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PubMed1993

Myofibroblast-like cells produce mRNA for type I and III procollagens in chronic active hepatitis.

In chronic active hepatitis the rate of collagen biosynthesis is largely determined by intracellular mRNA concentrations. To localize procollagen mRNA-producing cells, we investigated biopsy specimens from five patients with hepatitis B surface antigen-positive chronic active hepatitis and five patients without liver disease by in situ hybridization. We used type I and III procollagen cDNAs for transcription to (35S)-labeled probes. Parallel sections were stained with anti-actin monoclonal antibodies. Our results show that cells in which collagen synthesis is ostensibly enhanced can be localized by in situ hybridization of procollagen mRNAs. These cells were also anti-actin-positive in parallel sections and were localized in areas of inflammatory cell infiltration and necrosis. We conclude that myofibroblast-like cells may express procollagen mRNAs in chronic active hepatitis. Moreover, in situ hybridization may be a valuable diagnostic tool for providing additional morphologic information on the degree of fibrogenesis activity.

Actins↗
PubMed2000

[Effect of lipids on I and III procollagen mRNA expression of hepatic stellate cells].

OBJECTIVE: To study the effect of lipids on I and III procollagen mRNA expression of hepatic stellate cells (HSC). METHODS: HSC were isolated and cultured from the liver of Wistar rats by in situ perfusion with pronase and collagenase and the density gradient centrifugation with Nycodenz. HSC were incubated with triglyceride (25mg/L) and very low-density lipid (VLDL, 25 mg/L) for 10d, respectively. I and III procollagen mRNA expression of HSC was detected by Northern blot hybridization. RESULTS: After HSC was stimulated with triglyceride and VLDL, the expression of I and III procollagen mRNA increased obviously. CONCLUSION: Lipids may promote the expression of I and III procollagen mRNA, and may be associated with fatty liver and hepatic fibrogenesis.

Animals↗
PubMed2000

Expression of procollagen alpha 1 type III mRNA in rotator cuff tears.

The purpose of this study was to investigate the relationship among expression of procollagen alpha 1 type III mRNA, subacromial adhesion, and limited shoulder elevation in rotator cuff tears. Procollagen alpha 1 type III mRNA was analyzed in the torn supraspinatus tendons and synovial tissue of 42 patients with rotator cuff tears. As a control, four normal supraspinatus tendons of patients who had undergone shoulder surgery other than rotator cuff repair served as controls. Cells stained positively for procollagen alpha 1 type III mRNA were more abundant in the adhesive synovium of the subacromial bursa and in the torn supraspinatus tendons than in controls. Patients with complete-thickness tears who had undergone surgery less than 10 months after trauma were found to have more signal-positive cells than those later than 10 months. We conclude that the adhesive synovium of the subacromial bursa contains abundant cells stained positively for procollagen alpha 1 type III mRNA. We suggest that the production of type III collagen by these cells could contribute to the limitation of shoulder motion in patients with rotator cuff tears, and recommend release and excision of the excess adhesive synovium during rotator cuff surgery.

Adult↗
PubMed2000

Gene expression of procollagen alpha 1 (I) and alpha 1 (III) in partial-thickness tears of the deep pectoral tendon in chickens.

The purpose of this study was to evaluate the healing process of partial-thickness tears of the deep pectoral tendon in chickens which is considered to be equivalent to the human rotator cuff. The superior side (bursal-side) and the inferior side (joint-side) layers of the deep pectoral tendon of 80 adult chickens were lacerated in the same manner. The mRNA expression levels of alpha 1 (I) and alpha 1 (III) procollagen was at a level 2 weeks after laceration. The mRNA expression level of procollagen alpha 1 (I) was significantly higher than that of procollagen alpha 1 (III) in all experimental periods (p<0.05). The expression levels of procollagen-mRNAs were constitutively higher in bursal-side tears than in joint-side tears throughout the experimental periods (p<0.05), even 52nd weeks after laceration. These observations indicate that the bursal-side layer possesses higher healing ability than the joint-side layer in the chicken's deep pectoral tendon where subacromial impingement is absent, and that the remodeling of partial-thickness tears in chicken models needs more than one year.

