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At least 217 records · Page 12Linked to original sources
PubMed1998

Increased expression of heat-shock protein 47 is associated with overproduction of type I procollagen in systemic sclerosis skin fibroblasts.

Heat-shock protein 47 (HSP47) is a collagen-binding stress protein that is thought to act as a collagen-specific molecular chaperon during the biosynthesis and secretion of procollagen. In this study we examined the expression of HSP47 mRNA and protein in systemic sclerosis (SSc) skin fibroblasts. HSP47 mRNA and protein levels were significantly higher in fibroblast cultures from SSc patient-involved skin samples than in fibroblasts from normal skin from healthy individuals, as assessed by northern blot and immunoblot analyses, respectively. SSc cultured fibroblasts with increased levels of HSP47 mRNA and protein showed high expression of type I procollagen. By in situ hybridization, SSc skin had a higher number of fibroblasts with high HSP47 and procollagen alpha1(I) mRNA levels than normal skin, and the distribution of HSP47 mRNA was similar to that of procollagen alpha1(I) mRNA. We also investigated the effects of cytokines on the expression of HSP47 in normal cultured fibroblasts. Transforming growth factor-beta1 and interleukin-4 increased HSP47 mRNA and protein levels, whereas interferon-gamma reduced HSP47 expression. The same pattern of cytokine-regulated expression was observed for type I procollagen levels. These results indicate that HSP47 expression is closely associated with that of type I procollagen in skin fibroblasts, and that increased expression of HSP47 may be involved in the abundant production of type I procollagen by SSc fibroblasts.

Cells, Cultured↗
PubMed1997

Cleavage of type I procollagen by human mast cell chymase initiates collagen fibril formation and generates a unique carboxyl-terminal propeptide.

The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 microg/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-alpha1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibril-forming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.

Amino Acid Sequence↗
PubMed2000

Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae.

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.

Biopolymers↗
PubMed1995

Immunohistochemical and mutation analyses demonstrate that procollagen VII is processed to collagen VII through removal of the NC-2 domain.

Collagen VII is the major structural constituent of anchoring fibrils in the skin. It is synthesized as a procollagen that is larger than the collagen deposited in the tissue. In this study, we investigated the conversion of procollagen VII to collagen VII in human skin and in cutaneous cells in vitro and identified the propeptide using domain-specific antibodies. For this purpose, two bacterial fusion proteins containing unique sequences of the carboxy-terminal globular NC-2 domain of procollagen VII were prepared, and polyclonal antibodies raised against them. Immunoblotting showed that the anti-NC2 antibodies reacted with procollagen VII isolated from cultured keratinocytes, but not with collagen VII extracted from the skin. Immunohistochemical experiments with the NC-2 antibodies revealed a strong reaction in cultured keratinocytes, but the basement membrane zone of normal skin remained negative. The staining could not be rendered positive by chemical or enzymatic unmasking of potential hidden epitopes in the skin, indicating that most of the NC-2 domain is absent from normal skin. In contrast, a positive staining with NC-2 antibodies was observed in the skin of a patient with NC-2 antibodies was observed in the skin of a patient with dystrophic epidermolysis bullosa, who carried a 14-bp deletion at one of the intro-exon junctions of the collagen VII gene. This aberration led to an in-frame skipping of exon 115 from the mRNA and eliminated 29 amino acids from the NC-2 domain which include the putative cleavage site for the physiological processing enzyme, procollagen C-proteinase. The results indicate that in normal human skin, the removal of the NC-2 domain from procollagen VII precedes its deposition at the dermal-epidermal junction. Furthermore, they suggest that an aberration in the procollagen VII cleavage interferes with the normal fibrillogenesis of the anchoring fibrils.

Amino Acid Sequence↗
PubMed1996

Effect of low-power laser irradiation on procollagen synthesis in human fibroblasts.

