Structural Organization and Regulation of the Small Proline-rich Family of Cornified Envelope Precursors Suggest a Role in Adaptive Barrier Function*

Adriana Cabral, Patrick Voskamp, Anne-Marie Cleton-JansenDagger , Andrew South§, Dean Nizetic§, and Claude Backendorf

Department of Molecular Genetics, Leiden Institute of Chemistry, P. O. Box 9502, 2300 RA Leiden, The Netherlands, the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Nepsilon (gamma -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 alpha , 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).

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (-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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (15K):
[in this window]
[in a new window]
 
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).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Primers used in RT-PCR

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).


View larger version (67K):
[in this window]
[in a new window]
 
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.

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.


View larger version (23K):
[in this window]
[in a new window]
 
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).

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 (-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.


View larger version (34K):
[in this window]
[in a new window]
 
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.

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.


View larger version (42K):
[in this window]
[in a new window]
 
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (20K):
[in this window]
[in a new window]
 
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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: CE, cornified envelope; SPRR, small proline-rich protein; RT-PCR, reverse transcriptase-polymerase chain reaction; LD, long distance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Matoltsy, A. G., and Matoltsy, M. N. (1966) J. Invest. Dermatol. 46, 127-129[Medline] [Order article via Infotrieve]
2. Rice, R. H., and Green, H. (1977) Cell 11, 417-422[Medline] [Order article via Infotrieve]
3. Hohl, D. (1990) Dermatologica 180, 201-211[Medline] [Order article via Infotrieve]
4. Martinet, N., Kim, H. C., Girard, J. E., Nigra, T. P., Strong, D. H., Chung, S. I., and Folk, J. E. (1988) J. Biol. Chem. 263, 4236-4241[Abstract/Free Full Text]
5. Candi, E., Melino, G., Mei, G., Tarcsa, E., Chung, S. I., Marekov, L. N., and Steinert, P. M. (1995) J. Biol. Chem. 270, 26382-26390[Abstract/Free Full Text]
6. Steinert, P. M., and Marekov, L. N. (1995) J. Biol. Chem. 270, 17702-17711[Abstract/Free Full Text]
7. Steinert, P. M., and Marekov, L. N. (1997) J. Biol. Chem. 272, 2021-2030[Abstract/Free Full Text]
8. Steinert, P. M., and Marekov, L. N. (1999) Mol. Biol. Cell 10, 4247-4261[Abstract/Free Full Text]
9. Eckert, R. L., Yaffe, M. B., Crish, J. F., Murthy, S., Rorke, E. A., and Welter, J. F. (1993) J. Invest. Dermatol. 100, 613-617[Abstract]
10. Nemes, Z., Marekov, L. N., Fesus, L., and Steinert, P. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8402-8407[Abstract/Free Full Text]