Animals↗
PubMed1999

Study of activity of promoter from mouse alpha 2(I) procollagen gene.

OBJECTIVE: To clarify the segment and sequence in mouse alpha 2(I) procollagen gene which are responsible for high transcriptional activity during fibrogenesis. METHODS: This study was focused on further fractional analysis of 2 kb-length mouse alpha 2(I) procollagen gene promoter activity. Six chimeric genes were constructed in which various lengths of sequences between 2000 bp upstream of the start of transcription of the mouse alpha 2 (I) procollagen gene and 54 bp downstream of this site were fused to chloramphenicol acetyltransferase (CAT) reporter gene. These recombinant plasmids were transfected transiently to collagen-producing cells (NIH/3T3) and non-collagen-producing cells (COS7) with liposomal transfection method. The activities of putative promoters were observed and compared by means of CAT measurement in the transfected cells. RESULTS: The highest and partial cell specific CAT expression was observed in construction driven by -780 to bp fragment. The construction containing sequence deleted the proximal 500 bp from the transcription start site and part of exon I, and resulted in the lowest CAT expression. CONCLUSIONS: Some essential elements might exist in the 500 bp fraction proximal to transcription start site and part of exon I in mouse alpha 2(I) procollagen gene. The high potential promoter sequence between -780 bp from the start of transcription site and bp from this site is of great significance in our following study of searching for specific DNA-binding proteins in activated collagen-producing cells.

3T3 Cells↗
PubMed1999

Effect of integrin on procollagen synthesis by fibroblasts from scleroderma.

OBJECTIVE: To investigate the effect of integrin on fibroblasts from scleroderma in the production of procollagen. METHODS: Phosphorothioate modified antisense oligonucleotides were used to interfere with the expression of integrin alpha 5 or beta 1 subunit on fibroblasts from 10 cases of scleroderma, and then the changes of procollagen mRNA due to the decline in level of integrin were observed by using RT-PCR. RESULTS: Expression of integrin alpha 5 or beta 1 subunit was specifically inhibited by their corresponding antisense oligonucleotides. Fibroblasts with decreased expression of alpha 5 or beta 1 subunit produced lower level of procollagen alpha 1 (I), alpha 1 (III) mRNA in comparison to the control. CONCLUSION: Overproduction of procollagen may be inhibited at the level of transcription by lowering the expression of integrin alpha 5 or beta 1 subunit on fibroblasts in scleroderma.

Adult↗
PubMed2002

[Construction and in vitro activity of specific dual-ribozyme against alpha 1 (I) and (III) procollagen genes].

Overexpression of procollagen gene can cause the extraordinary increase of collagen's synthesis and therefore lead to the keloid and hypertrophic scar. To utilize ribozyme to suppress the expression of procollagen genes, a eukaryotic expression recombinant plasmid containing a dual-ribozyme gene against alpha 1 (I) and alpha 1 (III) procollagen genes was constructed. The ribozyme from in vitro transcription was incubated with target transcripts from recombinant plasmids which separately contained the fragments of the second exons of pro alpha 1 (I) and pro alpha 1 (III) collagen genes under various experimental conditions. The results showed that the dual-ribozyme could efficiently catalyze the specific cleavage of the target RNAs at 37 degrees C, 42 degrees C, 50 degrees C and Mg2+ concentration from 10 mmol/L to 20 mmol/L. This work provided a basis for further study on the ribozyme to suppress the expression of procollagen genes and control the cicatrization.

Base Sequence↗
PubMedDate not supplied

Liver stellate cells and aminoterminal peptide of type III procollagen in chronic hepatitis C treated with interferon.

