[Show abstract][Hide abstract] ABSTRACT: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs' lifecycle has been omitted.
We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types.
The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.
[Show abstract][Hide abstract] ABSTRACT: CCHCR1 (Coiled-Coil α-Helical Rod protein 1), within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene with the psoriasis associated risk allele CCHCR1*WWCC. Although its expression pattern in psoriatic skin differs from healthy skin and its overexpression influences cell proliferation in transgenic mice, its role as a psoriasis effector gene has remained unsettled. The 5'-region of the gene contains a SNP (rs3130453) that controls a 5'-extended open reading frame and thus the translation of alternative isoforms. We have now compared the function of two CCHCR1 isoforms: the novel longer isoform 1 and the previously studied isoform 3. In samples of Finnish and Swedish families, the allele generating only isoform 3 shows association with psoriasis (P<10(-7)). Both isoforms localize at the centrosome, a cell organelle playing a role in cell division. In stably transfected cells the isoform 3 affects cell proliferation and with the CCHCR1*WWCC allele, also apoptosis. Furthermore, cells overexpressing CCHCR1 show isoform- and haplotype-specific influences in the cell size and shape and alterations in the organization and expression of the cytoskeletal proteins actin, vimentin, and cytokeratins. The isoform 1 with the non-risk allele induces the expression of keratin 17, a hallmark for psoriasis; the silencing of CCHCR1 reduces its expression in HEK293 cells. CCHCR1 also regulates EGF-induced STAT3 activation in an isoform-specific manner: the tyrosine phosphorylation of STAT3 is disturbed in isoform 3-transfected cells. The centrosomal localization of CCHCR1 provides a connection to the abnormal cell proliferation and offers a link to possible cellular pathways altered in psoriasis.
PLoS ONE 11/2012; 7(11):e49920. DOI:10.1371/journal.pone.0049920 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite chronic inflammation, psoriatic lesions hardly ever progress to skin cancer. Aberrant function of the CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1, HCR) within the PSORS1 locus may contribute to the onset of psoriasis. As CCHCR1 is expressed in certain cancers and regulates keratinocyte (KC) proliferation in a transgenic mouse model, we studied its relation to proliferation in cutaneous squamous cell cancer (SCC) cell lines by expression arrays and quantitative RT-PCR and in skin tumors by immunohistochemistry. CCHCR1 protein was detected in the pushing border of SCC and lining basal cell carcinoma islands. Different from psoriasis, Ki67 had a similar expression pattern as CCHCR1. The most intense CCHCR1 staining occurred in areas positive for epidermal growth factor receptor (EGFR). Expression of CCHCR1 mRNA was upregulated 30-80% in SCC lines when compared to normal KCs and correlated positively with Ki67 expression. The most aggressive and invasive tumor cell lines (RT3, FaDu) expressed CCHCR1 mRNA less than non-tumorigenic HaCaT cells. Moreover, the tumor promoters okadaic acid and menadione downregulated CCHCR1 mRNA. We conclude that both in psoriasis and the early stages of KC transformation, CCHCR1 may function as a negative regulator of proliferation, but beyond a certain point in oncogenesis cannot control this phenomenon any longer.
PLoS ONE 02/2009; 4(6):e6030. DOI:10.1371/journal.pone.0006030 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the skin, expression of several matrix metalloproteinases (MMPs) occurs in response to tissue injury, tumorigenesis, angiogenesis, apoptosis, and inflammation. The recently cloned MMP-21 has been implicated in skin development and various epithelial cancers. In this study, we found that it is also expressed by differentiated keratinocytes (KCs) in various benign skin disorders, in which it was not associated with KC apoptosis or proliferation, and in organotypic cultures. Furthermore, MMP-21 was induced in keratinocytes in association with increased calcium and presence of the differentiation marker filaggrin. In stably transfected A431 and HEK293 cell lines, MMP-21 increased invasion of cells but did not associate with increased apoptosis, proliferation, or epithelial-to-mesenchymal transition. Of various agents tested in HaCaT cell cultures, only retinoic acid (10(-6) M) and staurosporine (2.5 x 10(-8) M) upregulated MMP-21 mRNA and protein expression, whereas tumor promoters, hormones, or dexamethasone were without effect. Our results suggest that MMP-21 may be an important protease in the terminal differentiation of keratinocytes.