The conflicting views of the effect of low-power laser (LPL) irradiation on procollagen synthesis have existed at the present time, whereas many clinical studies have tested usefulness of LPL irradiation for the wound healing. To evaluate the effect of LPL irradiation on the procollagen synthesis of human fibroblasts in vitro, LPL irradiation on human fibroblast was carried out using two different culture medium, serum-starved medium and fetal calf serum (FCS)-contained medium. In addition, to investigate the mechanism of the LPL on the procollagen synthesis of human fibroblasts, dexamethasone and methylene blue contained medium were used for inhibition of procollagen product at the pretranslational level and cGMP-mediated processes, respectively. Enhanced effect of LPL was consistently observed in the serum-starved medium (50% increase by a 3 min irradiation), not in the FCS-contained medium. The LPL enhanced effect was not blocked by dexamethasone (3% inhibition) but methylene blue (40% inhibition). Our data suggest that some factors in FCS might interfere with the enhanced effect of LPL on procollagen synthesis and the LPL might act as a direct stimulator of the procollagen synthesis. It seems probable that the LPL enhanced effects might be occurred at the translational level or at the pretranslational level, which is not affected by dexamethasone and cGMP, might be involved in the LPL enhanced effect of the procollagen synthesis in fibroblast.

Cells, Cultured↗
PubMed1987

Collagen gene expression in keloids: analysis of collagen metabolism and type I, III, IV, and V procollagen mRNAs in keloid tissue and keloid fibroblast cultures.

Regulation of collagen gene expression was studied in keloids and fibroblast cultures established from keloid biopsies from 9 patients. The collagen concentration in keloid tissue was not different from that in normal skin. The activities of 2 enzymes catalyzing intracellular collagen biosynthesis, prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT) were significantly elevated in the keloids, the mean increase in the former enzyme being 5-fold and in the latter 3-fold with respect to the controls. The mean procollagen production rate in the keloid fibroblasts was at the control level, with only 1 keloid cell line showing a procollagen synthesis rate higher than the mean value + 2 SD of the controls. The mean PH and GGT activities of the keloid fibroblasts were not elevated, but PH activity in 2 cell lines and GGT activity in 1 cell line were higher than the mean + 2 SD for the controls. Cellular type I, III, IV, and V procollagen mRNAs were measured by slot blot hybridization using specific human cDNA clones for the various collagen types. The amounts of type I, III, and V procollagen mRNAs corresponded to the ratios in which these collagen types are produced by fibroblasts. No synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. The total amount of type I and III procollagen mRNAs correlated significantly (p less than 0.01) with the procollagen synthesis rate measured after radioactive labeling of the cells in the keloid and control fibroblasts, indicating that collagen production in these cells is mainly controlled by regulating the final steady state levels of collagen mRNA. The results suggest that fibroblasts isolated from keloids often synthesize normal amounts of collagen.

Cells, Cultured↗
PubMed1976

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.

Amino Acid Sequence↗
PubMed1983

A monoclonal antibody specific for the amino terminal cleavage site of procollagen type I.

A monoclonal mouse IgG1 antibody was produced against the aminopropeptide of dermatosparactic sheep procollagen type I by using the hybridoma technique. Radioimmunoassays demonstrated an apparent affinity constant of 10(8) l X mol-1. The antibody reacted with a 19-amino-acid-long sequence spanning the procollagen N-proteinase cleavage site with stronger binding to structures contributed by the aminopropeptide. The antibody showed strong cross-reactions with similar antigens of bovine, human or chick origin but failed to react with the aminopropeptide of procollagen type III. Incubation of chick or sheep procollagen type I with stoichiometric amounts of antibody blocked the release from procollagen molecules of the aminopropeptide by procollagen N-proteinase. Thus, this antibody seems useful for studying various biological problems encountered in the conversion of procollagen.

Animals↗
PubMed1984

Procollagen is more stable in cellulo than in vitro.

The thermal denaturation of both intracellular and freshly secreted chick embryo tendon type I procollagen was investigated using susceptibility to proteolysis by trypsin and chymotrypsin as a probe for triple-helical conformation. Freshly secreted procollagen from the medium of matrix-free tendon cells in suspension or procollagen within the cells and in the pericellular environment melted at 45 degrees C. In contrast, if freshly secreted procollagen was subjected to the melting procedure after dialysis of the medium against 0.4 M NaCl, 0.1 M Tris HCl, pH 7.4 the protein melted at 42 degrees C, the melting temperature of purified procollagen dissolved in the same buffer. In each of these cases, the thermal denaturation profile was narrow, with a width of 1.0-1.5 degrees C. These results demonstrate that, in situ, procollagen is more stable toward thermal denaturation than was previously thought. This extra margin of thermal stability partially resolves the dilemma of how tissues are able to assemble triple-helical procollagen molecules at body temperatures that closely approach the melting temperature of the purified protein.

