Human Molecular Genetics, 2002, Vol. 11, No. 23 2867-2875
© 2002 Oxford University Press
A null mutation in the cystatin M/E gene of ichq mice causes juvenile lethality and defects in epidermal cornification
1Department of Dermatology, 2Department of Cell Biology, and 3Department of Human Genetics, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands
Received June 27, 2002; Accepted August 26, 2002
Genbank accession no. AY093591
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ABSTRACT |
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Cystatin M/E (CST6 ), a new member of the cystatin gene family, has a restricted expression pattern in humans, which is largely limited to cutaneous epithelia. Although cystatin M/E possesses two distinct biochemical properties, being a cysteine proteinase inhibitor and a substrate for transglutaminase, its physiological function is unknown. Here we report the isolation and characterization of the mouse Cst6 orthologue and the assignment of the chromosomal localization to the proximal end of mouse chromosome 19. This region corresponds to the locus of the spontaneous harlequin ichthyosis (ichq) mouse mutation, for which no causative gene has been identified so far. We found a nonsense mutation in the Cst6 gene of BALB/cJ–ichq/+ mice, which precludes the synthesis of functional protein. Immunohistochemistry confirmed the absence of cystatin M/E at the protein level in ichq/ichq mice. Mice that are homozygous for two null alleles display a hyperplastic, hyperkeratotic epidermis and abnormal hair follicles, and die between 5 and 12 days of age. In wild-type mice, cystatin M/E was found in the stratum granulosum and in the infundibulum of the hair follicle indicating that the anatomical site in the skin where cystatin M/E is normally expressed correlates with the abnormalities at the tissue level in ichq/ichq mice. Our data provide evidence that cystatin M/E is required for viability and for correct formation of cornified layers in the epidermis and hair follicles. The ichq mouse mutation may serve as a model for human type 2 harlequin ichthyosis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cystatins are natural and specific inhibitors of endogenous lysosomal cysteine proteinases (e.g., cathepsins B, C, H, K, L, and S) (1–3) and exogenous microbial cysteine proteinases (4,5). Cystatins are widely distributed in human tissues and body fluids and play an important role in regulating the activity of these proteinases (6). Mutations in the genes encoding the cystatin family members cystatin B and C cause neurological phenotypes in humans such as progressive myoclonus epilepsy (7) and the lethal autosomal dominant disease hereditary cystatin C amyloid angiopathy (HCCAA) (8). More generally, a disturbed balance between proteinases and their inhibitors can lead to irreversible damage as found in chronic inflammatory reactions (9) and tumor metastasis (10). Recently we reported that cystatin M/E, a new member of the cystatin gene family, has a tissue-specific expression pattern in humans, which is largely limited to cutaneous epithelia (11). Cystatin M/E is a 14 kDa secreted protein that shares only 35% homology with the human family 2 cystatins. It has a similar overall structure, such as a signal peptide and two intrachain disulfide bonds, but possesses the unusual characteristic of being a glycoprotein. This protein is only distantly related to the other known family members as reflected by the position of the CST6 gene. It resides on chromosome 11q13 (12), whereas all other family 2 cystatin genes are clustered in a narrow region on chromosome 20p11.2 (13). The CST6 encoded protein was initially identified as cystatin M by differential display as a down-regulated mRNA in metastatic breast tumour cells when compared to normal and primary breast tumour cells (14). It was proposed that loss of expression of cystatin M is likely to be associated with the progression of a primary tumour to a metastatic phenotype. Independently the same molecule was encountered by expressed sequence tag sequencing in cDNA libraries derived from epithelial cells and was designated cystatin E (15).
At this point, the function of cystatin M/E remained unknown. The secretion of cystatin M/E in sweat, its expression at the interface of the internal and external milieu (cornifying layers of epidermis and hair follicles) and the increased levels in inflammatory conditions strongly suggested that cystatin M/E is involved in host protection (16). Analysis of its obvious role as a cysteine proteinase inhibitor has so far only indicated a moderate inhibitory activity against cathepsin B (11,15), which is unlikely to be its main physiological function. Identification of potential endogenous or microbial target proteins could shed further light on the biological function of cystatin M/E and its presumed role in epidermal homeostasis.
