A novel homozygous nonsense mutation in the LAMC2 gene in patients with the Herlitz junctional epidermolysis bullosa.
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Abortion, Induced↗
Biomedical subjects
J Uitto
Publications and source records attributed to J Uitto.
Type VII collagen gene expression by human skin fibroblasts and keratinocytes in culture: influence of donor age and cytokine responses.
Type VII collagen is the predominant, if not the exclusive, component of the anchoring fibrils. In this study, we have examined the expression of the type VII collagen gene in human skin fibroblasts and keratinocytes in culture by Northern analyses and immunocytochemistry. Type VII collagen gene expression was greatly enhanced in all cell strains studied after stimulation by transforming growth factor-beta (TGF-beta). However, no definitive correlation between the donor age and the magnitude of TGF-beta response could be made. In contrast, the basal expression of the type VII collagen gene was shown to decrease in an age-dependent manner in fibroblasts. The pro-inflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) were shown to elevate type VII collagen mRNA levels in a dose-dependent manner. This response was inversely related to the donor age of the cell cultures. The attenuated response of cells from older individuals to TNF-alpha and IL-1 beta was specific for type VII collagen gene expression, because, in the same experiments, collagenase gene expression was strongly elevated by the two cytokines. Our data suggest that type VII collagen gene expression is subject to modulation by the cytokine network, which may play a role in controlling anchoring fibril assembly in normal skin and in pathologic conditions characterized by altered deposition of type VII collagen.
Adult↗
Collagen VII in severe recessive dystrophic epidermolysis bullosa: expression of mRNA but lack of intact protein product in skin and cutaneous cells in vitro.
The collagen VII gene, COL7A1, is the candidate gene for both the recessive and dominant forms of dystrophic epidermolysis bullosa (EBD). Collagen VII is a structural protein of the anchoring fibrils, which are rudimentary or altered in several subtypes of EBD. In severe recessive mutilating EBD, anchoring fibrils and collagen VII are not detectable in skin of most patients. To elucidate the underlying pathogenetic mechanisms, we analyzed collagen VII expression in cutaneous cells of six patients with this severe EBD subtype. Neither keratinocytes nor fibroblasts synthesized detectable amounts of collagen VII protein; however, Northern blot analysis revealed small amounts of normal-size collagen VII mRNA in both EBD and control fibroblasts. When the mRNA was amplified using reverse transcription-polymerase chain reaction, correct amplimers were present in all specimens. The results demonstrate that transcription of the COL7A1 gene occurs in these patients with severe mutilating EBD and suggest that post-transcriptional or post-translational events lead to absence of collagen VII protein from skin.
Adult↗
Genetic linkage mapping of heritable skin diseases: positional cloning versus the candidate gene approach.
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Chromosome Mapping↗
Enhanced elastin and fibrillin gene expression in chronically photodamaged skin.
Cutaneous aging consists of chronologic aging as well as actinic damage, referred to as photoaging. Most of the morphologic changes associated with an aged appearance result from actinic damage to the skin. The morphologic changes in sun-damaged skin are associated with accumulation of material having the staining characteristics of elastin, known as solar elastosis, in the superficial dermis. Previous studies have demonstrated the presence of elastin within areas of solar elastosis; however, little is known about the mechanisms leading to elastin accumulation in photoaged skin. In addition, fibrillin, the fibrillar component of elastic fibers, has been found in small amounts in solar elastosis. In this study we demonstrate increased elastin mRNA levels in photoaged skin, as well as increased elastin and fibrillin mRNAs in skin explant-derived fibroblasts using Northern hybridizations, compared with controls from sun-protected sites of the same individual. Increased elastin mRNA levels result from transcriptional upregulation of the gene, as demonstrated by transient transfections with a human elastin promoter/chloramphenicol acetyltransferase construct. Elevated mRNA levels were also correlated with increased elastin and fibrillin deposition in paired biopsy specimens from photodamaged and non-sun-exposed skin, as demonstrated by immunohistochemical staining. Thus, approaches to counteract transcriptional activation of elastin gene expression may be useful in preventing the changes associated with cutaneous photoaging.
Aged↗
Glucocorticosteroids up-regulate human elastin gene promoter activity in transgenic mice.
