Clinical and genetic features of 20 Japanese patients with vascular-type Ehlers-Danlos syndrome.

BACKGROUND: Vascular-type Ehlers-Danlos syndrome (vEDS) is a severe autosomal dominant inherited disorder resulting from mutations within the α1 type III collagen gene (COL3A1). The majority of published mutations are base changes leading to the substitution of single glycine residues within the triple-helical domain of type III collagen. Although clinical characteristics and mutations in the COL3A1 gene have been analysed for some patients from Europe and America, similar analyses have not yet been performed for Japanese patients with vEDS. OBJECTIVES: To analyse the genetic and phenotypic findings in Japanese patients with vEDS. METHODS: We analysed the clinical features of 20 unrelated individuals with vEDS. To quantify type III collagen production, the fibroblasts were cultured with (3) H-proline, and the radiolabelled collagenous proteins were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and fluorography. Mutations in COL3A1 were detected by sequence analysis of cDNA from patients' fibroblasts and subsequently by a genomic DNA sequence analysis. RESULTS: Thin and translucent skin with extensive bruising and hypermobility of the small joints were observed in about 90% of the patients, whereas the prevalence of serious clinical findings such as rupture/dissection/aneurysm of the arteries (30%) or rupture of the gastrointestinal tract (25%) was relatively low. Sequence analyses of the COL3A1 gene demonstrated heterozygous point mutations leading to glycine substitution in only nine patients (45%), while heterozygous splice-site mutations at the junction of the triple-helical exons were observed in the remaining 11 patients (55%). The average type III collagen production level in the cultured dermal fibroblasts was 14·6% of the normal value. The types of complication were not associated with specific mutations in COL3A1. CONCLUSION: The analysis in the present series revealed a low frequency of patients presenting with serious clinical findings such as arterial rupture/arterial dissection/aneurysm and perforation or rupture of the gastrointestinal tract, and revealed a higher prevalence of splice-site mutations at the junction of the triple-helical exons than of glycine substitution mutations in COL3A1.

A novel mutation of a leucine residue in coil 1A of keratin 9 in epidermolytic palmoplantar keratoderma.

Keratin 9 mutation was examined in a Japanese kindred of epidermolytic palmoplantar keratoderma (EPPK), which is a dominantly inherited autosomal disorder of keratinization characterized by diffuse thickening of the palms and soles and by epidermolytic hyperkeratosis histologically. We report herein a novel mutation, a C --> G transversion at nucleotide position 541 that converts a leucine residue (CTC) to a valine (GTC) at codon 159. As in all other reported cases of keratin 9 mutation in EPPK, this mutation lies within the highly conserved coil 1A of the rod domain, which is considered to play a role in the correct alignment of the coiled-coil molecules.

Increased collagen synthesis accompanying elevated m-RNA levels in cultured Werner's syndrome fibroblasts.

Although Werner's syndrome (WS) is a premature aging disease and its fibroblasts typically grow poorly in culture, WS may cause abnormalities in connective tissue metabolism that are seldom seen in normal aging, such as scleroderma-like skin. In a preliminary report, we described increased collagen synthesis in fibroblasts derived from two WS patients. The present study was undertaken to determine the degree of the regulation of collagen gene expression in dermal fibroblasts from two other patients. Overproduction of collagenase sensitive protein was observed in WS fibroblasts. Collagen m-RNA levels, that were determined by hybridization of RNA blots with specific cDNA were about 2 times greater than those in the control cells. These results suggest that control of collagen synthesis in WS fibroblasts is altered at the transcriptional level.

Regulation of collagen gene expression by transformed human fibroblasts: decreased type I and type III collagen RNA transcription.