The epidermis is a stratified epithelium that provides vital physical and mechanical barrier properties to the skin. The formation of human epidermis and its appendages is the result of a complex and tightly choreographed program of morphogenesis and differentiation. Recent studies have yielded a number of important insights into the mechanisms of these processes (17,18). The barrier function of human epidermis is largely provided by the cornified cell envelope (CE) (19), which is assembled by transglutaminase (TGase) cross-linking of several structural proteins. These cross-linked CE proteins include loricrin, small proline-rich proteins (SPRRs), calcium binding S-100 proteins, cytokeratins, filaggrin, SKALP/elafin, cystatin A, late envelope proteins (LEPs) and involucrin as well as several others (20–22). We have previously shown that cystatin M/E is a substrate for epidermal transglutaminases suggesting a role in the formation of the stratum corneum (11). The integrity of the stratum corneum is maintained by continuous renewal and shedding of old corneocytes at the skin surface. This process of desquamation is the final event in terminal differentiation of the epidermis and is likely to be regulated by the concerted action of proteolytic enzymes and their inhibitors (23,24). We hypothesized that cystatin M/E, being a proteinase inhibitor that is mainly expressed in differentiated keratinocytes of the interfollicular epidermis, may have an important role in this desquamatory process. Here we report that a null mutation in the mouse cystatin M/E gene (Cst6) causes the harlequin ichthyosis (ichq) phenotype in mice, characterized by abnormalities in cornification and desquamation, demonstrating an essential role for cystatin M/E in the final stages of epidermal differentiation and in hair follicle morphogenesis.
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RESULTS |
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Characterization and mapping of mouse cystatin M/E
To identify the mouse cystatin M/E gene, we designed oligonucleotide primers based on an EST sequence (GenBank accession no. AK003744) very likely encoding the mouse ortholog of human cystatin M/E. RT–PCR on mouse skin RNA yielded a single 475 bp PCR product that was subsequently used to screen a mouse bacterial artificial chromosome (BAC) genomic library. One positive clone (BAC271J15) was isolated, subcloned and sequenced. Mouse Cst6 (deposited in the GenBank database, accession no. AY093591) has an exon–intron structure identical to that of all other type 2 cystatins (6) and encodes a 149 amino acid protein (Fig. 1A). Alignment with its presumed human ortholog (GenBank Accession no. U62800) revealed 69% amino acid identity and 82% amino acid conservation (Fig. 1B). Homologies to other human type 2 cystatins are considerably less (<32% identity), corroborating the hypothesis that BAC271J15 harbors the mouse orthologous gene. Human CST6 has been assigned previously to chromosome 11q13 (12), which is syntenic to a region close to the centromere on mouse chromosome 19 (http://www.informatics.jax.org). We determined the chromosomal localization of mouse Cst6 by fluorescence in situ hybridization using clone BAC271J15 as a probe. As expected, Cst6 did indeed map to the proximal end of chromosome 19 (Fig. 1C).
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Expression of cystatin M/E in the mouse
Analysis of C57BL/6 mouse tissues revealed an expression pattern that is less restricted than observed previously in humans (11). RT–PCR on RNA from adult mice showed relatively high levels of cystatin M/E expression in skin, ileum, stomach, eye and cerebellum; moderate to low levels of expression were found in tongue, palatum, nasal cavity, colon, bladder, skeletal muscle, placenta and thymus. During embryogenesis, expression of cystatin M/E was observed from E16 onwards, which is a developmental stage where epidermal stratification is completed (Fig. 2A). Immunohistochemical analysis using affinity-purified antibodies against recombinant cystatin M/E (11) shows that the cystatin M/E protein is highly expressed in ciliated tracheal epithelium and in bronchial epithelium; lung tissue was completely negative, in accordance with the RT–PCR results (Fig. 2B and C). We also detected cystatin M/E expression in the photoreceptor outer segment of the retina (Fig. 2D), an observation that we have recently confirmed in human eye as well (unpublished results). This was an unexpected finding as we previously only detected high expression of cystatin M/E protein in epithelia and bodily secretions that were exposed to microorganisms (11), which initially suggested a role in host defense, analogous to other cystatin family members (5,25). Similar to our findings in humans we found expression of cystatin M/E in the epithelium of the nasal cavity (Fig. 2E), the infundibular epithelium of hair follicles (Fig. 2F) and in the stratum granulosum of interfollicular epidermis (Fig. 2G) (11).