Recent characterization of the human elastin gene identified three putative glucocorticoid responsive elements (GRE) within the 5'-flanking DNA. To test the functionality of these cis-elements, transgenic mice that express a human elastin promoter-reporter gene (CAT) construct in a tissue-specific manner were injected with triamcinolone acetonide (TMC) or dexamethasone (DEX), two glucocorticosteroids in clinical use. Subcutaneous injection of these glucocorticoids resulted in a marked, up to 28-fold, enhancement of the CAT activity in the skin at the site of injection. Similarly, intraperitoneal injection of DEX resulted in significant increases in the elastin promoter activity in various internal organs. Furthermore, incubation of skin fibroblast and aortic smooth muscle cell cultures established from the transgenic animals with TMC (10 ng/ml) resulted in marked increases in the elastin promoter activity. These studies demonstrate that glucocorticosteroids act as powerful up-regulators of human elastin promoter activity in transgenic mice.
Animals↗
Type VII collagen specifically binds fibronectin via a unique subdomain within the collagenous triple helix.
Type VII collagen is the major component of anchoring fibrils, structures within basement membranes beneath stratified squamous epithelium thought to mediate the adherence of the epidermis to the dermis of human skin. Type VII collagen has affinity for fibronectin. The interaction between type VII collagen and fibronectin is mediated through the collagen-binding domain on the amino terminus of fibronectin. Heretofore, the domain on the type VII collagen molecule that binds to fibronectin was not known. In this study, we mapped the binding site of fibronectin to a specific subdomain of the triple helical collagenous region of type VII collagen, immediately adjacent to the small carboxyl terminal non-collagenous domain. This fibronectin-binding site within the type VII collagen molecule lies between nucleotide residues 615 and 1161.
Amino Acid Sequence↗
Mouse 230-kDa bullous pemphigoid antigen gene: structural and functional characterization of the 5'-flanking region and interspecies conservation of the deduced amino-terminal peptide sequence of the protein.
The 230-kDa bullous pemphigoid antigen is a hemidesmosomal protein of the cutaneous basement membrane zone. The primary sequences deduced from full-length human cDNAs predict that this molecule consists of a central rod region and flanking globular domains. To get insight into regulation of the 230-kDa bullous pemphigoid antigen gene (BPAG1), and to evaluate evolutionary conservation of the amino-terminus of the protein, we screened a mouse genomic DNA library with a 0.3-kb cDNA corresponding to the 5' end of the human 230-kDa bullous pemphigoid antigen cDNA. A positive clone was isolated, and Southern analysis of the clone with the 0.3-kb cDNA allowed isolation of a 3.0-kb Hind III fragment containing the 5' end of the coding sequence. Alignment of the sequences of this subclone and human BPAG1 sequences revealed that this fragment contained 2466 bp of 5'-flanking DNA, upstream from the ATG translation initiation site, and 258 bp of translatable sequences that encode a putative polypeptide of 86 amino acids at the amino-terminus of the protein. This deduced polypeptide showed 91% homology with the corresponding human sequence. The TATAAA and CCAAT consensus sequences, as well as several putative cis-regulatory elements, were identified in the 5'-flanking region of the mouse DNA. To test the functional promoter activity of the 5'-flanking DNA, three mouse BPAG1 promoter/CAT reporter gene constructs, with the promoter segments spanning from -1133, -525, and -213 to -1, were developed. Transient transfections of mouse transformed keratinocytes (Pam 212 cells) with these constructs revealed clearly detectable CAT activities, indicating that the 5'-flanking region contains a functional promoter. Furthermore, these experiments suggested that the upstream sequences contain upregulatory elements, as well as elements that confer, at least in part, tissue specificity to the expression of the mouse 230-kDa BPA gene.
Amino Acid Sequence↗
Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes.
Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories, the simplex, junctional, and dystrophic forms, on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. Approaches of molecular biology have demonstrated that these three different forms of EB result from mutations in distinct genes: the simplex forms are due to mutations in the genes encoding keratins 5 and 14 expressed in basal keratinocytes; the junctional forms are associated with mutations in the kalinin/laminin 5 genes; and the dystrophic forms result from mutations in the type VII collagen gene (COL7A1). In this overview, we summarize our recent discoveries of pathogenic mutations in COL7A1, including premature termination codons that result in the severe, mutilating (Hallopeau-Siemens) type of recessive dystrophic EB and a glycine substitution in the collagenous region resulting in dominant dystrophic EB. Furthermore, we present evidence that implicates mutations in the kalinin/laminin 5 gamma 2 chain gene (LAMC2) in some forms of junctional EB. This information has provided the basis for DNA-based prenatal diagnosis during the first trimester of gestation, and sets the stage for the application of gene therapy to these devastating skin diseases in the future.
