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PubMed1987

An immunohistochemical and serum ELISA study of type I and III procollagen aminopropeptides in primary biliary cirrhosis.

By means of ELISA methodology, the aminopropeptides of Type I and Type III procollagen were measured in the serum of a group of patients with primary biliary cirrhosis. The corresponding liver biopsies were graded blindly for degrees of fibrosis and inflammation. When available, paraffin-embedded liver specimens underwent immunoperoxidase staining for mature Type I and III collagen as well as the aminopropeptides of Type I and III procollagen. Regardless of the degree of fibrosis or inflammation, serum levels of the aminopropeptide of Type I remained within normal limits. In contrast, serum levels of the aminopropeptide of Type III procollagen were elevated uniformly. Immunohistochemistry demonstrated that the aminopropeptide of Type III procollagen persists extracellularly. This finding may explain the previously reported relationship between levels of inflammation and serum levels of the Type III aminopropeptide.

Antibody Specificity↗
PubMed1987

Regulation of types I, III, and IV procollagen mRNA synthesis in glucocorticoid-mediated intestinal development.

Administration of dexamethasone (0.8 mg/kg) to 9-day-old rats once daily for 3 consecutive days caused precocious induction of adult specific disaccharidase activity in the small intestine. Maturation-specific disaccharidase activity was accompanied by decreased amounts of types I and III collagen and decreased procollagen type I and III mRNA levels. Conversely, type IV procollagen, fibronectin, and laminin amounts and their respective mRNA levels were increased. In vitro transcription of nuclei isolated from small intestine and colon of suckling rats indicated a decreased rate of synthesis of procollagen types I and III mRNAs and an increased rate of synthesis of procollagen type IV mRNAs and laminin mRNAs after dexamethasone treatment. The data suggest that glucocorticoids mediate a differential regulation of interstitial and basement membrane collagen gene expression in the developing rat intestine.

Animals↗
PubMed1986

Evidence for pretranslational regulation of collagen synthesis by procollagen propeptides.

We present, here, evidence for a pretranslational role of procollagen propeptides in the regulation of collagen synthesis. Amino- and carboxyl-terminal type I procollagen propeptides were isolated and purified from chick calvaria and tendon cultures. Human lung fibroblasts (IMR-90) were incubated in medium containing varying concentrations of propeptides. Amino-propeptides at 10 nM caused an 80% decrease in collagen synthesis compared to control. Higher concentrations of amino-propeptides did not decrease collagen synthesis further and no significant effect on non-collagen synthesis was found throughout the entire concentration range. Carboxyl-propeptides also inhibited collagen synthesis. At 10 nM, collagen synthesis was decreased by 30% and a concentration of 40 nM caused an 80% reduction. However, at the latter concentration non-collagen synthesis was also affected, decreasing by 20% relative to control. To assess possible pretranslational effects of propeptides, IMR-90 fibroblasts were treated with varying concentrations of each propeptide and levels of type I procollagen mRNA was determined by dot hybridization with a 32P-alpha 2(I) cDNA probe. Both propeptides caused significant concentration-dependent decreases in procollagen type I mRNA levels. At 10 nM, the amino-propeptide resulted in a 55% decrease in collagen mRNA levels while at 40 nM these levels decreased by 72% compared to control. Carboxyl-propeptides were also inhibitory, decreasing mRNA levels by 33% at 10 nM and 73% at 40 nM. Messenger RNA levels of a representative noncollagenous protein, beta-actin, were unaffected by either propeptide throughout the concentration range.

Cell Line↗
PubMed1979

Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures.

NH2-terminal extension peptides of type I and type III procollagens were isolated from dermatosparactic and normal fetal calfskin, respectively. Cell culture experiments showed that the globular domains of the tested procollagen peptides were biologically active but that peptides from the helical region of collagen had no effect. The peptides were added to the incubation medium of calf fibroblasts along with radioactive precursor amino acids, and the amount of newly synthesized collagen was determined. The experiments indicated that procollagen peptides exerted a feedback-like inhibitory effect specific for the synthesis of collagen. Neither degradation of collagen, hydroxylation of collagen alpha chains, nor synthesis of noncollagenous proteins were affected. Synthesis of type II collagen by calf chondrocytes was not reduced. In addition, it was shown that procollagen peptides from calf were equally effective when added to human fibroblast cultures, an observation that could be of considerable medical interest.