BACKGROUND/AIMS: Fibrogenesis plays a crucial role in development of cirrhosis, and liver stellate cells, activated to myofibroblasts expressing alpha-smooth muscle actin, are responsible for deposition of fibrous matrix; aminoterminal peptide of type III procollagen is a serum marker of active fibrogenesis. Interferon can slow ongoing fibrogenesis in chronic viral hepatitis, but it remains unclear whether the drug acts by a direct effect on stellate cells or by inhibiting the necro-inflammatory process. The aim of this study was to evaluate, in selected cases of chronic hepatitis C, whether changes in stellate cell expression induced by interferon correlated with changes in serum levels of procollagen or with clinical response to the therapy, in order to further investigate the mechanism of interferon's effect on fibrogenesis. METHODOLOGY: We studied 30 patients with chronic hepatitis C, treated with interferon and followed for more than 6 months. Before and after-treatment evaluation included histological scores for portal activity, lobular activity and fibrosis; immunohistochemical scores for alpha-actin expression by activated stellate cells in liver biopsy; and radioimmunoassay for procollagen in serum. According to the clinical response to interferon, the patients were subdivided into sustained responders, delayed relapsers, early relapsers and non-responders. RESULTS: We found that alpha-actin scores, portal and lobular activity scores and procollagen levels were all significantly lower after the treatment in responder patients, whereas in non-responders the after-interferon values were not different from the basal values. Moreover we found that the patients who were still clinical responders at the time of after-therapy evaluation, but relapsed subsequently (delayed relap-sers), still showed a pattern of "low fibrogenesis", like the sustained responders. On the contrary, the patients who had already relapsed at the time of after-interferon evaluation (early relapsers) showed a pattern of "high fibrogenesis", like the non-responders. CONCLUSIONS: Since all the patients had received a similar treatment for a similar period, our results suggest that fibrogenesis activity in a defined time is related to the clinical response present in that time, and is influenced by short-term variations in necro-inflammatory activity induced by interferon, rather than by interferon itself.

Actins↗
PubMed1991

Transgenic mice that express a mini-gene version of the human gene for type I procollagen (COL1A1) develop a phenotype resembling a lethal form of osteogenesis imperfecta.

A mini-gene version of the human gene for a pro-alpha 1(I) chain of type I procollagen (COL1A1) was prepared that contained -2.5 kilobases of the promoter region and the 5'- and 3'-ends of the gene but lacked a large central region containing 41 exons. The construct was modeled after a sporadic in-frame deletion of the human gene that produced a lethal variant of osteogenesis imperfecta, because it caused synthesis of shortened pro-alpha 1(I) chains that associated with normal pro-alpha 1(I) and pro-alpha 2(I) chains and caused degradation of both the shortened and normal pro-alpha chains through a process called procollagen suicide. The mini-gene was used to prepare transgenic mice. Eight of 15 transgenic mice expressed varying levels of the gene. All except one of the Fo founders were phenotypically normal, but several of the founders were apparently mosaic since they produced F1 progeny that died shortly after birth with a distinctive phenotype. The phenotype included extensive fractures of ribs and long bones similar to the fractures seen in lethal variants of osteogenesis imperfecta. Mice with the lethal phenotype expressed much higher levels of the mini-gene than transgenic mice without the lethal phenotype. Experiments with cultured skin fibroblasts from the transgenic mice demonstrated that shortened pro-alpha 1(I) chains synthesized from the mini-gene became disulfide-linked to pro-alpha 1(I) chains synthesized from the endogenous mouse gene. The results demonstrate that a mutated type I procollagen gene based on the model of procollagen suicide can be used to produce a severe phenotype of osteogenesis imperfecta that is genetically transmitted.

Animals↗
PubMed1990

Another mechanism for the defect in type III collagen accumulation in Ehlers-Danlos syndrome type IV: increased intracellular degradation of the procollagen.

The nature of type III collagen was examined in the skin and cultured skin fibroblasts from a patient with Ehlers-Danlos syndrome type IV. Although the culture medium contained a much lower amount of Type III collagen than the controls, the cells contained an apparently normal amount of Type III collagen. The patient's Type III procollagen showed no abnormalities in apparent molecular weight, the peptide length as examined by cyanogen bromide cleavage, the genomic DNA size including its C- and N-propeptide portion, mRNA size, or thermal stability; but a pulse-chase study revealed prolonged retention of the type III collagen in the cells. Degradation of Type III procollagen was induced by cell extracts but did not occur in the extracellular space and was inhibited in intact cells by the addition of ammonium chloride or leupeptin to the culture medium. Fluorescent staining showed a characteristic granular deposition of Type III procollagen in the peripheral region of the cytoplasm but no granular deposition of Type I procollagen. These results offer new insight into the mechanism of the decreased amount of Type III collagen in the tissue of patients with Ehlers-Danlos syndrome type IV.