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Mutation of Cst6 causes the harlequin ichthyosis phenotype in ichq/ichq mice
Bearing in mind the expression of cystatin M/E in skin and hair follicles and the localization on the proximal end of mouse chromosome 19, we examined the available literature on mutant laboratory mouse strains with abnormalities in epidermal differentiation or hair follicle morphology (26). On the basis of the literature we hypothesized that Cst6 could be a candidate gene for the ichq mouse phenotype that was recently described (27). We obtained breeding pairs of heterozygous BALB/cJ–ichq/+ mice and analysed the genomic sequence of the Cst6 locus in these mice. Additionally, Cst6 sequences of phenotypically wild-type and mutant offspring were examined. For this analysis, all three exons of Cst6 were amplified by PCR from mouse tail genomic DNA. We identified homozygosity for a single nucleotide deletion in exon 1 of the phenotypically mutant (ichq/ichq) mice (Fig. 3A). This 42delG deletion alters the reading frame, resulting in a premature stop codon at amino acid position 20 (Fig. 3B). In addition to the single nucleotide deletion, the mutated allele also carried two synonymous SNPs in exon 1 (C33T and C40T); the latter resulted in the loss of a StyI restriction site, which allowed convenient screening of the genotypes in the offspring.
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The homozygous mutant mice have no obvious phenotype at birth but from day 6 onwards they develop a layer of compact orthokeratotic scales, which first appear on the dorsal skin (Fig. 4A). These ichq/ichq mice die between day 5 and 12. For a detailed description of the macroscopic and microscopic appearance of the phenotype we refer to the work of Sundberg et al. (27). Immunohistochemical analysis confirmed the predicted absence of cystatin M/E at the protein level in ichq/ichq mice (Fig. 4B). Heterozygous mice and wild-type littermates displayed a normal cystatin M/E expression pattern (Fig. 4C–E) suggesting that the presence of cystatin M/E is essential for epidermal homeostasis and normal hair follicle morphogenesis.
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DISCUSSION |
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We found that a null mutation in the cystatin M/E gene of ichq mice is responsible for juvenile lethality and defects in epidermal cornification and desquamation, a phenotype that recapitulates most of the features of human type 2 harlequin ichthyosis (HI). Harlequin ichthyosis (MIM 242500) is a severe congenital skin disorder usually leading to a stillborn fetus or early neonatal death. Its clinical features at birth include ectropion, eclabium, ear dysmorphology and a thickened fissured epidermis (28). Histopathologically, HI is characterized by excessive epidermal and follicular hyperkeratosis. Biochemical and ultrastructural abnormalities have suggested genetic heterogeneity and division into three subtypes (29–31). The mouse ichq mutant, which presents as an ichthyosiform dermatitis, was recently described by Sundberg and co-workers as a model for HI (27). It was shown that the ichq mouse mutation phenotype has morphological and biochemical similarities to human type 2 HI, which include abnormally large mitochondria, absence of lamellar granules and a characteristic keratin expression pattern in the interfollicular epidermis. The human type 2 HI phenotype starts to develop in the hair canals and is subsequently expressed in the entire hairy skin (32). The type 2 HI phenotype is associated with keratinization and abnormal filaggrin metabolism in the hair suggesting that the hair canal plays an important role. Likewise, the ichq mice also demonstrated abnormal keratinization of hair follicles and the onset of the phenotype corresponded with emergence of hair fibers from follicles at 5 days of age. Homozygous ichq/ichq mice die between day 5 and 12, but the cause of death in these mice is not known (27). We speculate that, analogous to the situation in humans (33), disturbed thermoregulation or impaired breathing plays a major role. As HI is an extremely rare congenital and usually lethal disease in humans, its etiology and pathogenesis have been difficult to investigate. The identification of Cst6 as the causative gene in ichq mice provides a powerful tool to investigate the pathophysiology of this disease in humans. Importantly, it opens the way for direct screening of underlying mutations in affected individuals and thus may lead to genetic counseling possibilities. In addition, it provides the framework to identify functionally related genes that contribute to the heterogeneity of this disease.
We have previously demonstrated that cystatin M/E can be cross-linked to stratum corneum proteins by the action of TGases (11), a process that occurs in the final stage of maturation of the developing epidermis. Two other epidermal proteinase inhibitors were found to be anchored to structural proteins of the stratum corneum. SKALP/elafin was found in foreskin by direct protein sequencing (34) and cystatin A, the only member of the cystatin family that has so far been found in human epidermis, was shown to be part of the CE and acts as a TGase substrate (35). From this point of view, it could be possible that cystatin M/E is also a constituent protein of the CE and as such it could be indispensable for correct formation of the protective callus layer. Thus far, several studies have implicated at least 20 proteins in the assembly of CEs (36). Surprisingly, recent experiments on mice in which major CE constituents were knocked out (e.g., loricrin, envoplakin and involucrin), have failed to disrupt the barrier function (37–39). This suggests that there are compensatory mechanisms and additional unidentified components involved that maintain the skin barrier function. In view of these findings we think it is unlikely that the ichq phenotype is caused by the absence of cystatin M/E as a structural CE protein. Whereas the absence of CE structural proteins appears to be well tolerated, the mutation or absence of desmosomal or cytoskeletal proteins in differentiated keratinocytes often leads to severe pathology and disturbance of barrier function as witnessed by mutations in keratins (40), plakophilin (41), plakoglobin (42), desmoplakin (43) and desmoglein (44). Likewise, deficiency for regulatory enzymes as transglutaminase and steroid sulfatase leads to disease in humans (45,46). From this point of view it is tempting to speculate that cystatin M/E has a regulatory role in assembly and formation of the stratum corneum rather than a structural one.