Animals↗
PubMed1981

Coordinate transcriptional regulation of type I procollagen genes by Rous sarcoma virus.

Chicken embryo fibroblasts infected with a strain of Rous sarcoma virus containing a temperature-sensitive mutation in the gene coding for pp60src, a protein kinase, undergo changes in collagen synthesis within 4 h after a temperature shift. Cells shifted from the restrictive to the permissive temperature for transformation show decreasing levels of collagen synthesis and increasing levels of kinase activity; the reverse occurs when infected cells are shifted from the permissive to the restrictive temperature. Levels of type I procollagen mRNAs coding for pro alpha 1 and pro alpha 2 chains, measured by hybridization to nick-translated cloned alpha 1 and alpha 2 cDNA, decreased simultaneously soon after a reduction in temperature and reached a new steady state at about 50 h after the shift. In order to test for regulation at the transcriptional level, nuclei were isolated from normal and Rous sarcoma virus-transformed chicken embryo fibroblasts and allowed to transcribe in the presence of [alpha-32P]UTP. Procollagen mRNA sequences in newly synthesized and in total RNA from transformed cell preparations were both about 5-fold lower than the levels in normal cell preparations. We conclude that the coordinate decrease in procollagen mRNAs observed in Rous sarcoma virus-transformed chicken embryo fibroblasts is caused primarily by a decrease in the transcription of the type I procollagen genes, a decrease which is directly or indirectly mediated by the pp60src protein kinase.

Animals↗
PubMed1981

Correlation of collagen synthesis and procollagen messenger RNA levels with transformation in rat embryo fibroblasts.

A line of normal rat embryo fibroblasts was transformed with N-methyl-N'-nitro-N-nitrosoguanidine (a chemical carcinogen), SV40 and polyoma virus (two DNA viruses), and Rous sarcoma virus (an RNA tumor virus). In this study, we report a comparison of the levels of collagen synthesis and procollagen messenger RNA (mRNA) in 13 lines selected after transformation with one of these agents. Collagen synthesis and procollagen mRNA levels were compared with the degree of transformation determined from morphology, saturation density, growth in agarose, and tumorigenicity in nude mice. Each class of transformants had a characteristic level of collagen synthesis; this level correlated inversely with the degree of transformation of the rat embryo fibroblasts. In N-methyl-N'-nitro-N-nitrosoguanidine and SV40 transformants which were moderately transformed, collagen synthesis was hardly affected, but, in polyoma virus and Rous sarcoma virus transformants which were more severely transformed, collagen synthesis was 30 to 48% and 12 to 25%, respectively, of control levels. Type I procollagen mRNA activity measured in RNA from nine of the lines by an in vitro translation assay also decreased with increasing severity of transformation. Procollagen mRNA levels were reduced to about one-half of control levels in one SV40 transformant and to 17 to 23% of controls in polyoma virus and Rous sarcoma virus transformants. We conclude that, in this series of rat fibroblast lines, transformation with different agents resulted in characteristic levels of collagen synthesis and that collagen synthesis was most reduced in the cells which were most transformed by other criteria.

Animals↗
PubMed1982

Biosynthesis of procollagens and collagens by tissue explants and matrix-free cells from embryonic chick cornea.

Tendon and cornea of 17-day-old chick embryos primarily synthesize type I collagen as the major collagenous component nd type V collagen as a minor collagenous component. Matrix-free cells isolated from cornea and tendon synthesize and secrete mainly type I procollagen. A short lag period for the secretion of collagenous polypeptides into the medium of cell suspensions is observed. There is no significant difference in the synthesis and secretion of type I procollagen between matrix-free cells from tendon or cornea, except that cornea cells contain more free pro alpha 2(I) chains. The pro alpha 2(I) chain does not contain interchain disulfide linkages and can be separated from type I procollagen by ion exchange chromatography. However, no free pro alpha 2(I) chain can be detected in the medium of cell suspensions, suggesting that free pro alpha 2(I) is probably not secreted normally and may be rapidly degraded intracellularly. These results suggest that proper chain composition of type I procollagen is not regulated at the transcriptional or translational levels. Rather it is determined by the primary structure of the pro alpha chains and/or a posttranslational factor.

