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Biomedical subjects

A Hatamochi

Publications and source records attributed to A Hatamochi.

At least 19 recordsLinked to original sources
PubMed1994

Role of cytokines in controlling connective tissue gene expression.

Recently, the role of cytokines in controlling gene expression of connective tissue components has been increasingly emphasized. Many cytokines have been shown to have specific effects on gene expression of connective tissue components, and the roles of cytokines in controlling connective tissue metabolism during wound healing and in fibrosis have increasingly been discussed. In this article, the effects of cytokines on regulation of gene expression of connective tissue components, especially of type I collagen were described. We analysed transcriptional control of the alpha 1(I) collagen gene by TNF-alpha by means of DNA mediated transfection experiments using recombinant plasmids in which the promoter region of the human alpha 1(I) collagen had been fused to the chloramphenicol acetyl-transferase (CAT) gene, in human dermal fibroblasts. It was found that TNF-alpha reduced alpha 1(I) collagen transcription through at least up to -107 bp upstream of the human alpha 1(I) collagen promoter gene in dermal fibroblasts.

Animals↗
PubMed1994

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.

Actins↗
PubMed1994

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.

Adult↗
PubMed1994

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.

Adult↗
PubMed1992

Analysis of collagen gene expression by cultured fibroblasts in morphoea.

Collagen gene expression was studied in dermal fibroblasts derived from the inflammatory and sclerotic skin lesions of patients with localized or generalized morphoea. The levels of mRNA for type I collagen in early-passage fibroblasts derived from inflammatory lesions were higher than those obtained from the uninvolved skin, whereas those fibroblasts obtained from sclerotic lesions were unaltered. No alteration in type I collagen mRNA levels was observed in late-passage fibroblasts derived from the inflammatory lesions. The relative rate of collagen to total protein synthesis in early-passage fibroblasts derived from inflammatory lesions was higher than that of fibroblasts from uninvolved skin, while no alteration or a slight decrease was observed in fibroblasts from the sclerotic lesions. The data suggest that the inflammatory reactions induce increased collagen synthesis by fibroblasts in the skin in scleroderma.

Actins↗
PubMed1991

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.

Actins↗
PubMed1991

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.

Actins↗
PubMed1991

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.

Cell Line, Transformed↗
PubMed1991

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.

Collagen↗
PubMed1991

Analysis of methylation in the c-MYC gene in five human myeloma cell lines.

Genomic alterations of the human c-MYC gene were analysed in five human myeloma cell lines established in Kawasaki Medical School and compared with those of normal lymphocytes, Raji cells from Burkitt's lymphoma, and an Epstein-Barr virus positive lymphoblastoid cell line (LCL). Although no structural chromosome aberrations at 8q24, the c-MYC locus, were distinct, the mRNA level of c-MYC in these myeloma cell lines was 30-50-fold that in normal peripheral blood lymphocytes. Regarding the methylation of c-MYC, DNAs of the myeloma cell lines were digested with MspI plus EcoRI or HpaII plus EcoRI, and hybridized with three genomic 32P-labelled probes; the first, second and third exons of the human c-MYC gene, respectively. The extent of methylation in cytosine at a single CCGG site in the third exon substantially decreased in these myeloma cell lines as compared with that in normal tonsillar B, LCL and Raji cells. No significant differences in hypomethylation between these myeloma, normal B, LCL and Raji cells was detected in the first and second exon of c-MYC. These results suggest that the hypomethylation in the third exon of c-MYC might be related to the enhanced expression of c-MYC in these human myeloma cell lines.

Aged↗
PubMed1990

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.

Cells, Cultured↗
PubMed1989

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.

Adult↗
PubMed1989

Down-regulation of alpha 3(VI) chain expression by gamma-interferon decreases synthesis and deposition of collagen type VI.

Treatment of cultured human skin fibroblasts with increasing doses of gamma-interferon produces a distinct reduction of steady-state levels of the alpha 3 chain of collagen VI mRNA by about 60% but not of the alpha 1 and alpha 2 chain mRNAs. A similar decrease was also observed for collagen I and III mRNA while fibronectin mRNA remained at the same level. The decrease in alpha 3(VI) mRNA is accompanied by a reduced synthesis of collagen VI and by a reduced deposition of both collagen VI and fibronectin in urea-insoluble form in the cell matrix. No other gamma-interferon effects were observed for fibronectin biosynthesis. Immunoprecipitation of metabolically labeled collagen VI demonstrated a strongly reduced synthesis (by 65-80%) of intracellular alpha 3(VI) chains with no decrease found for alpha 1(VI) and alpha 2(VI) chains. All three chains were, however, found to be reduced in the culture medium. Pepsin treatment of immunoprecipitated collagen VI showed similar chain ratios for material in the culture medium obtained in the absence or presence of gamma-interferon. It indicates that correctly assembled heterotrimers of the composition [alpha 1(VI) alpha 2(VI) alpha 3(VI)] are formed and secreted also in the absence of an equivalent alpha 3(VI) chain synthesis but at a reduced rate. The data support previous predictions from sequence analyses [Chu et al. (1988) J. Biol. Chem. 263, 18,601-18,606] that collagen VI molecules composed of all three constituent chains are more stable than other assembly alternatives.

Actins↗
PubMed1989

Regulation of collagen VI expression in fibroblasts. Effects of cell density, cell-matrix interactions, and chemical transformation.

Collagen VI expression was studied in cultured human skin fibroblasts and mouse 3T3 cells using cDNA probes specific for alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. A 2-3-fold increase of these mRNAs was observed when fibroblasts were grown at increasing densities while only minimal changes occurred for the mRNA levels of collagens I and III, fibronectin, and beta-actin. Changes in mRNA correlated well with an increased production of corresponding proteins as determined by immunological assays. A comparable increase of alpha 1(VI) and alpha 2(VI) but not of alpha 3(VI) chain mRNAs was found for fibroblasts grown in a three-dimensional collagen gel after gel contraction. These conditions resulted, however, in a decrease of steady-state levels of collagens I and III and actin mRNAs. Transformation of 3T3 cells by phorbol ester did not change collagen VI mRNAs but caused a 3-5-fold reduction in mRNA levels for the other extracellular matrix proteins. These data strongly imply different regulatory mechanisms for the expression of collagen VI compared with collagens I and III and fibronectin. The differences may be correlated to changes in cell shape and reflect the requirement for collagen VI as a cell-binding protein.

Actins↗
PubMed1989

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on collagen synthesis of human skin fibroblasts in vitro.

The effect of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) on collagen metabolism by human diploid fibroblasts in confluent monolayer culture was examined. Recombinant TNF alpha reduced collagen mRNA levels 2-fold and stimulated collagenase mRNA levels 5-fold, while recombinant IFN gamma affected only collagen mRNA levels. The combination of TNF alpha (10 ng/ml) and IFN gamma (100 ng/ml) resulted in a much stronger (about 30-fold) reduction of collagen mRNA levels indicating that the two cytokines act synergistically. In contrast no such synergism was observed with respect to collagenase mRNA levels. The effect of TNF alpha and IFN gamma on collagen metabolism reported here indicates a complex interaction of different cytokines in the control of tissue remodeling that occurs during inflammation, repair, or atrophy.

Adult↗
PubMed1989

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.

Animals↗
PubMed1988

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.

Adrenal Cortex Hormones↗