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PubMed1999

Fibrogenesis and fibrolysis in collagenous colitis. Patterns of procollagen types I and IV, matrix-metalloproteinase-1 and -13, and TIMP-1 gene expression.

Collagenous colitis is characterized by the deposition of a superficial subepithelial collagenous layer, the pathogenesis of which is unknown. Because the excess matrix deposition is potentially reversible, a labile imbalance between fibrogenesis and fibrolysis may be suspected. Expression of procollagen alpha1(I) and alpha1(IV), matrix-metalloproteinase (MMP)-1 and -13, and tissue inhibitor of metalloproteinase (TIMP)-1 genes was semiquantitated by in situ hybridization on serial biopsies of 12 patients with collagenous colitis and compared to controls. Collagen types I, III, IV, and VI, tenascin, undulin/collagen XIV, and alpha-actin were localized by immunohistology. The superficial collagen layer stained strongly for collagen types I, III, and VI, and particularly for tenascin, but not for undulin. Elevated procollagen alpha1(I), procollagen alpha1(IV), and TIMP-1 transcript levels were found in alpha-actin-positive cells with linear distribution underneath the superficial collagenous layer, whereas MMP-1 RNA expression was variable and restricted to cell clusters. MMP-13 expression was undetectable. The patterns of procollagen alpha1(I)- and alpha1(IV)-specific labeling, combined with an intense tenascin- but absent undulin-specific staining, indicate deposition of an immature interstitial matrix that may be susceptible to degradation. The restricted MMP-1 RNA expression, counteracted by increased TIMP-1 expression, suggests locally impaired fibrolysis as a relevant factor in the pathogenesis of collagenous colitis.

Adult↗
PubMed1988

Relationship of type I procollagen to corticosteroid therapy in children with inflammatory bowel disease.

We determined the serum concentration of the C-terminal propeptide of type I procollagen (pColl-I-C) in 60 children and adolescents (ages 4 to 17 years) with inflammatory bowel disease (24 ulcerative colitis, 36 Crohn disease) and in seven children (ages 2 to 15 years) with nongastrointestinal disease (asthma) during varying regimens of corticosteroid therapy. Patients with inflammatory bowel disease were grouped according to disease severity (mild, and moderate to severe). Significantly lower pColl-I-C concentrations and growth velocities were found in each severity group among those subjects receiving daily corticosteroid therapy compared with those receiving alternate-day or no corticosteroid therapy (P less than 0.01). When daily corticosteroid therapy was initiated and then maintained for 7 to 14 days in 11 patients with exacerbation of inflammatory bowel disease clinical improvement resulted, but mean procollagen concentrations decreased significantly (P less than 0.001). In seven children with asthma receiving methylprednisolone intravenously, significant decreases in pColl-I-C concentrations were noted within 24 to 48 hours of therapy (P less than 0.001). These data indicate that serum procollagen values decrease during both short- and long-term daily administration of corticosteroid therapy. Longitudinal assessment of procollagen concentrations may provide rapid assessment of the effects of different corticosteroid regimens on growth.

Adolescent↗
PubMed1985

Copy number of the chicken type II procollagen gene.

The copy number of the type II procollagen gene has been determined by quantitative hybridization of cloned DNA to Southern blots of total cellular DNA from chicken. Two different DNA probes have been used. One contains the 289 base pair exon coding for the junction between the triple helical domain and the carboxyl-terminal propeptide and the other contains an 162 base pair exon coding for amino acids 712-765 of the triple helical domain. Both fragments hybridize to single bands present at one copy per haploid genome in genomic DNA from chicken liver or sternal cartilage after these DNAs are cleaved with either HinfI of PstI. When the hybridization stringency and washing conditions were modified sufficiently to stabilize hybridization of heteroduplexes with up to 31% mismatch, no additional hybridizing fragments were detected. These results show that there is not a second copy of this gene or a pseudogene which is more closely related to the type II procollagen gene than the alpha 1 type I procollagen gene. These studies suggest that the cartilage 3 alpha collagen chain, which appears to be very similar to the alpha 1 (II) collagen chain, is encoded by the type II procollagen gene but is differently processed than the major type II gene product, and that the alpha 1 (II) Minor chain identified in bovine type II collagen preparations most likely represents an allelic form of type II collagen rather than a product of a different gene.