The regulation of collagen gene expression in normal diploid human fetal fibroblasts (KMS-6 cells), and fibroblasts immortally transformed by treatment of KMS-6 with Co-60 gamma rays (KMST-6 cells) was compared to that of ones tumorigenically transformed by treatment of KMST-6 cells with Harvey murine sarcoma virus (KMST-6-Ras cells). Synthesized collagenous protein decreased to approximately 30% of that of normal fetal fibroblasts in both transformed cell lines, and the relative rate of collagen synthesis to total protein synthesis decreased about sixfold in KMST-6 cells and twelvefold in KMST-6-Ras cells. The m-RNA levels of type I collagen in both of these cell lines decreased to approximately 20% of that of the control fibroblasts, whereas type III collagen m-RNA levels decreased to only 9% of that of the control. The copy number of the collagen gene in both transformed cell lines was unaltered. The transcriptional rates of collagen alpha 1(I) and collagen alpha 1(III) in both cell lines decreased to 20% and 7% respectively of that of control. These data indicate that collagen synthesis was reduced at the transcriptional level in these transformed human fibroblasts.

Decreased collagenase expression in cultured systemic sclerosis fibroblasts.

One cause of the excessive deposition of collagen in systemic sclerosis is thought to be abnormal functioning of fibroblasts. The purpose of this study is to determine whether there is decreased expression of collagenase in systemic sclerosis fibroblasts. In this study, we analyzed collagen and collagenase expression in dermal fibroblasts derived from eight patients with systemic sclerosis and compared the findings with those from nine sex- and age-matched healthy subjects. Increased collagen synthesis accompanying enhanced mRNA levels was observed in two of eight strains, whereas all eight strains showed remarkable decreases in collagenase activity and production. There were no differences in the levels of collagenase mRNA between the systemic sclerosis strains and the normal strains. Results suggest that decreased collagenase expression is a characteristic of systemic sclerosis fibroblasts, and both increased collagen expression and decreased collagenase expression in systemic sclerosis fibroblasts may result in the excessive accumulation of collagen in patients with systemic sclerosis. It is also suggested that decreased collagenase expression is altered at translational and/or post-translational levels.

Collagenase gene expression in cutis laxa fibroblasts is upregulated by transcriptional activation of the promoter gene through a 12-0-tetradecanoyl-phorbol-13-acetate (TPA)-responsive element.

Our previous work demonstrated that collagenase mRNA levels are increased in fibroblasts derived from patients with cutis laxa (CL). To pursue the mechanism of the upregulation of collagenase expression, we investigated transcriptional levels of the collagenase gene in CL fibroblasts. Fibroblasts cultured from the skin of three congenital CL patients were studied. Northern blot hybridization revealed 2.8- to 7.3-fold increases in collagenase mRNA levels in CL fibroblasts compared with normal cells. Nuclear run-off experiments demonstrated that the transcription rate of the collagenase gene in nuclei isolated from the same cells was 5.1- to 10.2-fold higher in the CL fibroblasts than in the controls. Transient transfection of a normal collagenase promoter-CAT construct into the cells further showed significantly enhanced transcriptional activity in CL but not in normal fibroblasts. Experiments of transient transfection of deleted or small substituted collagenase promoter-CAT constructs indicated that collagenase transcription in CL fibroblasts was activated the TPA-responsive element site of the collagenase promoter gene. Although the levels of Jun and Fos gene expression did not differ from those observed in normal fibroblasts, AP-1-binding activity, as measured by the ability to bind to an oligonucleotide containing a TPA-responsive element, was significantly elevated in CL fibroblasts as compared with normal fibroblasts. These data suggest that collagenase expression is upregulated at the transcriptional level by endogenous activation of DNA binding of AP-1 in CL fibroblasts [corrected].

Decreased type VI collagen gene expression in cultured Werner's syndrome fibroblasts.

Gene expression of collagens VI, I, and III in Werner's syndrome was studied by measuring messenger RNA (mRNA) and protein production levels in four fibroblast strains from patients with Werner's syndrome and comparing them with age-matched healthy subjects. Levels of type VI collagen mRNA were decreased in all Werner's syndrome fibroblast strains and the decreases were in parallel in all three chains (alpha 1, alpha 2, and alpha 3) of type VI collagen. A coordinate increase of the alpha 1(I) and alpha 1(III) collagen mRNA levels was observed in three of the four Werner's syndrome fibroblast strains. However, no qualitative abnormality of these mRNA transcripts in Werner's syndrome fibroblasts were found by Northern blot analysis. Changes in type VI and type I collagen mRNA correlated well with production levels of corresponding proteins, as determined by immunologic assays. These data suggest that there are changes in expression of multiple connective tissue constituents in Werner's syndrome fibroblasts.