Animals↗
PubMed1982

Purification and characterization of procollagen mRNA from a heavy polysomal fraction.

A method is described for the purification of intact procollagen mRNA from heavy polysomal fractions isolated from 10 day-old chick embryos. Procollagen mRNA activity was characterized by gel electrophoresis under denaturing conditions and by its activity in in vitro cell-free translation systems derived from wheat germ and rabbit reticulocytes. 7-Methyl-guanosine monophosphate inhibited the translation of procollagen mRNA and globin mRNA to the same extent. Collagenase-sensitive polypeptides synthesized displayed the same electrophoretical mobilities as marker procollagen chains. The purity of the mRNA was 78% on the basis of ROt plot analysis of hybridization with its corresponding cDNA, using globin cDNA as the reference for 100%.

Animals↗
PubMed1981

Assembly and processing of procollagen V (AB) in chick blood vessels and other tissues.

The biosynthesis and processing of type V procollagens was investigated in chick embryo blood vessels labeled with radioactive amino acids. Monomeric, pepsin-sensitive pro alpha 1 V and pro alpha 2 V chains are slowly assembled into triple helically folded molecules. A small proportion of these procollagen V molecules contain interchain disulfide bridges, and the disulfide-linked heterodimer and heterotrimer (pro alpha 1 V)2pro alpha 2 V were found. A relatively fast conversion of procollagen V to p-collagen V is followed by slow change to collagen V. This time course is similar to the processing of procollagen III and is much slower than the rate of appearance of type I collagen in blood vessels. A combination of sedimentation and electrophoretic analyses was used to measure the relative size of the type V chains and to demonstrate attachment of noncollagenous peptides (Mr = 33,000) to p alpha 2 V by disulfide linkage. Similar quantitative pulse-chase studies were made with calvaria and crop. As the same unusual features of assembly and processing of type V chains were seen in muscle, bone, and blood vessels, we conclude that these are characteristic of type V collagen biosynthesis.

Animals↗
PubMed1993

[Identification of procollagen mRNA in chronic active hepatitis B].

In situ hybridization is a tool for staining intracellular procollagen mRNAs with specific probes. Our study shows the amounts of procollagen mRNAs of types I and III to be increased in liver biopsies of five patients with chronic active hepatitis B as compared with five healthy controls. Parallel staining employing anti-smooth-muscle-actin antibodies was able to identify myofibroblast-like cells at the same localization where procollagen mRNAs were found. Consequently, these transformed Ito-cells might be the procollagen-producing cells.

Actins↗
PubMed1994

Increased type I procollagen mRNA transcripts in the lungs of mice during the development of bleomycin-induced fibrosis.

In this study, in situ hybridization has been used to investigate the localization of type I procollagen messenger ribonucleic acid (mRNA) in normal lung, and in the lungs of mice during the development of bleomycin-induced pulmonary fibrosis. Lung fibrosis was induced by a single intratracheal instillation of bleomycin sulphate (6 mg.kg-1 body weight), and tissues examined at times up to 35 days thereafter. Tissue transcripts of alpha 2(I) procollagen mRNA were visualized after hybridization with 35S-labelled riboprobes. Hybridization signals were associated with alveolar interstitial cells throughout the normal lung, with additional areas of dense hybridization signals observed subpleurally. Three days following administration of bleomycin, there was no apparent change in the pattern of hybridization. By 10 days, foci of intense hybridization signals indicative of gene activation were observed associated with individual cells in the alveolar interstitium. At 21 days, the increase in hybridization signals appeared to be associated with greater numbers of cells rather than highly activated cells. These results demonstrate that procollagen genes are normally expressed in the mouse lung, and that during bleomycin-induced pulmonary fibrosis hybridization signals increase, suggesting that both enhanced gene expression by individual cells and increased numbers of cells expressing the type I procollagen gene are involved in the pathogenetic mechanism.

Animals↗
PubMed1993

[Localization of vigilin and type I, II procollagen mRNAs in chick embryos by in situ hybridization].