Assuming that cystatin M/E functions as an inhibitor of a hitherto unidentified proteinase, the mouse ichq mutant provides yet another example of a disturbed proteinase–antiproteinase balance causing faulty differentiation processes in the epidermis and hair follicle. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome (24), cathepsin C mutations in Papillon-Lefèvre syndrome (47), cathepsin L deficiency in furless mice (48) and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice (49). Previous studies in vitro and in vivo have shown that absence or saturation of inducible epidermal serine proteinase inhibitors such as SLPI (50) and SKALP/elafin (51,52) cause impaired wound healing and keratinocyte detachment. Although the absence of cystatin M/E induces some mild inflammation in the homozygous ichq/ichq mice, its major effect appears to be on the cornification or desquamation process rather than on inflammation (27). This matches the most striking clinical feature of HI in humans which is retention of cornified cells and the lack of stratum corneum desquamation. As the target proteinase of cystatin M/E is not known it is difficult to speculate on the exact mechanism. Identification of its putative target proteinase will be an essential step in the elucidation of the regulatory function of cystatin M/E, which is clearly the direction for future research.
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MATERIALS AND METHODS |
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Genomic cloning of mouse cystatin M/E
Based on a mouse EST sequence (GenBank AK003744) we designed two oligonucleotide primers that could amplify the cDNA encoding mouse cystatin M/E: primer MF1, 5'-ATGGAGCGTCCTCACTTCC-3'; and primer MR1, 5'-CCTGACTCTGTCACCCTGG-3' (positions are shown in Fig. 1). The reverse transcriptase (RT) reaction product of mouse skin (C57BL/6) was used for polymerase chain reaction amplification to obtain the cDNA of mouse cystatin M/E. PCR conditions were: 94°C for 6 min followed by 35 cycles of 94°C for 1 min, 53°C for 1 min and 72°C for 2 min. The 475 bp PCR product encoding mouse cystatin M/E cDNA was used to screen an RPCI-22 (129S6/SvEvTac) mouse BAC Library (53). One positive clone (BAC271J15) was isolated and characterized by restriction enzyme analysis and Southern blotting. Restriction fragments from BAC271J15 were subcloned into plasmid vector pZErO-2 (Invitrogen, Carlsbad, CA, USA) and inserts were sequenced at the DNA Sequencing Facility, Department of Human Genetics, UMCN, Nijmegen, the Netherlands. For inserts larger than 1 kb a transposon-based system (Epicentre, Madison, WI, USA) was used to allow complete sequencing. Resulting data were recorded, edited and assembled using the Clustal_X package (54), and analysed using ClustalW (http://searchlauncher.bcm.tmc.edu/) and BOXSHADE (http://www.ch.embnet.org).
Chromosomal localization
To determine the chromosomal localization of Cst6, fluorescence in situ hybridization (FISH) was performed on mouse metaphase chromosomes (55). Briefly, the BAC clone 271J15 and a mouse chromosome 19 paint were labeled with biotin-14-dATP (Life Technologies, Gaithersburg, MD, USA) and digoxigenin-11-dUTP (Roche, Mannheim, Germany), respectively, by nick translation and ethanol precipitated together with a 40-fold excess of mouse Cot-1 DNA (Life Technologies). Immunocytochemical detection of the hybridizing probe was achieved using fluorescein isothiocyanate (FITC)-conjugated avidin followed by goat-anti-avidin conjugated FITC. Mouse chromosome 19 paint was simultaneously detected by sheep-anti-digoxigenin conjugated Rhodamine and donkey-anti-sheep conjugated Texas Red. A Zeiss epifluorescence microscope was used for visual examination of the chromosome slides. Digital images were captured using a high-performance cooled CCD camera (Photometrics, Tucson, AZ, USA) coupled to a Macintosh Quadra 950 computer and analysed using imageTM F.I.S.H. software package (Oncor, Gaithersburg, MD, USA).