Cell Adhesion Molecules↗
Molecular pathology of the elastic fibers.
Elastic fibers form a network that contributes to the elasticity and resilience of tissues such as the skin. Histopathologic and ultrastructural abnormalities in the elastic fibers have been observed in several diseases of the skin and other tissues. Recent cloning of several genes involved in elastic fiber architecture has lead to the approach of the study of elastic fiber genodermatoses through molecular analysis. However, in genodermatoses, such as pseudoxanthoma elasticum, many of the genes encoding elastic fiber components have been excluded by genetic linkage analysis. In recent years, mutations in several of the genes encoding elastic fiber proteins have been demonstrated in other diseases. These include mutations in the fibrillin 1 gene in the Marfan syndrome, and genetic linkage of congenital contractural arachnodactyly to fibrillin 2, and, most recently, demonstration of abnormalities in the Menkes syndrome gene in X-linked cutis laxa. The first disorders to involve mutations in the elastin gene itself are, surprisingly, cardiovascular and neurobehavioral disorders, such as supravalvular aortic stenosis and Williams syndrome. These findings suggest that additional, as yet undiscovered, components of the elastic fiber network in the skin may hold the key to unraveling the molecular basis of the elastin-related genodermatoses.
DNA, Recombinant↗
Healing impairment of open wounds by skin irradiation.
BACKGROUND: Ionizing radiation is frequently used for the treatment of malignancy. Sequelae of therapeutic radiation frequently present clinical problems in the form of poor wound healing and easy injury of treated tissue in response to mild trauma. OBJECTIVE: We describe a radiation-impaired wound healing model in guinea pigs, developed to determine the effect of cutaneous irradiation on wound contraction. METHODS: Guinea pigs were anesthetized and a flap of skin was isolated and treated with x-rays to 18 Gy. Circular wounds of identical size were made in irradiated and control skin, and average wound size was recorded. RESULTS: Our results demonstrate that a statistically significant wound healing deficit is produced in open wounds by surface irradiation of skin. CONCLUSION: Radiation of skin results in slower healing of open wounds, and provides an in vivo system for evaluation of topical dressings and growth factors in radiation-impaired wounds.
Animals↗
Molecular biology of the 230-kD bullous pemphigoid antigen. Cloning of the BPAG1 gene and its tissue-specific expression.
The 230-kD bullous pemphigoid antigen is a hemidesmosomal protein of the cutaneous basement membrane zone. We have previously cloned overlapping cDNAs corresponding to the human 230-kD bullous pemphigoid antigen gene (BPAG1), located at the human chromosomal locus 6p11-12. Utilizing the cDNA clones, a genomic DNA lambda FIX II phage library was screened. Seven over-lapping genomic clones, spanning approximately 20 kb, were isolated. These clones were shown to contain the entire approximately 9-kb coding sequence of BPAG1, and it consisted of 22 separate exons which varied from 78 to 2,810 bp in size. Elucidation of 2.6 kb of 5'-flanking DNA was found to contain several putative transcriptional response elements, and development of promoter chloramphenicol acetyltransferase (CAT) reporter gene constructs allowed identification of putative cis-elements which confer keratinocyte-specific expression to the gene. In particular, a putative AP2-binding sequence (KRE2) in the position -(1,786-1778) was shown to be responsible for marked enhancement of the endogenous promoter, as well as of a heterologous thymidine kinase/CAT construct, activity in normal human keratinocytes. Normal human keratinocyte nuclear extracts contained a protein, designated as KTP1, which complexed with the KRE2 oligomer by gel mobility shift assays. UV cross-linking and Southwestern analysis suggested that KTP1 is a DNA-binding protein clearly distinct from AP2, and this protein may be responsible for the basal keratinocyte-specific expression of the BPAG1 gene.
Autoantigens↗
Elevated expression of type VII collagen in the skin of patients with systemic sclerosis. Regulation by transforming growth factor-beta.