Animals↗
PubMed1986

Partial purification and characterization of a procollagen C-proteinase from the culture medium of mouse fibroblasts.

A procollagen C-proteinase was purified about 100-fold from the medium of cultured mouse fibroblasts by a combination of ammonium sulfate precipitation, gel-filtration, and affinity chromatography on a column of Sepharose coupled to the carboxyl propeptide of type I procollagen. The purified enzyme did not exhibit other proteolytic activities, and it cleaved type I, II and III procollagens to produce the corresponding pN alpha chains and carboxyl propeptides as the only products. Amino acid sequencing of the first 14-18 residues at the N-terminus of the carboxyl propeptides generated by the enzyme from human pro alpha 1(I), pro alpha 2(I) and pro alpha 1(III) chains showed that the cleavage occurred at the physiological site, i.e. at the specific Ala-Asp bond in the pro alpha 1(I) and pro alpha 2(I) chains, and at the specific Gly-Asp bond in the pro alpha 1(III) chain. The pH optimum of the enzyme is 8.5 and its molecular weight as estimated by gel-filtration is about 125,000 daltons. The enzyme is inhibited by metal-chelators, various amines, dithiothreitol, N-ethylmaleimide and serum, but it is insensitive to pepstatin, leupeptin and serine proteases inhibitors. The enzyme differs from the C-proteinase described by Njieha et al. (Biochemistry 21:757-764, 1982), and the catheptic activities reported by Davidson et al. (Eur. J. Biochem 100:551-558, 1979) and Helseth and Veis (Proc. Natl. Acad. Sci. USA 81:3302-3306, 1984). The specificity of the enzyme is offered as evidence for a unique, C-proteinase, and its recovery from culture medium supports an extracellular location for procollagen processing.

Amino Acid Sequence↗
PubMed1986

Glucocorticoids change the ratio of type III to type I procollagen extracellularly.

Rat skin fibroblasts were treated with dexamethasone and labeled with radioactive proline for 2 hours. Intracellular and extracellular procollagens were immunoprecipitated with monospecific polyclonal antibodies. Both intracellular and extracellular types I and III procollagen were decreased coordinately by glucocorticoid treatment. The ratio of type III to type I procollagen was the same for control and dexamethasone-treated cell cultures. However, when rat skin fibroblasts were prelabeled with radioactive proline and chased in the presence of unlabeled proline the ratio of type III to type I extracellular procollagens remained constant in the glucocorticoid-treated cells and increased in control fibroblasts.

Animals↗
PubMed2001

Type I and type III procollagen gene expressions in the early phase of ligament healing in rabbits: an in situ hybridization study.

The purpose of this study is to observe type I and type III procollagen gene expressions in the healing ligament using in situ hybridization histochemistry. The rabbit medial collateral ligaments were incised and harvested at 3, 7, 14, and 28 days postoperatively. The healing ligament showed increased expression of both procollagen genes through this period compared with the unoperated ligament. The peak expression level was observed at 7 or 14 days for type I and at 7 days for type III, respectively. The strongest expression of both genes was detected in the scar tissue created between the ends of the old ligament. Although type III procollagen gene expression was observed almost only in the newly created scar tissue, type I procollagen gene was expressed not only in the scar tissue, but also at the ends of the previously normal ligament. These results suggest that type I collagen synthesis begins shortly after ligament injury and occurs at the ends of the injured ligament as well as in the scar tissue, and that type III collagen is largely synthesized in the scar tissue around one week after injury but continues being synthesized for at least four weeks after injury.

Animals↗
PubMed1997

Ethanol induces the expression of alpha 1(I) procollagen mRNA in a co-culture system containing a liver stellate cell-line and freshly isolated hepatocytes.