Collagen metabolism in cutis laxa fibroblasts: increased collagenase gene expression associated with unaltered expression of type I and type III collagen.

Collagen metabolism was studied in cutis laxa by analyzing collagen and collagenase gene expression in three dermal fibroblast strains from patients with congenital cutis laxa and comparing them with fibroblasts obtained from age-matched healthy subjects. Normal collagen synthetic activity was observed in the cutis laxa fibroblasts. An increased level of collagenase mRNA and unaltered levels of alpha 1(I) and alpha 1(III) collagen mRNA were found in all cutis laxa cell strains by dot blot hybridization. Reduced levels of elastin mRNA were also detected in these strains. However, no qualitative differences in these mRNA transcripts were detected between the control and cutis laxa fibroblasts by Northern blot analysis. Collagenase activity in fibroblast culture supernatants was then measured using fluorescein isothiocyanate (FITC)-labeled type I collagen. Increased collagenolytic activity in cutis laxa fibroblast culture supernatants was also found. These data suggest that increased collagenase expression of fibroblasts is related to the structural abnormality of dermal connective tissue in cutis laxa.

Localization of collagen alpha 1(I) gene expression during wound healing by in situ hybridization.

The cellular localization of collagen alpha 1(I) chain gene expression during wound healing in rats was investigated using in situ hybridization. Activation of collagen gene expression was first found within fibroblastic cells around the deep layers of the granulation tissue as early as 16 h post wounding. Heavily labeled cells were also detected near the intact wound edge and around hair follicles. At day 6 intense alpha 1(I) collagen gene expression was found within most cells of the granulation tissue and after day 8 most of the activity was localized to cells directly underneath the epidermis. 26 d after the induction of the wounds hardly any alpha 1(I) collagen gene expression could be demonstrated, which indicates a close, time-dependent control of collagen synthesis during repair processes.

Purification of a novel factor which binds to the mouse alpha 2 (I) collagen promoter.

We have identified and purified a DNA binding protein which specifically binds to a segment of the mouse alpha 2 (I) collagen promoter between -420 and -399 bp upstream of the start of transcription. Purification included heparin-agarose and sequence-specific DNA-affinity chromatography, followed by SDS-PAGE and renaturation of the DNA binding activity after elution from SDS-polyacrylamide gel. The DNA binding activity resides in two species of 42 kDa and 40 kDa, respectively. The levels of DNA binding activity of this factor, which has been tentatively designated as ColF1, are considerably higher in nuclear extracts of NIH-3T3 fibroblasts than in nuclear extracts from epidermal cells, lymphoid cells and transformed NIH-3T3 fibroblasts.

Collagenase gene expression in fibroblasts is regulated by a three-dimensional contact with collagen.

Collagenase activity in fibroblasts is regulated by cytokines and the interaction with the extracellular matrix. In this study we demonstrate that fibroblasts cultured within a three-dimensional collagen gel show a strong induction of collagenase gene expression. In addition to increased de novo synthesis most of the secreted enzyme was found to be activated leading to a high collagenolytic activity and complete degradation of collagen matrices after removal of fetal calf serum. Collagen I gene expression was found to be reduced under these conditions. These data suggest a specific modulation of cellular metabolism in response to contact with a three-dimensional collagenous matrix resulting in the divergent regulation of collagen and collagenase.

Influence of corticosteroids on chemotactic response and collagen metabolism of human skin fibroblasts.

Following chronic administration of corticosteroids in vivo, a number of complications occur, which mainly involve the metabolism of connective tissue cells. Therefore, several attempts have been made to develop corticosteroids, which show less pronounced side effects. Fibroblasts were kept in monolayer cultures and were exposed to corticosteroids demonstrating similar anti-inflammatory activity (prednicarbate, desoximetasone). Chemotaxis of fibroblasts was studied over 4 hr, protein and collagen synthesis were estimated using proteinchemical methods and also by dot blot hybridization. Corticosteroids used in a high dosage (10 microM) affected all biosynthetic capacities of the investigated fibroblasts. Protein synthesis and production of collagen types I and III were reduced and a similar decrease of mRNA levels for collagen type I could be found indicating an influence on the pretranslational control. In the same concentrations desoximetasone was much more active than prednicarbate. Fibroblast migration was dosage dependently inhibited from 10(-9) M to 10(-5) M for desoximetasone, while incubation with prednicarbate did not cause a reduction of the chemotactic response at concentrations lower than 10(-7) M. These data suggest that modifications of corticosteroids might result in a dissociation of some of their biological activities and can specifically influence their effects on biosynthetic capacities of fibroblasts.