Using a 35S-labeled RNA-RNA in situ hybridization technique, we found that vigilin mRNA was abundant in osteoblasts. It disappeared following the transition of osteoblasts to osteocytes. The amount of vigilin mRNA increased with proliferation and hypertrophy of chondrocytes. Fibroblasts contained moderate vigilin mRNA signals. Weak signals were observed in early chick embryos. Type I procollagen mRNAs were abundant in osteoblasts and fibroblasts of the periosteum, perichondrium, tendon and ligament. Type II procollagen mRNA was restricted to chondrocytes. Interestingly, a significant amount of pro alpha 1 (I) mRNA was observed in late proliferative and hypertrophic chondrocytes, which could not be blocked by an excess amount of unlabeled pro alpha 1 (II) antisense RNA. No signal was found in chondrocytes of human fetal cartilage. Vigilin mRNA had a similar distribution to type II procollagen mRNA in chondrocytes, and in other instances it showed a codistribution with type I procollagen mRNAs.

Animals↗
PubMed1993

Fetal antigen 2 (FA2): the aminopropeptide of the alpha 1 chain of human type I procollagen. A study on skin, tumour biology and bone metabolism.

Fetal antigen 2 (FA2) was purified from second trimester human amniotic fluid (AF) using immunospecific chromatography, gel filtration and reversed phase chromatography. Amino acid composition and sequence analyses of purified FA2 revealed identity to the amino acid sequence of the aminopropeptide of the alpha 1 chain of human type I procollagen. Gel filtration of AF as well as purified FA2 demonstrated two molecular forms of FA2, which during gel filtration were elucidated corresponding to MW of approximately 100 kDa and 30 kDa. The molecular forms were referred to as the high molecular component (FA2-HM) and low molecular component (FA2-LM), respectively. SDS-PAGE analysis (reducing and nonreducing conditions) showed a MW of 27 kDa for both FA2-HM and FA2-LM. Mass spectrometry analysis however revealed a MW of 14.343 +/- 3Da. Proteins with collagenous domains often reveal aberrant behaviour both in gel filtration and SDS-PAGE and this phenomenon may account for the apparent higher MW of FA2 observed using these techniques compared to mass spectrometry and amino acid composition/sequence analyses. A polyclonal, monospecific rabbit antibody raised against FA2 was used in immunohistochemical localization studies. Due to the obvious association of FA2 to the BM structure in adult and fetal skin, it was initially referred to as a BM associated component. However, FA2 was immunologically distinct from the well established BM components and moreover, the diffuse distribution of FA2 along the BM differed from linear localization of BM components (e.g. collagen type IV and laminin). The identification of FA2 as the aminopropeptide of the alpha 1 chain of human procollagen type I has the implication that in the immunohistochemical investigations the anti-FA2 may not distinguish between the free propeptide and procollagen type I. In the papillary dermal region of human adult skin type I collagen are arranged in loose fibrils to which the aminoterminal propeptide is still attached. Anti-FA2 may recognize epitopes in the type I procollagen present in this region, giving rise to the diffuse FA2 distribution along the BM. However, whether FA2 also appear as a free component in this region is unknown. In fetal skin where collagen is vividly layed down in the mesenchymal compartment staining with anti-FA2 revealed a diffuse mesenchymal distribution. Demonstration of FA2 within the cytoplasm of proliferating fibroblasts and diffusely in granulation tissue during wound healing suggest that FA2 was involved in the reparative processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1977

Impaired conversion of procollagen to collagen by fibroblasts and bone treated with tunicamycin, an inhibitor of protein glycosylation.

Tunicamycin, an inhibitor of lipid carrier-dependent protein glycosylation, was used in studies of procollagen synthesis, secretion, and proteolytic modification by chick cranial bones in organ culture and by chick tendon fibroblasts in tissue culture. Tunicamycin inhibited the incorporation of D-[2-3H]mannose into procollagen by greater than 90% whereas general protein synthesis and collagen synthesis were decreased by only 10 to 20%. The procollagen synthesized in the presence of tunicamycin was secreted normally and its immunological characteristics, as detected by an antiserum to the intact protein, were unchanged. However, tunicamycin caused an accumulation of biosynthetic intermediates containing disulfide-bonded COOH-terminal extensions in both cell and bone culture. Cleavage of NH2-terminal extensions was not detectably impaired. These findings provide additional support for the involvement of more than one enzyme in the limited proteolytic conversion of procollagen to collagen.