[Show abstract][Hide abstract] ABSTRACT: The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.
Human Molecular Genetics 05/2008; 17(7):1043-51. DOI:10.1093/hmg/ddm377 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously described striking molecular features including high frequency of membranous beta-catenin in subsets of familial colon cancers with as yet unknown predisposition. We hypothesized that such tumors might carry mutations in Wnt/beta-catenin target genes. Fibroblast growth factor 9 (FGF9) was an attractive target, as it maps to a common area of loss of heterozygosity (LOH) in colorectal carcinomas on 13q12.11. Here, we report, for the first time, the occurrence of FGF9 mutations in human cancers. We found a total of six distinct FGF9 mutations including one frameshift, four missense, and one nonsense, in 10 (six colorectal and four endometrial) out of 203 tumors and cell lines. The frameshift mutation was detected in five different tumors. Mapping of these mutations onto the crystal structure of FGF9 predicted that they should all lead to loss of function albeit through variable mechanisms. The p.R173K mutation should diminish ligand affinity for heparin/heparan sulfate, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations should negatively impact ligand's interaction with receptor, while p.G84E and p.E142X (FGF9(Delta142-208)) mutations should interfere with ligand folding. Consistent with these structural predictions, the p.V192M, p.D203G, and p.L188YfsX18 (FGF9(Delta205-208)) mutations impaired the ability of ligand to activate mitogen-activated protein kinase (MAPK) cascade in cultured cells expressing FGF receptors. LOH was observed in seven out of nine FGF9 mutant tumors, supporting the predicted loss of function. Interestingly, eight out of 10 (80%) of the FGF9 mutant tumors showed normal membranous beta-catenin expression and the absence of mutation in the beta-catenin gene (CTNNB1). These data suggest that FGF9 plays a role in colorectal and endometrial carcinogenesis.
Human Mutation 03/2008; 29(3):390-7. DOI:10.1002/humu.20653 · 5.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The HCR gene, officially called Coiled-Coil alpha-Helical Rod protein 1 (CCHCR1), located within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene for the risk effect. Recently, CCHCR1 was shown to promote steroidogenesis by interacting with the steroidogenic acute regulator protein (StAR). Here, we examined the role of CCHCR1 in psoriasis and cutaneous steroid metabolism. We found that CCHCR1 and StAR are expressed in basal keratinocytes in overlapping areas of the human skin, and CCHCR1 stimulated pregnenolone production in steroidogenesis assay. Overexpression of either the CCHCR1*WWCC risk allele or the non-risk allele enhanced steroid synthesis in vitro. Furthermore, the cytochrome P450scc enzyme was expressed in human keratinocytes and was induced by forskolin, a known activator of steroidogenesis, and forskolin also upregulated CCHCR1. CCHCR1 has an altered expression pattern in lesional psoriatic skin compared to normal healthy skin, suggesting its dysregulation in psoriasis. We found that the expression of CCHCR1 is downregulated twofold at the mRNA level in cultured non-lesional psoriatic keratinocytes when compared to non-psoriatic healthy cells. Our results also suggest a connection between CCHCR1 and vitamin D metabolism in keratinocytes. The expression of the vitamin D receptor (VDR) gene was lower in non-lesional psoriatic keratinocytes than in healthy cells. Furthermore, Vdr expression was downregulated in the keratinocytes of mice overexpressing the CCHCR1*WWCC risk allele when compared to keratinocytes from mice with the non-risk allele of CCHCR1. Finally, we demonstrate that other agents relevant for psoriasis and/or the regulation of steroidogenesis influence CCHCR1 expression in keratinocytes, including insulin, EGF, cholesterol, estrogen, and cyclosporin A. Taken the role of steroid hormones, including vitamin D and estrogen, in cell proliferation, epidermal barrier homeostasis, differentiation, and immune response, our results suggest a role for CCHCR1 in the pathogenesis of psoriasis via the regulation of skin steroid metabolism.