Mitochondrial A7445G mutation in two pedigrees with palmoplantar keratoderma and deafness.

A New Zealand and a Scottish pedigree with maternally inherited sensorineural deafness were both previously shown to carry a heteroplasmic A7445G mutation in the mitochondrial genome. More detailed clinical examination of the New Zealand family showed that the hearing loss was progressive, with the severity of the overall loss and the frequencies most affected differing markedly between individuals of similar age, and showed that many relatives also had palmoplantar keratoderma. Review of the literature demonstrated three other large families with presumed autosomal dominant inheritance of palmoplantar keratoderma and hearing loss. In a United Kingdom pedigree the syndrome was transmitted by female and male parents, an inheritance pattern which made mitochondrial inheritance unlikely; however, in a Turkish and a Japanese pedigree the affected individuals were all maternally related. Subsequent analysis of the Japanese pedigree documented the same A7445G mitochondrial mutation as was previously found in the New Zealand and Scottish pedigrees. Other mitochondrial sequence variants previously reported in the New Zealand or Scottish pedigrees were absent from the Japanese pedigree which suggests that the A7445G mutation arose independently in all three pedigrees. To our knowledge palmoplantar keratoderma has not previously been associated with mitochondrial defects; however, the current findings suggest that the A7445G mutation is associated not only with progressive hearing loss but also with palmoplantar keratoderma. The penetrance and expressivity of both symptoms varied considerably between individuals in the Scottish and New Zealand Studies which suggests that additional environmental and/or genetic factors are involved.

Effect of histamine on collagen and collagen m-RNA production in human skin fibroblasts.

Direct effects of histamine on collagenous and non-collagenous protein synthesis by human skin fibroblasts were studied. Fibroblasts derived from human skin were incubated with various concentrations of histamine. Collagen and non-collagenous protein synthesis were measured by incorporation of 3H-proline. Both collagen synthesis measured as protein-bound hydroxyproline and non-collagenous protein synthesis measured as protein-bound proline increased in the presence of histamine at concentrations of 10(1)-10(2) micrograms/ml. Total RNA was extracted and m-RNA levels of various proteins were estimated by dot blot analysis, and densitometrically quantified. The levels of alpha 1(I) collagen and beta-actin m-RNA were clearly increased at the same concentrations. m-RNA levels of alpha 1(III) collagen were also increased but the rate was lower than that of alpha 1(I) collagen. No alteration of beta-tubulin m-RNA level was observed at the same concentrations. These results demonstrate that stimulation of collagen synthesis by histamine is pretranslationally controlled.

Effects of cyclosporin A on cell proliferation and collagen production by human skin fibroblasts.

Cyclosporin A (CSA) is a potent immunosuppressive drug that has been used clinically for the treatment of organ rejection after transplantation as well as for patients with a wide variety of immune-mediated disorders. CSA has recently been reported to be effective in systemic sclerosis, which is a disease of the connective tissues leading to fibrosis of the skin and other involved organs. In this study, we investigated whether CSA affects the cell proliferation and collagen synthesis of human skin fibroblasts. CSA inhibited the DNA synthesis and cell growth of cultured fibroblasts at concentrations of 10(-8) M to 10(-5) M in a dose-dependent manner. The production of both collagen and non-collagenous protein at both the mRNA and protein levels was not affected by 10(-8) to 10(-6) M CSA, but was decreased in the presence of 10(-5) M CSA. These results suggest that CSA may inhibit the proliferation of fibroblasts, but not their synthesis of collagenous and non-collagenous proteins at therapeutic concentrations.

