pulsed-field gel electrophoresis as
previously described (21), allowing the establishment of a contiguous
restriction map. All EcoRI bands, identified by
hybridization of human DNA with an SPRR2 probe (16, 21), were detected
on the various cosmids, and sequence analysis established the presence of seven SPRR2 genes. SPRR1A, SPRR1B, and
SPRR3 genes have been described (40-42). A probe for
SPRR4 was derived from an expressed sequence tag (EST)
library and obtained by RT-PCR of epidermal RNA. Superposition of the
sequencing data with the contiguous restriction map provided the
physical map of the SPRR locus, with the exact position and the
transcriptional orientation of the different genes. Gene orientation
and intergenic distances were verified by long-distance (LD) PCR (Fig.
1).
Cell Culture--
Normal human keratinocytes were cultured as
previously described (43) and induced to terminally differentiate by
using the stratification assay (43). Shortly, monolayers of basal cells ( Expression Studies--
Frozen tissues obtained from the
Department of Pathology (Leiden University Medical Center) were
homogenized, and total RNA was isolated by the Trizol method (Life
Technologies, Inc.). RNA from skin, esophagus, and uterus were also
purchased from Invitrogen. Total RNA (0.4 µg) was reverse transcribed
using Super RT (SphaeroQ) and random hexamer primers (Amersham
Pharmacia Biotech). PCR was performed for 25, 30, and 35 cycles with 20 pmol of gene-specific primers and 0.4 units of AmpliTaq DNA polymerase
(PerkinElmer Life Sciences). The specificity of the various primer
mixes was determined by using the various cosmids or derived plasmids
containing a single SPRR gene and verified by sequencing (data not
shown). The absence of DNA contamination in RNA preparations could be easily controlled as several primer combinations bridged an intron. The
positions of the respective primers in the SPRR sequences and the size
of the PCR fragments are indicated in Table I.
The Refined Physical Map and Transcriptional Orientation Identify
Two Distinct SPRR Groups--
Fig. 1
shows the physical map of the complete SPRR locus, which was determined
by analyzing a cosmid contig with 8 different restriction enzymes (see
"Experimental Procedures"). The cluster, previously localized to a
170-kilobase region on human chromosome 1q21 (16, 21), comprises 11 genes: SPRR1A, SPRR1B, 7 SPRR2 genes
(A-G), SPRR3, and the recently
identified SPRR4
gene.2 Long distance PCR
(LD-PCR) was performed to confirm the relative position and orientation
of the various genes (stippled lines). The transcriptional
orientation of the various members (indicated by arrows) is
not random and allows the splitting of the SPRR cluster into two
groups. One group consists of SPRR1A, SPRR1B, SPRR3, and SPRR4, which are placed in a proximal
region and are transcriptionally oriented from centromere to telomere.
The other group comprises the seven SPRR2 genes, clustered in a
100-kilobase region, all oriented in the same direction but opposite to
group 1 genes.
Group-specific Differences in the Diversity of SPRR Protein
Structures--
The subdivision of SPRR genes into two groups is also
justified by the comparison of the predicted amino acid sequences (Fig. 2A). Group 1 genes
(SPRR1A, SPRR1B, SPRR3, and
SPRR4) are characterized by a long amino terminus, an
8-amino acid repeat motif and a short carboxyl terminus, whereas group
2 genes (all SPRR2) have a short amino terminus, a 9-amino acid repeat
motif, and a more extended carboxyl terminus. In the central repeats of
the various proteins, more diversity exists among group 1 proteins,
which show differences in both number of repeats and consensus sequence
of the repetitive unit. These differences justify the classification of
the four group 1 genes into three classes (SPRR1, SPRR3, and SPRR4). On the contrary, the seven group 2 proteins are characterized by a much
higher homogeneity, as each member contains three repeats of the same
nonamer consensus. Hence, all group 2 genes belong to a single class,
SPRR2. In Fig. 2B the central repetitive domains of SPRR2
genes have been aligned. Although repeats 1, 2, and 3 of a single gene
have different consensi at the nucleotide level (mainly because of
variations in the wobble position), each of the three repeats is highly
conserved among the various members. This indicates that during
evolution, repeat duplication has preceded gene duplication and was
maintained hereafter in each gene. The seven group 2 proteins (Fig.