To study the fibrogenic action of ethanol in vitro we used a co-culture system of freshly isolated hepatocytes and a liver stellate cell line (CFSC-2G) developed in our laboratory. Our results show that in this co-culture system ethanol induces the expression of alpha 1(I) procollagen mRNA in a dose- and time-dependent manner. This effect of ethanol was due to its metabolism by alcohol dehydrogenase since 4-methylpyrazole prevented the ethanol-mediated increase in alpha 1(I) procollagen mRNA. Ethanol was more efficient than acetaldehyde in inducing alpha 1(I) procollagen mRNA expression and its effect was protein synthesis-independent. Transfection of either hepatocytes or liver stellate cells with a reporter gene, chloramphenicol acetyl transferase (CAT), driven by 3700 bp of the mouse alpha 1(I) procollagen promoter demonstrated that only LSC expressed significant CAT activity and that this activity was enhanced by ethanol. Overall, our results suggest that this co-culture system is a useful model to study alcohol-induced fibrogenesis in vitro and that mechanisms other than acetaldehyde formation may also play an important role in alcohol-induced fibrogenesis.

Acetaldehyde↗
PubMed1992

Abnormalities in the biosynthesis of type III procollagen in cultured skin fibroblasts from two patients with multiple aneurysms.

We examined the synthesis of collagenous proteins by cultured skin fibroblasts taken from 14 patients with an abdominal aortic aneurysm and either an aneurysm at a second site (8 patients) or a first order relative with an abdominal aortic aneurysm (6 patients). Fibroblasts were labeled with [3H] proline and, following pepsin digestion of media proteins, the ratio of type I/III collagen was examined by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). With the exception of two patients, the ratio of type I/III collagen in the media of fibroblasts from aneurysm patients was similar to control values (6 controls). In two of the patients, the type I/III collagen ratio was greater than 3 standard deviations from the mean of both control ratios and those of other aneurysm patients. mRNA levels coding for type III procollagen, however, were normal in both patients. Patient #1 (ME) showed reduced type III procollagen on SDS-PAGE analysis of intracellular proteins. Intracellular and media type III procollagen levels were normal in patient #2 (HR), but media type III collagen was reduced by over 50% after digestion with a combination of trypsin and alpha-chymotrypsin for 5 minutes at 36 degrees C. Control type III collagen was only reduced after digestion at 39 degrees C. These data suggest an altered thermal stability of the type III collagen trimer synthesized by this patient, probably due to a mutation in the amino acid sequence. The data presented in this paper suggest that some forms of common abdominal aortic aneurysms may be caused by mutations in the gene coding for type III procollagen.

Aged↗
PubMed1997

Cloning of equine type II procollagen and the modulation of its expression in cultured equine articular chondrocytes.

The complete nucleotide sequence of equine type II procollagen has not been previously reported, and equine-specific probes have not been available. We report the complete sequence and discuss the molecular characteristics of equine type II procollagen mRNA which was cloned from a cDNA library prepared from mRNA isolated from equine articular chondrocytes. The coding sequence (4257 bp) was 92.4% homologous to the cDNA of the human sequence, and the propeptide was 97% identical to the human sequence. We demonstrated that when equine chondrocytes are grown in phenotypically-maintained cultures, the expression of type II procollagen is linked to the age of the animal. Additionally, in cultures of young equine chondrocytes, IL1-beta and TNF-alpha both reduced the expression of type II procollagen mRNA in a dose responsive manner.

Amino Acid Sequence↗
PubMed1980

Inhibitors of procollagen N-protease. Synthetic peptides with sequences similar to the cleavage site in the pro alpha 1(I) chain.

A series of peptides was synthesized with amino acid sequences identical with the cleavage site at which the procollagen N-protease cleaves the N-terminal propeptide from the pro alpha 1 chain of type I procollagen. Peptides up to 11 residues in length did not serve as substrates for the enzyme, an observation consistent with the demonstration that the N-protease will not cleave denatured procollagen or dissociated pro alpha chains. Several of the peptides, however, served as effective inhibitors of the cleavage of procollagen. Comparison of the inhibitor activities of peptides of varying lengths suggested that the L-phenylalanine found three residues to the left of the cleavage site was important for inhibitor activity. This suggestion was confirmed by synthesis of analogues of inhibitory peptides in which L-phenylalanine was replaced by D-phenylalanine, tyrosine, lysine, aspartic acid, or glycine.

Animals↗
PubMed2001

Transgenic mice with inactive alleles for procollagen N-proteinase (ADAMTS-2) develop fragile skin and male sterility.