A hallmark of systemic sclerosis (SSc) is the development of tissue fibrosis. Excessive production of several connective tissue components normally present in the dermis, including type I, III, V, and VI collagens as well as fibronectin and proteoglycans, is a consistent finding in the skin of SSc patients. Type VII collagen is a major constituent of anchoring fibrils, present in the skin at the dermal-epidermal basement membrane zone. TGF-beta has been shown to upregulate the expression of the type VII collagen gene. In this study, we assessed the expression of type VII collagen and TGF-beta in the skin of patients with SSc. Indirect immunofluorescence showed an abundance of type VII collagen in the patients' skin, including the dermis. Ultrastructural analysis of SSc skin revealed an abundance of fibrillar material, possibly representing type VII collagen. The increased expression of type VII collagen epitopes was accompanied by the elevated expression of immunodetectable TGF-beta 1 and TGF-beta 2. Dermal fibroblasts cultured from the affected individuals showed a statistically significant (P < 0.02) increase in the expression of type VII collagen at the mRNA level, as detected by reverse transcription-PCR with a mutated cDNA as an internal standard, and increased deposition of the protein as assessed by indirect immunofluorescence. Thus, type VII collagen is abundantly present in SSc patients' dermis, a location not characteristic of its normal distribution, and its aberrant expression may relate to the presence of TGF-beta in the same topographic distribution. The presence of type VII collagen in the dermis may contribute to the tightly bound and indurated appearance of the affected skin in SSc patients.
Adult↗
Interleukin-10 modulates type I collagen and matrix metalloprotease gene expression in cultured human skin fibroblasts.
IL-10, originally isolated from mouse helper T cells, is a cytokine with regulatory functions on a number of interleukins. In this study we show that recombinant human IL-10 affects the expression of several genes involved in extracellular matrix synthesis and remodeling in human dermal fibroblast cultures. As judged by Northern blot analyses, type I collagen gene expression was downregulated, while collagenase and stromelysin gene expression were markedly enhanced by IL-10. No effect on tissue inhibitor of metalloproteases mRNA levels was noted. Transient transfections of skin fibroblasts with type I collagen promoter/chloramphenicol acetyl transferase reporter gene constructs showed downregulation by IL-10, suggesting inhibition at the transcriptional level. When compared with control cultures, incubation with IL-10 resulted in a decrease in immunostaining of fibroblast cultures with antibodies to human type I collagen. In contrast, immunostaining of such IL-10-treated cultures with antibodies to human collagenase resulted in an increase in immunostaining. This study suggests a role for IL-10 in the breakdown and remodeling of the extracellular matrix.
Blotting, Northern↗
Mutations in the non-helical linker segment L1-2 of keratin 5 in patients with Weber-Cockayne epidermolysis bullosa simplex.
Keratins are the major structural proteins of the epidermis. Analyzing keratin gene sequences, appreciating the switch in keratin gene expression that takes place as epidermal cells commit to terminally differentiate, and elucidating how keratins assemble into 10 nm filaments, have provided the foundation that has led to the discoveries of the genetic bases of two major classes of human skin diseases, epidermolysis bullosa simplex (EBS) and epidermolytic hyperkeratosis (EH). These diseases involve point mutations in either the basal epidermal keratin pair, K5 and K14 (EBS), or the suprabasal pair, K1 and K10 (EH). In severe cases of EBS and EH, mutations are found in the highly conserved ends of the alpha-helical rod domain, regions that, by random mutagenesis, had already been found to be important for 10 nm filament assembly. In order to identify regions of the keratin polypeptides that might be more subtly involved in 10 nm filament assembly and to explore the diversity in mutations within milder cases of these diseases, we have focused on Weber-Cockayne EBS, where mild blistering occurs primarily on the hands and feet in response to mechanical stress. In this report, we show that affected members of two different W-C EBS families have point mutations within 1 residue of each other in the non-helical linker segment of the K5 polypeptide. Genetic linkage analyses, the absence of this mutation in > 150 wild-type alleles and filament assembly studies suggest that these mutations are responsible for the W-C EBS phenotype. These findings provide the best evidence to date that the non-helical linker region in the middle of the keratin polypeptides plays a subtle but significant role in intermediate filament structure and/or intermediate filament cytoskeletal architecture.
Alleles↗
Recurrent nonsense mutations within the type VII collagen gene in patients with severe recessive dystrophic epidermolysis bullosa.