Journal of Molecular Medicine 07/2007; 85(6):589-601. DOI:10.1007/s00109-006-0155-0 · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The PSORS1 locus is the consistently replicated genetic risk factor for psoriasis. Clinical associations with the main marker allele of PSORS1, HLA-Cw6, have been addressed in a number of studies, but clinical associations have not been used as a way to distinguish the effects of the neighbouring candidate genes in PSORS1. Our results show that HLA-Cw6 and CCHCR1 risk allele associations with clinical features of psoriasis are predictably highly similar in a Finnish nationwide cohort of 379 psoriasis patients. The clinical profiling of a small group of patients (n=34) who were HLA-Cw6- but CCHCR1*WWCC positive suggested that no great differences existed between them and HCR-Cw6- patients. HCR+ genotype (as well as Cw6+ genotype) correlated for the first time positively with female sex and, in contrast with previous studies, negatively with disease severity. Presence of psoriatic arthritis was more pronounced in HCR-psoriasis (as well as in Cw6- psoriasis). Clinical profiling may be a useful approach to distinguishing genetic effects of candidate genes even within a locus in sufficiently large cohorts.
[Show abstract][Hide abstract] ABSTRACT: In a nationwide study, we identified a total of 59 patients diagnosed with primary pulmonary hypertension (PPH) in Finland between the years 1987 and 1999. These data support a minimum estimate for a PPH population prevalence of 5.8 cases/million with an incidence of 0.2-1.3 cases/million/year. The male-to-female ratio among the patients was 1:4, while 7% (4/59) of the PPH probands had a known family history of the disorder. Familial or sporadic PPH showed no geographic clustering to any region of Finland. Sequencing of the coding regions and exon-intron boundaries of the bone morphogenetic protein receptor type 2 (BMPR2) identified heterozygous BMPR2 mutations in 12% (3/26) of the sporadic and 33% (1/3) of the familial patients. All four mutations were different, and two of those have been previously reported in other populations. Pathogenic defects in BMPR2 include a novel missense mutation (c.2696G>C encoding R899P), located within the receptor intracellular cytoplasmic domain whose function has been poorly characterized. Our analysis demonstrates that this mutant, while localizing to the cell surface, does not impact on SMAD-mediated (mothers against decapentaplegic homolog) intracellular signaling, but leads to constitutive activation of the p38(MAPK) pathway. The absence of a founder mutation in a genetically homogeneous population, such as the Finns, suggests that all identified BMPR2 mutations have to be rather young while the ancestral (if any) mutations have been lost either due to repetitive genetic bottlenecks or due to significant negative selection. Hum Mutat 26(2), 1-6, 2005. (c) 2005 Wiley-Liss, Inc.
Human Mutation 08/2005; 26(2):119-24. DOI:10.1002/humu.20200 · 5.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The anion transporters SLC26A6 (PAT1) and SLC26A7, transporting at least chloride, oxalate, sulfate and bicarbonate, show a distinct expression and function in different mammalian species. They are expressed in kidney, but their exact localization in human kidney has not been studied. We therefore examined SLC26A6 and A7 expression in human kidneys.
The localization of SLC26A6 and A7 in different segments of human nephrons was studied by RT-PCR and immunohistochemistry by comparing to the tubular markers PNRA, CD10, Tamm-Horsfall antigen, high molecular weight cytokeratin, CK7, AQP2 and H(+)V-ATPase.
In human kidney, SLC26A6 is expressed in distal segments of proximal tubules, parts of the thin and thick ascending limbs of Henle's loops, macula densa, distal convoluted tubules and a subpopulation of intercalated cells of collecting ducts. SLC26A7 is expressed in extraglomerular mesangial cells and a subpopulation of intercalated cells of collecting ducts.