Activation of fibroblast proliferation by Werner's syndrome fibroblast-conditioned medium.

The effects of Werner's syndrome (WS) fibroblast-conditioned medium on cell proliferation and collagen production of normal fibroblasts were studied using four WS fibroblast strains. The conditioned medium from the WS fibroblasts brought about activation of normal fibroblast proliferation, whereas, that from late passage normal fibroblasts and fibroblasts from aged donors' skin did not. Collagen and non-collagenous protein synthesis in normal fibroblasts were increased by the addition of conditioned medium from the WS fibroblasts, but the relative rates of collagen synthesis and non-collagenous protein synthesis were unaltered. These results suggest that activation of fibroblast proliferation by conditioned medium is one of the characteristics of WS fibroblasts.

Effects of tretinoin tocoferil on gene expression of the extracellular matrix components in human dermal fibroblasts in vitro.

Recently, it has been reported that tretinoin tocoferil (TT), a synthesized ester-bond compound of all-trans-retinoic acid and alpha-tocopherol, accelerates the formation of granuloma and is effective in promoting experimental open skin wound healing. To investigate whether TT affects the gene expression of extracellular matrix components of human dermal fibroblasts, we measured the mRNA levels of various extracellular matrix components of fibroblasts incubated with TT using specific cDNA probes. The mRNA levels of elastin increased up to 30% of the controls and those of collagen III and VI up to 60%. The mRNA levels of collagen I and fibronectin remarkably increased up to 90% of the controls. These results suggest that the stimulatory effect of TT on the gene expression of many extracellular matrix components might be one of the mechanisms of its promotion of wound healing.

Increased expression of type VI collagen genes in cutis laxa fibroblasts.

Type VI collagen gene expression in cutis laxa was studied by measuring messenger RNA (mRNA) and protein production levels in four fibroblast strains from patients with congenital cutis laxa and comparing them with those in fibroblasts obtained from age-matched healthy subjects. Levels of type VI collagen mRNA were increased in all cutis laxa fibroblast strains and the levels of alpha 1 (VI) and alpha 3 (VI) chain mRNAs increased in parallel. Increases in type VI collagen mRNAs correlated well with production levels of the corresponding proteins, as determined by immunological assay. These results suggest that increased type VI collagen gene expression is one of the characteristics of cutis laxa dermal fibroblasts and that this abnormality may be related to the skin changes in cutis laxa.

Diffuse palmoplantar keratoderma with deafness.

Two brothers with diffuse palmoplantar keratoderma (Thost-Unna type) also were deaf. Of the 38 members of the patients' family, five had a similar disorder and ten had only hearing loss. The mode of inheritance of the dermatosis is regarded as autosomal dominant and is diagnostically distinguished from the other dermatoses associated with the disturbance of keratinization and deafness. To our knowledge, this is the second report of diffuse palmoplantar keratoderma (Thost-Unna type) with deafness, which is considered to be a new variant of the keratodermatoses.

Epidermal cell proliferation following an active arthus reaction in the guinea pig.

Active Arthus reactions were provoked by injections of 100 micrograms horseradish peroxidase (HRP), 10 micrograms HRP and 100 micrograms bovine serum albumin (BSA) into the skin of sensitized guinea pigs. Labeling indices (LI) of epidermal basal cells were measured 1, 4, 8, 24, 48 and 72 h later by the in vivo 3H-thymidine labeling technique, and compared with those obtained with injections of antigens into the skin of non-sensitized guinea pigs. From 1-8 h after the induction of an active Arthus reaction, the LI of epidermal basal cells of the skin injected with 100 micrograms HRP decreased to a remarkably low value. On the other hand, those obtained with the reaction against 10 micrograms HRP were significantly high. At 24 h after the reaction, LI were as high as those obtained in non-sensitized guinea pigs with control intradermal injections, though the former persisted high until 48 h after the injection. In addition, decreased LI of the epidermal basal cells were observed in the skin 4 h after intradermal injections of immune complexes. It was suggested that DNA synthetic activity of the epidermis increases in a mild active Arthus reaction, while the activity may be suppressed in a severe active Arthus reaction up to 8 h after provocation.