2C) are highly homologous. For instance, 2B
differs from 2A by 1 amino acid and from 2D and
2E by 2 residues. Notably, all amino acids previously
identified as being involved in transglutaminase-mediated cross-linking
during CE formation (11, 14) are conserved in all SPRR2 proteins
(red residues).
Fig. 3 provides a global view of the
sequence conservation among group 2 genes (black plot). The
highest similarity is found in exon 2 (94%) and corresponds to the
amino terminus of the protein (from position 1250 to 1400).
Nevertheless, high sequence conservation is not restricted to the
coding sequence, because in SPRR2B and 2E
(red plot) a 550-base pair region, with 100% identity,
extends from the intron to the coding sequence (positions 850-1400).
The various promoters revealed an average homology of ~70%. The
major differences are between positions 200 and 300 bases and are due to a deletion in SPRR2B.
Differential Expression among Single SPRR Genes--
The lower
sequence conservation within the promoters of both group 1 (42) and
group 2 prompted us to monitor the specific expression pattern for each
gene. Initially, we analyzed RNA from various human tissues by
hybridization with class specific probes (results not shown). Besides
the expected expression in various stratified squamous epithelia (27,
35), some SPRRs were also detected in tissues that (normally) do not
contain these epithelia (uterus, bladder, ovary, and trachea). Uterus,
ovary, and 3 stratified squamous epithelia, namely skin, esophagus, and
cervix, were chosen for single gene analysis. Likewise, expression in a
well established in vitro system, which permits the study of
keratinocyte terminal differentiation (45), was also analyzed.
Because of the high homology within the SPRR family, gene specific
semi-quantitative RT-PCR was carried out to characterize the relative
expression patterns for individual genes (Fig.
4). The analysis of calcium-mediated
in vitro keratinocyte differentiation ( Individual SPRR Genes Respond Selectively to UV
Irradiation--
To analyze the response of the SPRR gene family to
external damaging insults, we have treated human keratinocyte cultures with UV light and measured the expression of individual SPRRs (Fig.
5). In two independent experiments,
various members reacted selectively to this DNA-damaging agent. Whereas
SPRR4, 2C, and 2G are consistently
induced, a certain degree of variability is observed between individual
experiments in the case of 2B, 2D, and
2F. SPRR1A, 1B, 3,
2A, and 2E do not respond to UV irradiation. The
variability in SPRR2B, 2D, and 2F
induction is likely because of small differences in cell density, which
are difficult to control at the start of the experiment, but might
affect gene expression (46). UV irradiation did not affect the
expression of involucrin. Overall these results indicate that
individual SPRR genes are differentially expressed, although only a
limited amount of biological samples (five different human tissues and
in vitro-cultured keratinocytes) were analyzed, and the
effect of a single external agent (UV) was studied.
The cornified envelope (CE) has a vital role in the barrier
function of stratified squamous epithelia. Recent biochemical studies
suggest that SPRR proteins are the major modulators of the
biomechanical properties of cornified envelopes (12-15). Among all CE
precursor proteins identified to date, SPRRs are the only ones that are
encoded by a gene family. Although all genes have a common ancestor
(16), the present analysis indicates that the family can be divided
into two distinct subgroups with separate evolutionary histories.
Whereas group 1 genes have clearly diverged in protein structure, group
2 genes are characterized by a highly conserved coding sequence.
Darwinian selection (recently reviewed in Ref. 47) is not likely to be
the driving force behind this conservation, as most wobble
positions, including those which are specific for each of the
three repeats (Fig. 2B), have been strongly preserved among
all group 2 genes. The nature of the process responsible for this high
similarity is revealed by comparing SPRR2B and
SPRR2E (Fig. 3). An identical 550-base pair long region, flanked by non-identical DNA, points to gene conversion as the implicated mechanism. Gene conversion is a process of homologous recombination, which can be defined as a non-reciprocal transfer of
information between two sequences. As one sequence can be converted into the other one this process can result in the homogenization of
gene families (reviewed in Ref. 47).