Ammonium Chloride↗
PubMed1990

Phenotypic heterogeneity in osteogenesis imperfecta: the mildly affected mother of a proband with a lethal variant has the same mutation substituting cysteine for alpha 1-glycine 904 in a type I procollagen gene (COL1A1).

A proband with a lethal variant of osteogenesis imperfecta (OI) has been shown to have, in one allele in a gene for type I procollagen (COL1A1), a single base mutation that converted the codon for alpha 1-glycine 904 to a codon for cysteine. The mutation caused the synthesis of type I procollagen that was posttranslationally overmodified, secreted at a decreased rate, and had a decreased thermal stability. The results here demonstrate that the proband's mother had the same single base mutation as the proband. The mother had no fractures and no signs of OI except for short stature, slightly blue sclerae, and mild frontal bossing. As a child, however, she had the triangular facies frequently seen in many patients with OI. On repeated subculturing, the proband's fibroblasts grew more slowly than the mother's, but they continued to synthesize large amounts of the mutated procollagen in passages 7-14. In contrast, the mother's fibroblasts synthesized decreasing amounts of the mutated procollagen after passage 11. Also, the relative amount of the mutated allele in the mother's fibroblasts decreased with passage number. In addition, the ratio of the mutated allele to the normal allele in leukocyte DNA from the mother was half the value in fibroblast DNA from the proband. The simplest interpretation of the data is that the mother was mildly affected because she was a mosaic for the mutation that produced a lethal phenotype in one of her three children.

Alleles↗
PubMed1985

Complete primary structure of the human alpha 2 type V procollagen COOH-terminal propeptide.

Recently we presented the partial covalent structure of a type V collagen chain. Analysis of amino acids 796-1020 in the human alpha 2(V) Gly-X-Y region showed strong conservation of charged positions with the interstitial collagens but also revealed substitutions unique to type V. To gain more information about this procollagen and primarily to resolve the ambiguous nature of the 3' noncollagenous propeptide, we sequenced several cDNA clones coding for amino acids adjacent to the carboxyl end of the alpha chain. Here we report the complete primary structure of the alpha 2(V) COOH-terminal propeptide. In general, the latter sequence (270 residues) bears a greater degree of similarity to those of the interstitial rather than the basement membrane procollagens. Compared to the interstitial procollagens, however, more divergence has occurred in alpha 2(V) surrounding the conserved N-asparaginyl-linked carbohydrate attachment site at residues 171-173, and alpha 2(V) possesses an additional potential glycosylation site (Asn-Lys-Thr) located in a hypervariable region near the NH2 terminus. Although certainly premature to form any rigid hypothesis, a pattern emerges that may be characteristic of alpha 2 versus alpha 1 chains. Both the alpha 2(I) and alpha 2(V) telopeptides are devoid of a lysine, which in alpha 1 chains forms an interchain cross-link with residue 87 of the collagenous region. Also in contrast to the interstitial alpha 1 carboxyl propeptides is the absence in alpha 2(I) and alpha 2(V) of a cysteine that probably participates in an interchain disulfide bond. Therefore, one can speculate that those alpha 2 chains, represented only once in procollagen trimers, may not be under the same selective pressure as alpha 1 chains to maintain certain residues responsible for stabilizing the triple helical molecules.

Amino Acid Sequence↗
PubMed1986

Increased collagen biosynthesis and increased expression of type I and type III procollagen genes in tight skin (TSK) mouse fibroblasts.