11. Candi, E., Melino, G., Sette, M., Oddi, S., Guerrieri, P., and Paci, M. (1999) Biochem. Biophys. Res. Commun. 262, 395-400[CrossRef][Medline] [Order article via Infotrieve]
12. Candi, E., Tarcsa, E., Idler, W. W., Kartasova, T., Marekov, L. N., and Steinert, P. M. (1999) J. Biol. Chem. 274, 7226-7237[Abstract/Free Full Text]
13. Steinert, P. M., Candi, E., Kartasova, T., and Marekov, L. (1998) J. Struct. Biol. 122, 76-85[CrossRef][Medline] [Order article via Infotrieve]
14. Steinert, P. M., Kartasova, T., and Marekov, L. N. (1998) J. Biol. Chem. 273, 11758-11769[Abstract/Free Full Text]
15. Tarcsa, E., Candi, E., Kartasova, T., Idler, W. W., Marekov, L. N., and Steinert, P. M. (1998) J. Biol. Chem. 273, 23297-23303[Abstract/Free Full Text]
16. Gibbs, S., Fijneman, R., Wiegant, J., van Kessel, A. G., van De Putte, P., and Backendorf, C. (1993) Genomics 16, 630-637[CrossRef][Medline] [Order article via Infotrieve]
17. Kartasova, T., van Muijen, G. N., van Pelt-Heerschap, H., and van de Putte, P. (1988) Mol. Cell. Biol. 8, 2204-2210[Medline] [Order article via Infotrieve]
18. Kartasova, T., and van de Putte, P. (1988) Mol. Cell. Biol. 8, 2195-2203[Medline] [Order article via Infotrieve]
19. Marenholz, I., Volz, A., Ziegler, A., Davies, A., Ragoussis, I., Korge, B. P., and Mischke, D. (1996) Genomics 37, 295-302[CrossRef][Medline] [Order article via Infotrieve]
20. Mischke, D., Korge, B. P., Marenholz, I., Volz, A., and Ziegler, A. (1996) J. Invest. Dermatol. 106, 989-992[Abstract]
21. South, A. P., Cabral, A., Ives, J. H., James, C. H., Mirza, G., Marenholz, I., Mischke, D., Backendorf, C., Ragoussis, J., and Nizetic, D. (1999) J. Invest. Dermatol. 112, 910-918[Abstract/Free Full Text]
22. Volz, A., Korge, B. P., Compton, J. G., Ziegler, A., Steinert, P. M., and Mischke, D. (1993) Genomics 18, 92-99[CrossRef][Medline] [Order article via Infotrieve]
23. Song, H. J., Poy, G., Darwiche, N., Lichti, U., Kuroki, T., Steinert, P. M., and Kartasova, T. (1999) Genomics 55, 28-42[CrossRef][Medline] [Order article via Infotrieve]
24. Backendorf, C., and Hohl, D. (1992) Nat. Genet. 2, 91[Medline] [Order article via Infotrieve]
25. Kartasova, T., Parry, D. A. D., and Steinert, P. M. (1995) J. Invest. Dermatol. 104, 611
26. Abraham, J. M., Wang, S., Suzuki, H., Jiang, H. Y., Rosenblum-Vos, L. S., Yin, J., and Meltzer, S. J. (1996) Cell Growth Differ. 7, 855-860[Abstract]
27. Hohl, D., de Viragh, P. A., Amiguet-Barras, F., Gibbs, S., Backendorf, C., and Huber, M. (1995) J. Invest. Dermatol. 104, 902-909[Abstract]
28. Jarnik, M., Kartasova, T., Steinert, P. M., Lichti, U., and Steven, A. C. (1996) J. Cell Sci. 109, 1381-1391[Abstract/Free Full Text]
29. Kartasova, T., Darwiche, N., Kohno, Y., Koizumi, H., Osada, S., Huh, N., Lichti, U., Steinert, P. M., and Kuroki, T. (1996) J. Invest. Dermatol. 106, 294-304[Abstract]
30. Lohman, F. P., Medema, J. K., Gibbs, S., Ponec, M., van de Putte, P., and Backendorf, C. (1997) Exp. Cell Res. 231, 141-148[CrossRef][Medline] [Order article via Infotrieve]
31. Gibbs, S., Lohman, F., Teubel, W., van de Putte, P., and Backendorf, C. (1990) Nucleic Acids Res. 18, 4401-4407[Abstract]