Expression analysis and immunohistochemistry
Adult and embryonic tissues from C57BL/6 mice were obtained from the Central Animal Laboratory, University of Nijmegen, The Netherlands. The following tissues were studied with respect to cystatin M/E expression: skin, tongue, palatum, nasal cavity, sole of the foot, esophagus, ileum, colon, stomach, lung, trachea, bronchus, ureter, kidney, bladder, pancreas, liver, heart, spleen, skeleton muscle, lymph node, uterus, placenta, prostate, testis, tail, eye, cerebrum, cerebellum, adrenal gland and thymus. We collected mouse embryos starting at day 10 post coitum. Mouse tissues were rinsed in PBS, fixed for 4 hours in buffered 4% formalin and embedded in paraffin wax. All material was cut in 7 µm sections, deparaffinized, rehydrated and preincubated with 20% normal goat serum. Immunohistochemical staining was further performed as previously described using affinity-purified polyclonal rabbit anti-human cystatin M/E antibodies (11).
RNA isolation and RT–PCR amplification
Total RNA from mouse tissues was isolated using TRIzol Reagent (Life Technologies). Oligo-dT primed first strand cDNA was generated from total RNA with Moloney murine leukemia virus Range H- reverse transcriptase (Boehringer, Mannheim, Germany). The reverse transcriptase reaction products were used for PCR amplification to detect the cDNA of mouse cystatin M/E and the housekeeping gene mouse acidic ribosomal phosphoprotein P0 (Arbp) (56). We used oligonucleotide primers that could amplify a part of the mouse cystatin M/E cDNA including exon 2 and exon 3: primer MF2, 5'-TACTACCTGACTTTGGA CATAG-3'; and primer MR1 (positions are shown in Fig. 1). The oligonucleotide sequences to generate mouse Arbp cDNA were 5'-GTGTGAGGTCACTGTGCC-3' and 5'-ACCGAATC CCATATCCTC-3'. PCR-reactions were carried out as described above. Annealing temperatures were 53°C when using the cystatin M/E primers and 47°C for the Arbp primers. PCR products were analysed by agarose gel electrophoresis.
Ichq mice
The harlequin ichthyosis (ichq) mouse mutation arose spontaneously in 1989 in a colony of BALB/cJ mice at The Jackson Laboratory (27). We obtained BALB/cJ–ichq/+ mice from The Jackson Laboratory and bred them to produce the phenotypically mutant (ichq/ichq) and wild-type (+/+ and ichq/+) mice used here. Animals were housed in specific-pathogen-free facilities at the Central Animal Laboratory, University of Nijmegen, The Netherlands. All animal protocols were approved by the University of Nijmegen Institutional Animal Care and Use Committee.
Mutation analysis
We designed primers from intronic sequences flanking all three exons of mouse Cst6 to amplify genomic DNA of the phenotypically mutant (ichq/ichq) and wild-type (+/+ and ichq/+) mice. DNA was PCR-amplified under the following conditions: 94°C for 6 min followed by 35 cycles of 94°C for 1 min, 53°C for 1 min, and 72°C for 2 min. Primers were: exon1F, 5'-CATCTCTGGTTCTTACACTGC-3'; exon1R, 5'-GCAGCTCTCTGTTTATCTCC-3'; exon2F, 5'-GGAAAGCA TAGACACACAGG-3'; exon2R, 5'-ACTGGTCAGGATAGAC AAGC-3'; exon3F, 5'-GAGTGCAGAAGAGAGACTGG-3'; and exon3R, 5'-CAGATTTATTGCAACAGACG-3'. PCR products were analysed upon electrophoresis and the DNA was purified by use of a PCR purification kit (Qiagen, Valencia, CA, USA). Both strands of the DNA fragments were sequenced and analysed. The nucleotide sequence of the Cst6 coding region in wild-type (+/+) BALB-cJ mice appeared to be identical to wild-type mice with other genetic backgrounds (Sv129, C3H, BL6, and pBA/1).
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ACKNOWLEDGEMENTS |
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We thank Hennie N.E.C. ten Dam for assistance with photography. NKI (Dutch Cancer Institute) Amsterdam kindly provided the mouse chromosome 19 paint. This work was financially supported by grant 902.11.092 from the Netherlands Organization for Scientific Research (NWO).
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +31 0243617245; Fax: +31 0243541184; Email: p.zeeuwen{at}derma.azn.nl
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