Our results show that in human kidney SLC26A6 and A7 have a distinct, partially overlapping expression in distal segments of nephrons. The distribution partly differs from that found previously in rodent kidneys.
[Show abstract][Hide abstract] ABSTRACT: Genetic susceptibility for psoriasis is regulated to the greatest extent by the PSORS1 locus. Three psoriasis-associated susceptibility alleles have been identified within it, namely, HLACw6, HCR*WWCC and CDSN*5, but strong linkage disequilibrium between them has made it difficult to distinguish their individual genetic effects, and animal models to study their effects are not known. To study the function of HCR, we engineered transgenic mice with either a non-risk allele of HCR or the HCR*WWCC risk allele under the control of the cytokeratin-14 promoter. These choices were motivated by the apparently dominant effect of PSORS1 on psoriasis susceptibility and the physiological expression of HCR in basal keratinocytes. Transgenic mice appeared phenotypically normal and histologically their skin was indistinguishable from wild-type mice. Expression studies using Affymetrix arrays suggested that the HCR risk allele has specific functional consequences relevant to the pathogenesis of psoriasis. Comparison of gene expression changes between non-risk and risk allele mice revealed similarities to previous observations in human psoriatic skin, including upregulation of cytokeratins 6, 16 and 17 in risk allele mice. We also observed changes in the expression of genes associated with terminal differentiation and formation of the cornified cell envelope. Our results support the concept that HCR may constitute an essential gene in the PSORS1 locus. These observations are also compatible with a model that a susceptibility gene for psoriasis induces changes that are contributory but not sufficient by itself to produce the clinical phenotype.
Human Molecular Genetics 09/2004; 13(15):1551-61. DOI:10.1093/hmg/ddh178 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that HCR is a good candidate gene for psoriasis based on its location in the PSORS1 locus, predicted secondary structure change of the associated allele, and expression pattern. To understand better the function of HCR, we studied how HCR expression is altered in hyperproliferative skin diseases other than psoriasis and in cancers. We examined also its regulation by different cytokines, growth factors, and antipsoriatic agents using quantitative RT-PCR (TaqMan) analysis and its location by immunostaining of keratinocyte cultures. Compared to psoriasis, HCR protein had a different distribution in chronic dermatitis, pityriasis rubra pilaris, mycosis fungoides, and chronic skin ulcers. In three of six grade III squamous cell carcinomas of the skin, four of four adenocarcinomas of the lung, and two of two ductal breast adenocarcinomas, positive cytoplasmic staining in cancer cells was detected. As in psoriasis, Ki67 did not colocalize with HCR. In cell cultures, HCR staining was detected perinuclearly in the cytoplasm and in the nuclei, suggesting that the protein may have a role in both compartments. A 2-fold downregulation of HCR mRNA expression was observed on stimulation with interferon-gamma. Based on the observations that HCR is detected in cancers of epithelial origin in Ki67-negative areas and that interferon-gamma downregulates its expression, we suggest it to have an antiproliferative function.
[Show abstract][Hide abstract] ABSTRACT: Human matrix metalloproteinase-21 (MMP-21), the newest member of the MMP gene family, has been suggested to play an important role in embryogenesis and tumor progression and to be a target of the Wnt, Pax, and Notch signaling pathways. Here we report detection of MMP-21 by RT-PCR in mouse embryos aged 10.5, 12.5, 13.5, and 16.5 days, as well as in various adult murine organs. In both humans and mice, MMP-21 protein was detected in the epithelial cells of developing kidney, intestine, neuroectoderm, and skin but not in normal adult skin using immunohistochemistry with two unrelated antibodies. However, it was present in invasive cancer cells of aggressive subtypes of basal and squamous cell carcinomas, although it was not expressed in skin disorders characterized by mere keratinocyte hyperproliferation. Of several cytokines tested, transforming growth factor-beta1 induced MMP-21 in vitro in HaCaTs and keratinocytes as judged by real-time quantitative TaqMan PCR. Although suprabasal differentiating keratinocytes expressed MMP-21 in developing skin in vivo, MMP-21-positive keratinocytes were detected by immunohistochemistry in both low and high calcium cultures. MMP-21 expression was not up-regulated by ras transformation in HaCaT cell lines (HaCaT, A5, II-4, and RT3); in skin and colon cancers, its expression did not associate with apoptosis, beta-catenin transactivation, or epithelial MMPs-9 and -10. However, MMP-21 protein was found in the same regions as MMP-7 but not in the same cells. Our results suggest that during development, MMP-21 expression is temporally and spatially tightly controlled. Unlike many classical MMPs, it is present in various normal adult tissues. Among epithelial MMPs, MMP-21 has a unique expression pattern in cancer.