Whereas the chromosomal organization and the protein structures of
SPRRs clearly distinguish group 1 and group 2 genes, such subdivision
is not evident when examining the expression patterns of individual
genes. In fact, the major finding of this work is that all human SPRR
genes, irrespective of the group or class they belong to, are under the
control of specific and selective regulatory processes. Apparently,
during the evolution of the SPRR gene family, creation of regulatory
diversity was more important than diversification in protein structure.
This implies that the control of protein dosage must be of major
importance for the function of these genes.
Our RT-PCR analysis corroborates and extends earlier studies using
class-specific DNA/RNA probes, antibodies, or CE peptide sequencing,
which have not allowed the detection of gene-specific differences
within one class. The high expression of SPRR3 in esophagus
and its absence from epidermis (27), as well as the elevated expression
of SPRR1 in internal epithelia (48) have previously been
observed. An interesting novel observation is the preferential
expression of SPRR2G and SPRR4 in skin.
Generally, it appears that genes that are well expressed in external
"dry" epithelia (skin) are lower in internal "wet" epithelia
and vice versa. Especially the preferential expression of
SPRR2F in ovary is noteworthy. Expression of specific SPRR2
genes in murine uterus (23) and the presence of an SPRR homolog in
cultured Chinese hamster ovary (CHO) cells (49) have previously been
reported. At present, there is no satisfactory explanation for SPRR
expression in these organs, which do not contain stratified squamous
epithelia. It has been suggested that the presence of SPRRs in
non-squamous epithelia might reflect a predisposition to undergo
squamous metaplasia (23, 50). Alternatively, SPRR genes could be
involved in other forms of programmed cell death (apoptosis), which is
known to occur in these tissues (51, 52). A recent inspection of
bladder epithelium with specific antibodies revealed SPRR1
and SPRR3 expression in the most superficial (umbrella)
cells.3 Hence, a more
thorough investigation, which is beyond the focus of this paper, will
be imperative to assess the relevance of SPRR expression in these tissues.
Whereas differential expression of individual SPRR genes is likely to
reflect the specific barrier requirements of different epithelia, the
UV experiment underlines the importance of barrier adaptation following
external insults. UV responsiveness of SPRRs is not a novel finding,
because they were originally isolated in our laboratory as UV inducible
genes (17). The novelty resides in the fact that specific members of
this gene family are selectively induced by UV light. Consequently,
induction of SPRR4, 2C, and 2G is not
caused by a global effect of UV irradiation on the process of terminal
differentiation, during which most SPRRs are induced (Fig. 4). This
view is also supported by the finding that involucrin expression is not
modulated after UV irradiation (Fig. 5). These results indicate that,
besides providing resistance and flexibility to very specialized
tissues, SPRRs might fulfill a major role in the adaptation of
epithelial barriers to a large variety of external and endogenous stimuli.
Recent evidence has indeed linked SPRR expression with barrier
formation during mouse development (53). Within the cornified cell
envelope, which constitutes a major determinant of the protective barrier, SPRRs have a specialized role as they function as
cross-bridging agents, which either interconnect or adjoin other CE
precursor proteins. Both the structure and the concentration of the
various SPRR proteins are believed to affect the biomechanical
properties of the CE (25). It is possible that even small changes in
amino acid composition can influence these parameters. Whereas the use of one specific class is probably dictated by tissue specific requirements, adaptation to external signals is likely to be more efficient by varying the concentration of a given SPRR protein. Both
mechanisms are however by no means exclusive. Indeed, as various
epithelia are exposed to specific insults, some correlation between
tissue expression and responsiveness to a given agent can be expected.
As such, the finding that SPRR2G and SPRR4, which are preferentially expressed in the epidermis, are also responsive to
UV irradiation is not surprising.
External insults can be numerous and can differ largely between
different epithelia (e.g. UV irradiation for the epidermis, tobacco smoke, or food-derived chemicals for oral epithelia, acid reflux for esophagus). By taking into account this large diversity of
external insults, which might request barrier function adaptation, it
is unlikely that all these signals are channeled to a single regulatory
promoter region. A gene family, coding for highly homologous proteins,
regulated by specialized promoters, responding to both inducing and
repressing signals, is likely to allow fine-tuning of the barrier, to
guarantee optimal protection to the organism.