32. Garmyn, M., Yaar, M., Boileau, N., Backendorf, C., and Gilchrest, B. A. (1992) J. Invest. Dermatol. 99, 743-748[Abstract]
33. Fujimoto, W., Marvin, K. W., George, M. D., Celli, G., Darwiche, N., De Luca, L. M., and Jetten, A. M. (1993) J. Invest. Dermatol. 101, 268-274[Abstract]
34. Ishida-Yamamoto, A., Iizuka, H., Manabe, M., O'Guin, W. M., Hohl, D., Kartasova, T., Kuroki, T., Roop, D. R., and Eady, R. A. (1995) Arch. Dermatol. Res. 287, 705-711[Medline] [Order article via Infotrieve]
35. Xu, X. C., Mitchell, M. F., Silva, E., Jetten, A., and Lotan, R. (1999) Clin. Cancer Res. 5, 1503-1508[Abstract/Free Full Text]
36. Yaar, M., Eller, M. S., Bhawan, J., Harkness, D. D., DiBenedetto, P. J., and Gilchrest, B. A. (1995) Exp. Cell Res. 217, 217-226[CrossRef][Medline] [Order article via Infotrieve]
37. Saunders, N. A., Smith, R. J., and Jetten, A. M. (1993) Biochem. Biophys. Res. Commun. 197, 46-54[CrossRef][Medline] [Order article via Infotrieve]
38. Nizetic, D., Monard, S., Young, B., Cotter, F., Zehetner, G., and Lehrach, H. (1994) Mamm Genome 5, 801-802[Medline] [Order article via Infotrieve]
39. Ivens, A., and Little, P. (1995) in DNA Cloning 3 A Practical Approach (Glover, D. , and Hames, B., eds) , pp. 1-47, IRL Press, Oxford
40. Sark, M. W., Fischer, D. F., de Meijer, E., van de Putte, P., and Backendorf, C. (1998) J. Biol. Chem. 273, 24683-24692[Abstract/Free Full Text]
41. An, G., Tesfaigzi, J., Chuu, Y. J., and Wu, R. (1993) J. Biol. Chem. 268, 10977-10982[Abstract/Free Full Text]
42. Fischer, D. F., Sark, M. W., Lehtola, M. M., Gibbs, S., van de Putte, P., and Backendorf, C. (1999) Genomics 55, 88-99[CrossRef][Medline] [Order article via Infotrieve]
43. Fischer, D. F., Gibbs, S., van De Putte, P., and Backendorf, C. (1996) Mol. Cell. Biol. 16, 5365-5374[Abstract]
44. Belt, P. B., Groeneveld, H., Teubel, W. J., van de Putte, P., and Backendorf, C. (1989) Gene (Amst.) 84, 407-417[Medline] [Order article via Infotrieve]
45. Rheinwald, J. G., and Green, H. (1977) Nature 265, 421-424[Medline] [Order article via Infotrieve]
46. Lee, Y. S., Yuspa, S. H., and Dlugosz, A. A. (1998) J. Invest. Dermatol. 111, 762-766[Abstract]
47. Li, W.-H. (1997) Molecular Evolution , Sinauer Associates, Inc.
48. Lee, C. H., Marekov, L. N., Kim, S., Brahim, J. S., Park, M. H., and Steinert, P. M. (2000) FEBS Lett. 477, 268-272[CrossRef][Medline] [Order article via Infotrieve]
49. Tesfaigzi, J., and Carlson, D. M. (1996) Exp. Cell Res. 228, 277-282[CrossRef][Medline] [Order article via Infotrieve]
50. Jetten, A. M., De Luca, L. M., Nelson, K., Schroeder, W., Burlingame, S., and Fujimoto, W. (1996) Mol. Cell. Endocrinol. 123, 7-15[CrossRef][Medline] [Order article via Infotrieve]
51. Gosden, R., and Spears, N. (1997) Br. Med. Bull. 53, 644-661[Abstract]
52. Salamonsen, L. A., Kovacs, G. T., and Findlay, J. K. (1999) Baillieres Best Pract. Res. Clin. Obstet. Gynaecol. 13, 161-179[CrossRef][Medline] [Order article via Infotrieve]
53. Marshall, D., Hardman, M. J., and Byrne, C. (2000) J. Invest. Dermatol. 114, 967-975[Abstract/Free Full Text]
54. Segre, J. A., Bauer, C., and Fuchs, E. (1999) Nat. Genet. 22, 356-360[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.