[Show abstract][Hide abstract] ABSTRACT: We have characterized a novel human matrix metalloproteinase (MMP-21) from human placenta DNA complementary to RNA (cDNA). The 569 amino acid translation of the cDNA includes all the typical features of an MMP family member, namely a signal sequence, a prodomain with a PRCGVPD motif, a zinc-binding catalytic domain with an HEIGHVLGL sequence, and a hemopexin-like domain flanked by two cysteine residues. Furthermore, MMP-21 has a furin activation sequence, but no transmembrane sequence nor a cytoplasmic domain. As in Xenopus laevis and Cynops pyrrhogaster there is an additional insertion of approximately 30 amino acids between the prodomain and the catalytic domain, which is poorly conserved between the species and is in human MMP-21 especially proline rich. The MMP-21 gene has seven exons and is located in chromosome 10. This new MMP is the human orthologue for XMMP and CyMMP expressed during gastrulation of X. laevis and C. pyrrhogaster, respectively. A 2.5 kb messenger RNA was observed in fetal liver by Northern analysis. By reverse transcription-polymerase chain reaction, MMP-21 is expressed in various human fetal and adult tissues as well as in cancer cell lines. MMP-21 protein can also be detected in malignancies such as ovarian and colon carcinomas by immunohistochemical staining. Our findings suggest that MMP-21 functions in embryogenesis and tumor progression.
[Show abstract][Hide abstract] ABSTRACT: Ectodermal dysplasias are a large group of rare genetic disorders with developmental abnormalities in skin, teeth, hair and nails. Many of them are clinically serious and impair the life of patients. The cloning of the gene for the most common of them, X-linked anhidrotic ectodermal dysplasia, in 1996 opened the door to dissect novel developmental pathways at the molecular level. Since then, several new genes and proteins with novel functions have been identified.
Trends in Molecular Medicine 06/2002; 8(5):197-200. DOI:10.1016/S1471-4914(02)02342-0 · 9.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PSORS1, near HLA-C, is the major genetic determinant of psoriasis. We present genetic and structural evidence suggesting a major role for the HCR gene at the PSORS1 locus. Genotyping of 419 families from six populations revealed that coding single-nucleotide polymorphisms of HCR formed a conserved allele HCR*WWCC that associated highly significantly with psoriasis and with the HLA-Cw6 allele in all populations. Because of strong linkage disequilibrium between HLA-Cw6 and HCR*WWCC, the two genes could not be genetically distinguished by this sample size. However, the variant HCR allele was predicted to differ in secondary structure from the wild-type protein. HCR protein expression in lesional psoriatic skin differed considerably from that observed in normal skin. These results provide strong evidence for the HCR*WWCC allele as a major genetic determinant for psoriasis, probably by a mechanism impacting on keratinocyte proliferation.