The Tight Skin (TSK) mouse is a mutant strain that displays connective tissue abnormalities characterized by excessive accumulation of collagen in skin, subcutaneous tissues, and some internal organs such as the heart. Increased collagen biosynthesis by skin organ cultures from affected mice has been previously demonstrated, but the mechanisms responsible have not been identified. In order to examine the molecular alterations responsible for the increased production of this protein, normal and TSK mouse dermal fibroblast cell lines were established, and studies of collagen biosynthesis and expression of Types I and III procollagen genes were performed. Secondary cultures of 5 normal and 5 TSK mice dermal fibroblasts were incubated in media containing 10% fetal calf serum and 50 micrograms/ml ascorbic acid and after labeling with [14C]proline for 72 h the amount of [14C]hydroxyproline synthesized was determined. The results showed that TSK mice dermal fibroblasts produced significantly greater amounts of [14C]hydroxyproline than their normal counterparts (118 +/- 28.3 X 10(-2) versus 53.7 +/- 21.9 X 10(-2) dpm/micrograms of DNA; p less than 0.004). Subsequently, the expression of three procollagen genes in normal and TSK mice fibroblasts was analyzed by Northern blot hybridization of polyadenylated RNA to the human cDNA clones alpha 12, Hf 32, and RJ 5 which are specific probes for transcripts of alpha 1(I), alpha 2(I), and alpha 1(III) procollagen chains, respectively. It was found that TSK mice fibroblasts consistently displayed increased levels (up to 5-fold) of all three collagen transcripts while beta-actin mRNA levels were unchanged. The results demonstrate that TSK mice dermal fibroblasts produce excessive amounts of collagen in culture concomitant with a dramatic increase in the expression of Types I and III procollagen genes.

Animals↗
PubMed1987

Phenotypic plasticity of extracellular matrix gene expression in cultured hamster lung fibroblasts. Regulation of type I procollagen and fibronectin synthesis.

Enhanced synthesis and accumulation of interstitial collagens in the lungs of bleomycin-treated animals have been ascribed to drug-induced alterations in the cellular phenotype as judged by preferentially elevated rates of transcription of collagen and fibronectin genes (Raghow, R., Lurie, S., Seyer, J. M., and Kang, A. H. (1985) J. Clin. Invest. 76, 1733-1739). To explore the biosynthetic contribution of mesenchymal cells to the fibrotic process we cultured hamster lung fibroblasts from animals at 0, 1, 2, and 4 weeks after bleomycin treatment and compared their ability to synthesize type I collagen and fibronectin. Our analyses involved (i) measurements of the rates of procollagen and fibronectin synthesis in relation to the total protein synthesis, (ii) quantitative analyses of the steady-state levels of mRNAs coding for type I procollagen chains and fibronectin by Northern hybridization, and finally (iii) a semiquantitative comparison of the steady-state levels of type I procollagen transcripts by in situ hybridization to recombinant cDNA probes. We show that in the earliest stages of the growth in vitro, the patterns of type I procollagen and fibronectin biosynthesis by cells in primary culture closely corresponded with those found in the intact lung. With increasing duration in culture, the distinctive phenotypes of cells from control as well as fibrotic lungs changed steadily; the biosynthetic profiles of fibroblasts cultured for 10 days from untreated or bleomycin-treated lungs were nearly identical. Based on these data, we conclude that the biosynthetic phenotype of fibroblasts, as judged by the parameters of collagen and fibronectin synthesis, appears to be reversible. The characteristic biosynthetic phenotype of fibroblasts, in the interactive milieu of the intact lung (both control and bleomycin-treated), is maintained by cellular and/or humoral factor(s) which are removed or inactivated by the procedures used for isolation and in vitro culture of fibroblasts.

Animals↗
PubMed1989

A single base mutation that substitutes serine for glycine 790 of the alpha 1 (III) chain of type III procollagen exposes an arginine and causes Ehlers-Danlos syndrome IV.

Previous observations (Stolle, C.A., Pyeritz, R.E., Myers, J.C., and Prockop, D.J. (1985) J. Biol. Chem. 260, 1937-1944) indicated that fibroblasts from a proband with dominantly inherited Ehlers-Danlos syndrome type IV synthesized type III procollagen with a structural defect near the collagenase cleavage site at amino acid 781 and near the trypsin-sensitive site at 789. The type III procollagen was unusually sensitive to proteinases and cleaved by trypsin into a three-quarter fragment at 0 degrees C. Here we demonstrate that the mutation in the type III procollagen gene is a single base mutation that converts the codon for glycine at amino acid 790 of the alpha 1(III) chain to a codon for serine. The mutation probably makes the procollagen molecule unusually sensitive to proteases because it causes local unfolding of the triple helix and exposes the adjacent arginine residue. The results provide the first indication that not all glycine substitutions in the triple helices of fibrillar collagens are equivalent in terms of their effects of the biological function of the molecule.