The identification of two groups of UV inducible genes
(dependent/independent on the culture conditions) within the SPRR2 class indicates that at least two different UV responsive signaling pathways selectively target specific members of the gene family. Other
signal transduction cascades, initiated by other external or endogenous
agents, are likely to regulate other family members. Our previous
finding that the SPRR2A promoter, which is not affected by
UV light (Fig. 5), is under the control of an interferon-stimulated response element (ISRE) (43), not present in other SPRR2 genes (Fig.
6), supports such a view.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(
-glutamyl)lysine isopeptide bonds catalyzed mainly
by transglutaminases 1 and 3 (4, 5). The assembly of the CE starts with
the formation of a scaffold constituted of involucrin and envoplakin
near the desmosomes. Subsequently, other reinforcing proteins, such as cystatin
, elafin, loricrin, and SPRRs (6-9) are added to complete the CE structure, which serves as an attachment platform for specific lipids (10). Biochemical evidence has suggested that the
characteristics of the CE related to toughness, strength and
flexibility, exhibited by different stratified squamous epithelia, are
dictated by SPRR proteins (11-15).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ca2+ conditions) were induced to stratify for 48 h
by adding Dulbecco's modified Eagle's medium containing 5% serum,
without growth factors (+Ca2+ conditions). UV-C irradiation
(30 J/m2) was applied to the monolayers before the addition
of +Ca2+ medium. RNA was isolated according to Ref. 44.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Contiguous map of the entire SPRR locus
identifying two groups and four subclasses of genes. A cosmid
contig covering the whole SPRR locus was analyzed with 8 restriction
enzymes. The SPRR 1, 2, 3, and 4 subclasses are indicated by different
ovals. Arrows define the transcriptional
orientation of each gene. Fragments hybridizing with the different SPRR
probes are indicated with a small open square beneath each
restriction bar. Fragments amplified by long-distance PCR
(LD-PCR) are indicated with stippled lines. Regions with
more than 20 kilobases could not be resolved by LD-PCR
(SPRR3/1B and 2F/2C).
Primers used in
RT-PCR
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Fig. 2.
Structural features of SPRR group 1 and 2 proteins. A, amino acid sequence comparison of amino-
and carboxyl-terminal domains. The number of reiterated repeats and the
number of amino acids present in each repeat motif are represented.
Amino acids identical in both groups are indicated in red.
Light- or dark-gray backgrounds indicate
amino acid identity within group 1 or group 2, respectively.
Asterisks indicate stop codons. B, comparison of
the nucleotide sequences of SPRR2 repetitive domains. Repeat specific
nucleotide substitutions are indicated in red.
Class-specific differences are represented in bold.
C, amino acid conservation among SPRR2 proteins. The amino
termini, the 3 internal repeats (R1, R2, and R3) and the carboxyl
termini of the seven SPRR2 proteins are compared. Amino acids involved
in the transglutaminase cross-linking reaction are represented in
red, whereas sequence differences between the various
members are in bold.
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Fig. 3.
Similarity analysis of human SPRR2 genomic
sequences. The genomic structure of SPRR2 genes is schematically
indicated. Exon 2 comprises the whole coding sequence (CDS)
and the 3'-untranslated region (UTR). The similarity plot of
the seven SPRR2 genes is represented in black, whereas the
red plot compares SPRR2B and SPRR2E.
The analysis was carried out with the plot-similarity program of GCG
(Wisconsin Package Version 10.0).
Ca2+
and +Ca2+) revealed that all SPRRs are induced during this
process, except for SPRR2F. In stratified squamous epithelia
distinctions in gene expression between the different tissues were
observed. Only SPRR2G and SPRR4 are
preferentially expressed in skin. All other SPRRs show higher
expression levels in mucosal-stratified squamous epithelia, but with
tissue-specific modulation (e.g. compare the relative expression of SPRR2B, 2C, and 2D in
esophagus and cervix). SPRR1A and 3 are present
in ovary, SPRR2D is found in uterus, and 2B, 2E, and 2F in both uterus and ovary. Especially,
the high expression of SPRR2F in ovary is remarkable.
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Fig. 4.
RT-PCR analysis of gene-specific SPRR
expression profiles. RT-PCR products were derived from RNA of the
indicated sources and amplified during 25, 30, and 35 cycles.