Animals↗
PubMed1986

Incorporation of sulphate into type III procollagen by cultured human fibroblasts. Identification of tyrosine O-sulphate.

Confluent cultures of normal human skin fibroblasts were labelled overnight with [35S]sulphate, and the incorporation of the isotope into type III procollagen, secreted into the medium, was verified by radioimmunoassay and immunoprecipitation after removing the heavily sulphated proteoglycans by anion-exchange chromatography. Type III procollagen and its pro and pN alpha chains were visualized in fluorographs of the immunoprecipitates. The labelled procollagen could be isolated by a combination of ion-exchange chromatography and gel filtration and was found to contain tyrosine O-sulphate, which was identified by thin-layer electrophoresis after Ba(OH)2 hydrolysis. The regions sulphated in the type III procollagen molecule were susceptible to pepsin digestion. Digestion with purified bacterial collagenase at +37 degrees C produced a labelled fragment that was recognized by antibodies against the aminoterminal propeptide of type III procollagen, indicating that the sulphated tyrosine residues are located either in this propeptide or in the non-helical telopeptide region of the type III collagen molecule proper. Sulphation of tyrosine residues is a new post-translational modification in procollagen, which could be involved in the regulation of the processing of type III procollagen into collagen and thus affect the formation of collagen fibres.

Cells, Cultured↗
PubMed2000

Procollagen IIC-peptide as a marker for assessing mechanical risk factors of knee osteoarthritis: effect of obesity and varus alignment.

OBJECTIVE: To ascertain by cross sectional examination whether the concentration of procollagen IIC-peptide in joint fluid significantly correlates with mechanical risk factors of knee osteoarthritis (OA), such as obesity (body mass index) and varus alignment (lateral femorotibial angle). METHODS: The concentrations of procollagen IIC-propeptide in synovial fluid were measured by a sandwich enzyme immunoassay of 65 patients with the same radiological stage of primary knee OA-that is, Ahlbäk stage I. The relations between procollagen IIC-peptide and body mass index and lateral femorotibial angle were examined using simple regression analysis and multiple regression analysis. RESULTS: Significant positive correlations were found between procollagen IIC-propeptide concentrations and body mass index (r=0.479, p<0.0001), and between procollagen IIC-propeptide concentrations and lateral femorotibial angle (r=0.375, p=0.0021). Significant correlations were also found by multiple regression analysis. The multiple correlation coefficient of body mass index and femorotibial lateral angle to the procollagen IIC-propeptide concentrations was 0.547 (p<0.0001). CONCLUSIONS: The findings suggest that synthesis of type II collagen by chondrocytes is enhanced by abnormal mechanical stress, in this case obesity and varus alignment. It is concluded that procollagen IIC-propeptide concentrations in joint fluid are a useful marker of early OA.

Biomarkers↗
PubMed1994

Cadmium selectively inhibits fibroblast procollagen production and proliferation.

Chronic inhalation of cadmium fumes has been associated with the development of emphysema, a disease characterized by extensive disruption of lung connective tissue. Cadmium is also an important contaminant of tobacco and may play a role in cigarette smoking-related emphysema. In this paper we investigated the effect of nontoxic doses of cadmium chloride (CdCl2) on fibroblast procollagen production and proliferation, key features of connective tissue repair following injury. CdCl2 inhibited fibroblast procollagen production in a dose-dependent manner in two different cell lines. For fetal rat fibroblasts, maximal effects were observed at 10 microM CdCl2, with values reduced by 82 +/- 6% (mean +/- SE, n = 6, P < 0.01) relative to control cells. In contrast, noncollagen protein synthesis by these cells was enhanced in the presence of CdCl2. In human fetal lung fibroblasts (HFL1), maximal inhibition of procollagen production (83 +/- 2%, P < 0.01) was observed at 40 microM CdCl2, whereas noncollagen protein synthesis was unaffected. In both cell lines the inhibition of procollagen production was shown to be due to decreased procollagen synthesis and an increase in the proportion of newly synthesized procollagen degraded. Cadmium also affected fibroblast proliferation in response to 2% serum, with values for fetal rat cells depressed by 17 +/- 4, 72 +/- 2, and 86 +/- 4% (all P < 0.01) compared with controls at 1, 5, and 10 microM CdCl2, respectively. These data show that cadmium selectively inhibits fibroblast procollagen production and also attenuates their mitogenic response to serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

c-Jun-dependent inhibition of cutaneous procollagen transcription following ultraviolet irradiation is reversed by all-trans retinoic acid.