Arginine↗
PubMed1989

Early increases in pulmonary mRNA encoding procollagens and transforming growth factor-beta in mice sensitive to cyclophosphamide-induced pulmonary fibrosis.

Pulmonary fibrosis was induced 7 weeks after a single i.p. injection of cyclophosphamide (200 mg/kg b.wt.) in BALB/c mice; C57Bl/6 mice were unaffected. There was a corresponding strain variation in the effects of cyclophosphamide on levels of pulmonary mRNA encoding alpha 2I and alpha 1III procollagen, and transforming growth factor-beta. In BALB/c mice, the ratios of alpha 2I and alpha 1III procollagen mRNA to polyadenylated RNA were increased 1 week after cyclophosphamide injection. No increases in levels of either procollagen mRNA occurred in C57Bl/6 mice. The ratio of fibronectin mRNA to polyadenylated RNA was elevated to a similar extent in both murine strains during the 1st week after cyclophosphamide treatment. The pulmonary content of transforming growth factor-beta mRNA and its ratio to polyadenylated RNA increased 2-fold at 1 and 2 weeks in BALB/c but not C57Bl/6 mice. Thus, collagen accumulation in cyclophosphamide-sensitive mice is preceded by increased pulmonary alpha 2I and alpha 1III procollagen mRNA. The early strain selective elevation of transforming growth factor-beta mRNA in response to cyclophosphamide suggests a role, in vivo, for transforming growth factor-beta in drug-induced pulmonary fibrosis.

Animals↗
PubMed1986

Induction of procollagen processing in fibroblast cultures by neutral polymers.

In cultures of dermal fibroblasts, procollagen and the intermediates pC- and pN-collagen accumulated in the culture medium with little further processing to collagen. When polyethylene glycol (PEG) or other neutral polymers were added to fibroblast culture medium, no collagen or procollagen was found in the medium, but all the collagen was associated with the cell layer. The type I procollagen was fully processed to collagen with an initial transient accumulation of pN-collagen, and the processed collagen formed covalently cross-linked dimers. The presence of pepsin-sensitive COOH-terminal telopeptides and the accumulation of pN-collagen in PEG-treated cultures of dermatosparactic fibroblasts indicated that it was likely that processing occurred via the correct in vivo propeptidase activities. At the levels used in this study, PEG did not have any toxic effect during the incubation period on the fibroblasts in culture, since the amount of total protein synthesis was not altered by addition of PEG to cultures. However, the level of collagen production was reduced to about half, indicating that there was increased degradation or that the released collagen propeptides or the accumulation of processed collagen in association with the cells exerted a feedback regulation on collagen synthesis. Addition of neutral polymers to the culture medium provided a simple means of achieving complete and accurate processing of procollagen which more closely resembled the in vivo process.

Animals↗
PubMed1986

Procollagen III peptide in bronchoalveolar lavage fluid. A potential marker of altered collagen synthesis reflecting pulmonary disease in sarcoidosis.

Type III procollagen N-terminal peptide was not detectable in bronchoalveolar lavage fluid from healthy volunteers but was present in fluid from the majority of patients with pulmonary sarcoidosis (N = 110); the mean concentration was 0.6 micrograms/liter returned fluid, range less than or equal to 0.2 to 19.5 or, expressed in relation to the amount of albumin recovered, 9.5 mg/gm albumin (range less than or equal to 1 to 45). The serum concentrations in the patients with sarcoidosis were normal. Significant inverse correlations were found between lavage fluid procollagen peptide and vital capacity (p less than 0.001), forced expiratory volume (p less than 0.01), and diffusion capacity (p less than 0.01). Lavage fluid procollagen peptide was also related to pulmonary radiological findings (p less than 0.001) and serum levels of angiotensin converting enzyme (p less than 0.001). These findings support the hypothesis that procollagen peptide in lavage fluid is a potential marker of activated pulmonary fibroblasts or an expanded fibroblast mass associated with interstitial lung fibrosis.

Adult↗