Transgenic mice were prepared with inactive alleles for procollagen N-proteinase (ADAMTS-2; where ADAMTS stands for a disintegrin and metalloproteinase with thrombospondin repeats). Homozygous mice were grossly normal at birth, but after 1-2 months they developed thin skin that tore after gentle handling. Although the gene was inactivated, a large fraction of the N-propeptides of type I procollagen in skin and the N-propeptides of type II procollagen in cartilage were cleaved. Therefore the results suggested the tissues contained one or more additional enzymes that slowly process the proteins. Electron microscopy did not reveal any defects in the morphology of collagen fibrils in newborn mice. However, in two-month-old mice, the collagen fibrils in skin were seen as bizarre curls in cross-section and the mean diameters of the fibrils were approx. half of the controls. Although a portion of the N-propeptides of type II procollagen in cartilage were not cleaved, no defects in the morphology of the fibrils were seen by electron microscopy or by polarized-light microscopy. Female homozygous mice were fertile, but male mice were sterile with a marked decrease in testicular sperm. Therefore the results indicated that ADAMTS-2 plays an essential role in the maturation of spermatogonia.

Alleles↗
PubMed2006

TIMP-3 inhibits the procollagen N-proteinase ADAMTS-2.

ADAMTS-2 is an extracellular metalloproteinase responsible for cleaving the N-propeptides of procollagens I-III; an activity necessary for the formation of collagenous ECM (extracellular matrix). The four TIMPs (tissue inhibitors of metalloproteinases) regulate the activities of matrix metalloproteinases, which are involved in degrading ECM components. Here we delineate the abilities of the TIMPs to affect biosynthetic processing of procollagens. TIMP-1, -2 and -4 show no inhibitory activity towards ADAMTS-2, in addition none of the TIMPs showed inhibitory activity towards bone morphogenetic protein 1, which is responsible for cleaving procollagen C-propeptides. In contrast, TIMP-3 is demonstrated to inhibit ADAMTS-2 in vitro with apparent Ki values of 160 and 602 nM, in the presence of heparin or without respectively; and TIMP-3 is shown to inhibit procollagen processing by cells.

ADAM Proteins↗
PubMed1985

Primary structure of the telopeptide and a portion of the helical domain of chicken type II procollagen as determined by DNA sequence analysis.

A comparison of the nucleotide sequences of three new cDNA clones for chicken type II procollagen with the sequences of the other three types of chicken fibrillar procollagens reveals that the most conserved regions correlate with the positions of hydroxyproline, hydroxylysine, cysteine and lysine residues. On the basis of replacement-site-divergence calculations it is concluded that alpha 1(II) and alpha 1(I) procollagens diverged later than alpha 1(I) and alpha 2(I) procollagens.

Amino Acid Sequence↗
PubMed1987

Epidermal growth factor increases collagen production in granulation tissue by stimulation of fibroblast proliferation and not by activation of procollagen genes.

The effects of epidermal growth factor (EGF) on granulation-tissue formation and collagen-gene expression were studied in experimental sponge-induced granulomas in rats. After daily administration of 5 micrograms of EGF into the sponge, total RNA was extracted from the ingrown granulation tissue at days 4 and 7 and analysed by Northern hybridization for the contents of mRNAs for types I and III procollagens. EGF treatment increased procollagen mRNA, particularly at day 4. To determine whether this elevation was due to increased proliferation of collagen-producing fibroblasts or to activation of collagen-gene expression in these cells, fibroblast cultures were started from granulation tissue and treated with EGF. These experiments confirmed that EGF is a potent mitogen for granuloma fibroblasts in a dose-dependent manner. The effect of EGF treatment on radioactive hydroxyproline production in cultured cells was inhibitory. The decreased rate of collagen synthesis was also indicated by decreased amounts of procollagen mRNAs. The results suggest that the stimulation of wound healing and collagen production by EGF is due to increased fibroblast proliferation, and not to increased expression of type I and III procollagen genes.

Animals↗
PubMed1981

Reduced secretion of structurally abnormal type I procollagen in a form of osteogenesis imperfecta.