The generalized mutilating form of recessive dystrophic epidermolysis bullosa (i.e., the Hallopeau-Siemens type; HS-RDEB) is a life-threatening disease characterized by extreme mucocutaneous fragility associated with absent or markedly altered anchoring fibrils (AF). Recently, we reported linkage between HS-RDEB and the type VII collagen gene (COL7A1), which encodes the major component of AF. In this study, we investigated 52 unrelated HS-RDEB patients and 2 patients with RDEB inversa for the presence, at CpG dinucleotides, of mutations changing CGA arginine codons to premature stop codons TGA within the COL7A1 gene. Eight exons containing 10 CGA codons located in the amino-terminal domain of the COL7A1 gene were studied. Mutation analysis was performed using denaturing gradient gel electrophoresis of PCR-amplified genomic fragments. Direct sequencing of PCR-amplified products with altered electrophoretic mobility led to the characterization of three premature stop codons, each in a single COL7A1 allele, in four patients. Two patients (one affected with HS-RDEB and the other with RDEB inversa) have the same C-to-T transition at arginine codon 109. Two other HS-RDEB patients have a C-to-T transition at arginine 1213 and 1216, respectively. These nonsense mutations predict the truncation of approximately 56%-92% of the polypeptide, including the collagenous and the noncollagenous NC-2 domains. On the basis of linkage analysis, which showed no evidence for locus heterogeneity in RDEB, it is expected that these patients are compound heterozygotes and have additional mutations on the other COL7A1 allele, leading to impaired AF formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Transcriptional interactions of transforming growth-factor-beta with pro-inflammatory cytokines.
BACKGROUND: Inflammation and tissue injury are characterized by a massive infiltration of mononuclear cells. These pro-inflammatory cells, which are the precursors of an inflammatory response by the immune system, secrete a variety of cytokines and growth factors that alter the biosynthetic repertoire of the resident connective tissue cells. Specifically, expression of connective tissue matrix metalloproteinases, such as stromelysin and interstitial collagenase, is enhanced, together with the expression of chemoattractants for leukocytes, such as interleukin-8 (IL-8). These events lead to increased connective tissue degradation. We have examined the growth factor regulation of expression in cultured fibroblasts of the prototypic pro-inflammatory factors interstitial collagenase and IL-8. RESULTS: We demonstrate that transforming growth factor-beta (TGF-beta) does not interfere with cytokine-induced IL-8 gene expression, nor does it affect the activity of NF-kappaB-driven promoters. In contrast, TGF-beta down-regulates collagenase gene expression through the induction of the jun-B proto-oncogene. Jun-B is a negative regulator of c-jun, which mediates cytokine activation of collagenase gene expression through its action as a component of the AP-1 transcription factor. CONCLUSION: Our data suggest that TGF-beta may attenuate the deleterious events that occur in inflammation by preventing cytokine-induced extracellular matrix degradation, although it does not affect cytokine-induced recruitment of pro-inflammatory cells. Furthermore, our data suggest a potential therapeutic use for jun-B, which may be a candidate for gene therapy in disease states that are characterized by excessive connective tissue degradation.
Journal Article↗
Complete coding sequence, intron/exon organization, and chromosomal location of the gene for the core I protein of human ubiquinol-cytochrome c reductase.
Core I protein is a nuclear-encoded component of the ubiquinol-cytochrome c reductase complex of the mitochondrial respiratory chain. We have located the gene for the human core I protein in the p21 region of chromosome 3, just upstream of the COL7A1 gene which encodes type VII collagen. The core I gene, which has been sequenced in its entirety, is comprised of 10,417 base pairs, from the major transcription start site to the polyadenylation signal, and contains 13 exons. The predicted polypeptide contains 480 amino acids, of which the first 34 are predicted to constitute a typical mitochondrial leader peptide containing 6 positively charged arginine residues. The predicted human protein shows significant homology with core I protein from Saccharomyces cerevisiae, rather high homology (64% similarity, 46% identity) with the processing enhancing protein, which functions as core I protein in Neurospora crassa, and, surprisingly, highest homology with the small subunit of the mitochondrial processing peptidase of rat (74% similarity, 55% identity). The predicted human sequence is 87% identical to the reported bovine core I sequence predicted from cDNA cloning, up to residue 298, but the two predicted sequences are widely divergent after that point.
Amino Acid Sequence↗