The aged appearance of skin following repeated exposure to solar ultraviolet (UV) irradiation stems largely from damage to cutaneous connective tissue, which is composed primarily of type I and type III collagens. We report here that a single exposure to UV irradiation causes significant loss of procollagen synthesis in human skin. Expression of type I and type III procollagens is substantially reduced within 24 hours after a single UV exposure, even at UV doses that cause only minimal skin reddening. Daily UV exposures over 4 days result in sustained reductions of both type I and type III procollagen protein levels for at least 24 hours after the final UV exposure. UV inhibition of type I procollagen synthesis is mediated in part by c-Jun, which is induced by UV irradiation and interferes with procollagen transcription. Pretreatment of human skin in vivo with all-trans retinoic acid inhibits UV induction of c-Jun and protects skin against loss of procollagen synthesis. We have reported previously that UV irradiation induces matrix-degrading metalloproteinases in human skin and that pretreatment of skin with all-trans retinoic acid inhibits this induction. UV irradiation, therefore, damages human skin connective tissue by simultaneously inhibiting procollagen synthesis and stimulating collagen breakdown. All-trans retinoic acid protects against both of these deleterious effects and may thereby retard premature skin aging.

Dose-Response Relationship, Radiation↗
PubMedDate not supplied

Expression of Sox9 and type IIA procollagen during ocular development and aging in transgenic Del1 mice with a mutation in the type II collagen gene.

PURPOSE: To study the expression and distribution of transcription factor Sox9 and type IIA procollagen in the developing and aging eyes of normal and transgenic Dell mice carrying pro(alpha)1(II) collagen transgenes with a short deletion mutation, which cause ocular abnormalities in this mouse line. METHODS: The eyes of Del1 mice were studied on embryonic days E14.5, E16.5 and E18.5, and at the ages of 4 and nine months, using their nontransgenic littermates as controls. Sox9 and pro(alpha)1(IIA) collagen were detected by RNase protection assay and immunohistochemistry. RESULTS: RNase protection assay revealed Sox9 transcripts in the eyes of Del1 and control mice during development and aging. The mRNA for type IIA procollagen had a similar temporal expression pattern. On embryonic days E14.5, E16.5 and E18.5, Sox9 was located by immunohistochemistry in the nuclei and type IIA procollagen in the extracellular space of the developing retina. During growth and aging, the ocular expression of Sox9 mRNA and the immunohistochemical reaction for Sox9 antibody diminished, concomitant with the reduction in type II procollagen mRNA. However, at the age of nine months, levels of Sox9 and type IIA procollagen mRNAs were higher in the degenerating eyes of Del1 and control mice. CONCLUSIONS: The similarities in the temporo-spatial distribution of Sox9 and type IIA procollagen suggest that this transcription factor is involved in the activation of type II collagen expression in the eye, as has been demonstrated in prechondrogenic mesenchyme and immature cartilage. The increased production of Sox9 and type IIA procollagen in the aging retina and vitreous is analogous to degenerating articular cartilage where attempted tissue repair has also been observed.

Aging↗
PubMed1988

Procollagen intermediates during tendon fibrillogenesis.

The purpose of this study was to correlate ultrastructural features of tendon collagen fibrils at various stages of development with the presence of procollagen, pN-collagen, pC-collagen, and the free amino propeptides and carboxyl propeptide of type I procollagen. Tendons from 10-, 14-, and 18-day chicken embryos reveal small, well-defined intercellular compartments containing collagen fibrils with diameters showing a unimodal distribution. At 21 days (hatching) and 9 days (post hatching) and at 5 weeks (post hatching), the compartments are larger, less well-defined, and there is multimodal distribution of tendon fibril diameters. Procollagen and the intermediates pN-collagen and pC-collagen are present in tendons up to 18 days. Thereafter there is a marked reduction in procollagen, whereas the intermediates persist throughout all stages of development. Similarly, free amino propeptides and carboxyl propeptides of type I procollagen were found at all stages. The amino propeptide of type III procollagen was restricted to the peritendineum until 7 weeks post hatching. At that time, a network of fibrils containing the amino propeptide of type III procollagen was seen delineating well-circumscribed compartments of collagen fibrils throughout the entire tendon. This study supports the notion that pN- and pC-collagen have an extracellular role and participate in collagen fibrillogenesis.