Osteogenesis imperfecta is a clinically and genetically heterogeneous group of inherited connective tissue disorders in which bone fragility is the predominant feature. Cultured dermal fibroblasts from one patient with the lethal perinatal form of osteogenesis imperfecta secrete type I procollagen at a rate half that of normal cells. Short-term labeling experiments and treatment with alpha,alpha'-dipyridyl (which prevents posttranslational prolyl and lysyl hydroxylation) demonstrated that these cells produce two distinct pro alpha 1(I) chains, which are synthesized at the same rate. Analysis of cyanogen bromide peptides indicated that the two chains differ in their primary structures. Thus, structural abnormalities in type I procollagen prevent this molecule from being secreted normally, resulting in an anomalously low ratio of type I procollagen to other extracellular matrix molecules. While the lethal perinatal form of osteogenesis imperfecta may be heterogeneous, we propose that the underlying pathogenesis of at least one form is decreased secretion of type I procollagen.

Cells, Cultured↗
PubMed1985

Restricted homology between human alpha 1 type IV and other procollagen chains.

Screening of a human fibroblast cDNA library with a mouse type IV procollagen clone resulted in one 1.05-kilobase isolate that was used to identify a 1.7-kilobase clone overlapping the former by less than 150 nucleotides. EcoRII digestion revealed that the larger clone exhibited the pattern characteristic of DNA coding for a collagenous sequence. Blot hybridization to RNA from mouse F9 stem cells and from these cells treated with retinoic acid and N6, O2'-dibutyryl-cAMP showed induction of type IV mRNA. DNA sequencing and comparison of the derived amino acids with the reported protein data demonstrated that the clones encode part of the alpha 1 chain of human type IV procollagen. Alignment of alpha 1 (IV) with other human procollagens showed minimal but detectable homology. A small cluster of charged residues in the alpha chain is partially shared by type IV. In close proximity is an interruption in the alpha 1 (IV) Gly-Xaa-Yaa region corresponding to the 3' end of a unique proline-free, and therefore also less rigid, area in other collagen triple helices. Analysis of the carboxyl-terminal alpha 1 (IV) peptide showed a repeat symmetry possibly dictated by the six cysteines in each half of the structure. The position of five cysteines in addition to four tyrosine/tryptophan groups allowed a correlation to be drawn between the 3' noncollagenous type IV region and the larger, highly conserved carboxyl propeptides of other human procollagens. Such similarities in the different chains may define functional domains conserved throughout evolution.

Animals↗
PubMed1991

Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia.

A minigene version of the human gene for type II procollagen (COL2A1) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro alpha chains that associate with normal pro alpha chains and thereby cause degradation of the shortened and normal pro alpha chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing the minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro alpha 1(II) chains that were disulfide-linked to normal mouse pro alpha 1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.

Animals↗
PubMed1997

cDNA cloning and expression of bovine procollagen I N-proteinase: a new member of the superfamily of zinc-metalloproteinases with binding sites for cells and other matrix components.

Procollagen N-proteinase (EC 3.4.24.14) cleaves the amino-propeptides in the processing of type I and type II procollagens to collagens. Deficiencies of the enzyme cause dermatosparaxis in cattle and sheep, and they cause type VIIC Ehlers-Danlos syndrome in humans, heritable disorders characterized by accumulation of pNcollagen and severe skin fragility. Amino acid sequences for the N-proteinase were used to obtain cDNAs from bovine skin. Three overlapping cDNAs had an ORF coding for a protein of 1205 residues. Mammalian cells stably transfected with a complete cDNA secreted an active recombinant enzyme that specifically cleaved type I procollagen. The protein contained zinc-binding sequences of the clan MB of metallopeptidases that includes procollagen C-proteinase/BMP-1. The protein also contained four repeats that are homologous to domains found in thrombospondins and in properdin and that can participate in complex intermolecular interactions such as activation of latent forms of transforming growth factor beta or the binding to sulfatides. Therefore, the enzyme may play a role in development that is independent of its role in collagen biosynthesis. This hypothesis was supported by the observation that in some tissues the levels of mRNA for the enzyme are disproportionately high relative to the apparent rate of collagen biosynthesis.

Amino Acid Sequence↗