Amino Acids↗
PubMed1996

Localization of type I procollagen gene expression in silica-induced granulomatous lung disease and implication of transforming growth factor-beta as a mediator of fibrosis.

We have used the silica-induced model of pulmonary injury in the rat to study the pattern of collagen expression in granulomatous lung inflammation. A single intratracheal instillation of silica into adult rats resulted in granulomatous inflammation leading to fibrosis and alveolar proteinosis. The development of disease in these animals was characterized over a 27-day period after treatment by means of histological, biochemical, and molecular analyses. Biochemical analyses indicated that significant increases in the weights of silicotic lungs were due to elevated amounts of DNA and total protein. Analysis of hydroxyproline content showed a 15-fold increase in this amino acid in silicotic lungs, confirming the development of a fibrotic reaction. In situ hybridization for type I procollagen mRNA displayed increased gene expression in the parenchyma, conducting airways, and vasculature of silicotic rats. Within the parenchyma, type I procollagen was expressed uniquely within granulomatous lesions. Immunohistochemistry indicated type I procollagen was being expressed by an alpha-smooth muscle actin-negative population of cells. Immunolocalization of extra-cellular transforming growth factor-beta showed coincident temporal and spatial overlap with type I procollagen expression, implicating this cytokine as a mediator of collagen gene expression in this model.

Animals↗
PubMed1998

[Procollagen III in bronchoalveolar lavage fluid of patients with allergic alveolitis and sarcoidosis].

In 12 patients with sarcoidosis and 11 patients with allergic alveolitis concentration of procollagen III peptide in bronchoalveolar lavage fluid (in bronchial and alveolar fraction) was estimated using RIA method. In studied populations procollagen III levels were higher in comparison to control. In patients with allergic alveolitis and with DLCO < 60% pred. procollagen III peptide concentrations in BAL fluid were significantly higher than in patients with sarcoidosis. In patients with allergic alveolitis a positive correlation between BAL lymphocytes number and procollagen III peptide concentration was observed.

Adolescent↗
PubMed1985

Collagen biosynthesis in systemic scleroderma: regulation of posttranslational modifications and synthesis of procollagen in cultured fibroblasts.

Activities of prolyl hydroxylase (PH), lysyl hydroxylase (LH), and the collagen glycosyltransferases and the extent of the posttranslational modification of lysine residues in newly synthesized collagen were studied in fibroblast cultures obtained from 9 scleroderma patients. The rate of procollagen synthesis had increased more than 3-fold in 3 scleroderma fibroblast lines, but had not changed to the same extent in the others, even though these did not differ from the "high-producers" histologically, clinically, or immunohistologically. The activities of PH and LH correlated significantly with the rate of procollagen synthesis in the same cell lines (p less than 0.001), but the glycosyltransferase activities were not elevated in the scleroderma fibroblasts. Further studies nevertheless indicated that the extent of the posttranslational modification of lysine residues had not significantly changed in the procollagen synthesized by any of the scleroderma fibroblasts investigated.

Adult↗
PubMed2000

Design and synthesis of acidic dipeptide hydroxamate inhibitors of procollagen C-proteinase.

Procollagen C-proteinase (PCP) is essential for the cleavage of procollagen to collagen in the extracellular matrix of animals and is, therefore, of major relevance to studies of ectopic deposition of collagen during fibrosis. In this study, we describe the design and synthesis of acidic side chain hydroxamate dipeptide inhibitors of PCP having IC50 values in the range 0.1-10 microM that mimic the location of aspartic acid residues in the P1' and P2' positions (i.e. immediately C-terminal) of the PCP cleavage site in procollagen. Assays of PCP using purified human type I procollagen (a natural substrate of PCP) showed that the structure activity relationship of the inhibitors was improved with a glutamic acid mimic at the P1' position. The results also showed that the presence of an acidic side chain at the P2' position was not necessary for PCP inhibition. Marimastat and BB3103, which are highly effective inhibitors of matrix metalloproteinases and ADAMS proteinases, respectively, did not inhibit PCP.

Amino Acid Sequence↗