Human Molecular Genetics 04/2002; 11(5):589-97. DOI:10.1093/hmg/11.5.589 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anhidrotic ectodermal dysplasia (EDA) is an X-linked disorder characterized by abnormal development of ectoderm and its appendices. The EDA gene encodes different isoforms of ectodysplasin, a transmembrane protein. The two longest isoforms, ectodysplasin-A1 and -A2, which differ by an insertion of two amino acids, are trimeric type II membrane proteins with an extracellular portion containing a short collagenous domain and a TNF ligand motif in the C-terminal region. We show that ectodysplasin is released from cells to the culture medium. Deletion constructs were used to localize the cleavage site and show that the putative recognition sequence of a furin-like enzyme is needed for the cleavage. Some EDA patients have missense mutations affecting this recognition sequence, suggesting that cleavage has biological significance in vivo. EDAR, a recently cloned member of the TNFR family and the product of the downless gene, is able to co-precipitate ectodysplasin, confirming that they form a ligand-receptor pair. In situ hybridization and immunostaining studies show that ectodysplasin and EDAR are expressed in adjacent or partially overlapping layers in the developing human skin. We conclude that as a soluble ligand, ectodysplasin is able to interact with EDAR and mediate signals needed for the development of ectodermal appendages.
Human Molecular Genetics 05/2001; 10(9):953-62. DOI:10.1093/hmg/10.9.953 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stromelysin-2 is a matrix metalloproteinase that degrades in vitro several protein components relevant to wound repair such as collagens III and IV, gelatin, nidogen, laminin-1, proteoglycans, and elastin. Furthermore, it can activate other matrix metalloproteinases, such as collagenase-1 (matrix metalloproteinase-1) and collagenase-2 (matrix metalloproteinase-8), as well as 92 kDa gelatinase. The aim of this study was to determine in a large variety of wounds (normally healing dermal and mucosal wounds, suction blisters, ex vivo cultures, diabetic, decubitus, rheumatic, and venous ulcers) and keratinocyte cultures, which factors contribute to stromelysin-2 expression and how it is induced in relation to other matrix metalloproteinases. Our results show that stromelysin-2 mRNA and protein are upregulated later (at 3 d) than matrix metalloproteinase-1 in normally healing wounds and ex vivo explants, in which stromelysin-2 is invariably expressed by keratinocytes migrating over dermal matrix. The number of keratinocytes expressing stromelysin-2 was greatest in chronic inflamed diabetic and venous ulcers compared with rheumatoid and decubitus ulcers, six of which had no signal. In keratinocyte cultures, tumor necrosis factor-alpha, epidermal growth factor, and transforming growth factor-beta1 induced stromelysin-2 expression as measured by quantitative reverse transcriptase-polymerase chain reaction, whereas different matrices did not upregulate the mRNA. Immunostaining demonstrated stromal transforming growth factor-beta1 in contact with the stromelysin-2-positive keratinocytes. Our results suggest that stromelysin-2 expression is important for the normal repair process and is upregulated by cytokines rather than cell-matrix interactions. Stromelysin-2 is most likely to participate in the remodeling of the newly formed basement membrane, and is not overexpressed in retarded wound healing.
[Show abstract][Hide abstract] ABSTRACT: Anhidrotic ectodermal dysplasia (EDA) is a human genetic disorder of impaired ectodermal appendage development. The EDA gene encodes isoforms of a novel transmembrane protein, ectodysplasin. The sequence of the longest isoform includes an interrupted collagenous domain of 19 Gly-X-Y repeats and a motif conserved in the tumor necrosis factor (TNF)-related ligand family. In order to understand better the function of the ectodysplasin protein molecule and its domains, we have studied the processing and localization of wild-type and mutated isoforms in transfected human fetal kidney 293 and monkey kidney COS-1 cells. Similar to other members of collagenous membrane proteins and members of TNF-related ligands, ectodysplasin is a type II membrane protein and it forms trimers. The membrane localization of ectodysplasin is asymmetrical: it is found on the apical and lateral surfaces of the cells where it co-localizes with cytoskeletal structures. The TNF-like motif and cysteines found near the C-terminus are necessary for correct transport to the cell membrane, but the intracellular and collagenous domains are not required for the localization pattern. Our results suggest that ectodysplasin is a new member in the TNF-related ligand family involved in the early epithelial-mesenchymal interaction that regulates ectodermal appendage formation.
Human Molecular Genetics 11/1999; 8(11):2079-86. · 6.39 Impact Factor