Ca2+ and +Ca2+ indicates RNA from
keratinocytes grown in vitro in the absence or presence of
calcium, respectively.
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Fig. 5.
Selective induction of individual SPRR genes
after UV irradiation of keratinocyte cultures. Stripped monolayers
of undifferentiated keratinocytes were irradiated (30 J/m2,
UV-C) or mock-irradiated and induced to differentiate by the addition
of +Ca2+ medium (see "Experimental Procedures"). RNA
was isolated 48 h later and analyzed by gene-specific RT-PCR (35 PCR cycles). The results presented are from two independent experiments
(A and B).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Regulatory elements in SPRR2 promoters.
Transcription factor binding sites previously identified in the
SPRR2A promoter (43) are represented and were placed in the
other promoters by sequence homology. Dotted lines indicate
deletions. Single point mutations are represented in bold.
TATA boxes and an initiator sequence (INR) in
SPRR2C are indicated. ZNF, binding site for
Krüppel-like zinc-finger factors; ETS, binding site
for the Ets family of transcription factors; ISRE,
interferon-stimulated response element; Octamer, binding
site for Oct transcriptional regulators; AP-1, binding site
for Jun/Fos factors.
Previous work from our laboratory has focused on the promoter regions of specific members of the SPRR1, 2, and 3 classes and has revealed that integration of signals transmitted via various signaling pathways plays an essential role in the regulation of these genes (43). Such a strict regulation is a prerequisite for efficient barrier function adaptation. Indeed, ablation of the Klf4 transcription factor, one of the regulators of SPRR2 expression, results in severe barrier deficiency in the mouse (54). Differential regulation of SPRR promoters relies on variations in the precise position of specific cis-elements within the global promoter context. This variation was recognized as a major factor in determining stimulus specific expression (40, 42). As a matter of fact, diversification of control elements is also seen in the promoter regions of the highly homologous SPRR2 genes (Fig. 6), in concert with their differential regulation. Differences include the deletion of an element (AP-1 site in 2B; ETS site in 2F; octamer and ZNF site in 2G), the replacement of one element by another one (ISRE/ETS in 2A; TATA/initiator in 2C) and single point mutations in binding sites (AP-1 sites in 2D and 2G). This diversification in regulatory elements is likely to affect both the binding of specific transcription factors and their mutual cooperativity (43). For instance, the absence of ETS sites in SPRR2F might explain the loss of regulation of this gene during in vitro keratinocyte differentiation (Fig. 5; Ref. 43). Whether the same change is also responsible for the unexpected high expression of this gene in non-squamous epithelia of the uterus and ovary is not yet known.
In conclusion, we propose that the two structurally different groups of
human SPRR genes provide on one hand specific resistance to very
specialized tissues, whereas allowing on the other hand adaptation to a
plethora of variable physiological and environmental insults. On this
basis, the structural organization of the SPRR gene family reflects the
functional duality with which epithelial barriers are confronted to
guarantee optimal protection to the organism.
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ACKNOWLEDGEMENTS |
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We thank Drs. D. Hohl (Lausanne) and T. Kartasova (Bethesda) for critically reading the manuscript and Dr. V. T. H. B. M. Smit (Dept. of Pathology, LUMC) for providing human tissue. Drs. J. Brouwer and P. van de Putte are acknowledged for stimulating discussions and A-M Borgstein for technical assistance. The hospitality of Dr. E. Bakker (Dept. of Clinical Genetics, LUMC) was appreciated.
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FOOTNOTES |
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* This research was supported by Grant BMH4-CT96-0319 of the European Community and by the J. A. Cohen Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Laboratory of Molecular Genetics, Gorlaeus Laboratories, P. O. Box 9502, 2300 RA Leiden, The Netherlands. Tel.: 31 71 527 4409; Fax: 31 71 527 4537; E-mail: Backendo@chem.leidenuniv.nl.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M100336200
2 A, Cabral, A., Sayin, S., de Winter, D., Fischer, S. Pavel, and C. Backendorf, manuscript in preparation.
3 A., Cabral, A., Sayin, S., de Winter, D., Fischer, S. Pavel, and C. Backendorf, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CE, cornified envelope; SPRR, small proline-rich protein; RT-PCR, reverse transcriptase-polymerase chain reaction; LD, long distance.
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