Animals↗
PubMed1996

Evolution of circulating C-terminal propeptide of type I procollagen in patients with chronic renal failure pre and post renal transplantation.

The carboxyterminal propeptide of type I procollagen is a biochemical marker of type I collagen synthesis. We evaluated circulating carboxyterminal propetide of type I procollagen levels in patients with terminal renal failure before and after kidney transplantation. Serum carboxyterminal propeptide of type I procollagen, osteocalcin, total alkaline phosphatase, intact parathyrin, creatinine, calcium and phosphate levels were determined in 20 patients, before and 15, 30, 90 and 180 days after surgery. Serum creatinine and intact parathyrin concentrations showed a significant decrease after kidney transplantation. Immunosuppressive treatment consisted of low dose prednisone, cyclosporin and antilymphoblast globulin. In our group, only 5 patients (25%) showed serum carboxyterminal propeptide of type I procollagen levels higher than normal before kidney transplantation. At 15 and 30 days, carboxyterminal propeptide of type I procollagen concentrations showed a decrease, while at 90 and 180 days there was a significant increase above the normal range (p = 0.006; ANOVA). Serum osteocalcin and total alkaline phosphatase levels increased significantly at the same time. We found a significant correlation between carboxyterminal propetide of type I procollagen and osteocalcin at 15 and 30 days after kidney transplantation. We conclude that the significant increase in carboxyterminal propeptide of type I procollagen levels after kidney transplantation reflect an increase in bone turnover. The low doses of steroids employed do not seem to have a significant inhibitory effect on collagen synthesis.

Adolescent↗
PubMed1990

Cyclosporin A stimulates transcription and procollagen secretion in tubulointerstitial fibroblasts and proximal tubular cells.

The brief study described in this report was undertaken to determine whether cyclosporin A had any direct effect on the expression of tubulointerstitial procollagens in cultured renal cells. Our findings indicate that murine tubulointerstitial fibroblasts secreted significantly more procollagen type I after the addition of cyclosporin A, whereas syngeneic proximal tubular cells expressed significantly more types I and IV procollagen after cyclosporin stimulation. These increases in procollagen gene product correlated concordantly with changes in the levels of cytoplasmic mRNA with procollagen-specific cDNA probes. Transfection of these fibroblasts and proximal tubular cells with chimeric gene constructs containing enhancer/promoter elements for alpha2(I) and alpha 1(IV) procollagen linked to a chloramphenicol acetyltransferase reporter gene indicates that the stimulatory effect of cyclosporin on procollagen expression depends, at least to some extent, on an increase in transcriptional activity.

Animals↗
PubMed1998

[Study on in situ expression of type I and III procollagen mRNAs in different parts of keloid and normal skin].

OBJECTIVE: Based on the histomorphologic differences of three parts of keloid, i.e., invasive, proliferative and senile regions, the study was carried out to explore the differences of expression of type I and type III procollagen mRNAs in the three different parts of keloid and normal skin. METHOD: Four samples of keloid from four patients and two samples of normal skin were collected. The expression of pro alpha 1 (I) and pro alpha 1 (III) procollagen mRNAs in keloid and normal skin was investigated by in situ hybridization techniques. RESULT: Both type I and III procollagen mRNAs levels increased in keloid, especially type I, resulting in elevated ratios of type I to III procollagen mRNA. In keloid, expressive intention of type I and type III procollagen mRNAs was higher in the papillary layer than in the reticular layer. Also the expressive intention of type I and type III in keloid was higher in invasive and proliferative regions than in the senile region. However, expressive intention of type I and type III procollagen mRNAs in the invasive region of keloid was similar to that in the proliferative region. CONCLUSION: The differences of expression of type I and type III procollagen mRNAs in different pathological regions of keloid and normal skin is one cause of forming different pathological regions in keloid.

Adult↗