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PubMed2003

Transforming growth factor-beta induces secretion of activated ADAMTS-2. A procollagen III N-proteinase.

The metalloproteinase ADAMTS-2 has procollagen I N-proteinase activity capable of cleaving procollagens I and II N-propeptides in vitro, whereas mutations in the ADAMTS-2 gene in dermatosparaxis and Ehlers-Danlos syndrome VIIC show this enzyme to be responsible in vivo for most biosynthetic processing of procollagen I N-propeptides in skin. Yet despite its important role in the regulation of collagen deposition, information regarding regulation and substrate specificity of ADAMTS-2 has remained sparse. Here we demonstrate that ADAMTS-2 can, like the procollagen C-proteinases, be regulated by transforming growth factor-beta 1 (TGF-beta 1), with implications for mechanisms whereby this growth factor effects net increases in formation of extracellular matrix. TGF-beta 1 induced ADAMTS-2 mRNA approximately 8-fold in MG-63 osteosarcoma cells in a dose- and time-dependent, cycloheximide-inhibitable manner, which appeared to operate at the transcriptional level. Secreted ADAMTS-2 protein induced by TGF-beta 1 was 132 kDa and was identical in size to the fully processed, active form of the protease. Biosynthetic processing of ADAMTS-2 to yield the 132-kDa form is shown to be a two-step process involving sequential cleavage by furin-like convertases at two sites. Surprisingly, purified recombinant ADAMTS-2 is shown to cleave procollagen III N-propeptides as effectively as those of procollagens I and II, whereas processing of procollagen III is shown to be decreased in Ehlers-Danlos VIIC. Thus, the dogma that procollagen I and procollagen III N-proteinase activities are provided by separate enzymes appears to be false, whereas the phenotypes of dermatosparaxis and Ehlers-Danlos VIIC may arise from defects in both type I and type III collagen biosynthesis.

ADAM Proteins↗
PubMed1997

Alteration of the kinetics of type I procollagen synthesis in human osteosarcoma cells by 1,25-dihydroxyvitamin D3.

The kinetics of type I procollagen synthesis in a human osteosarcoma cell line, MG 63, were investigated after treatment with 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3), a hormonal inducer of phenotypic differentiation. Pulse label and chase experiments demonstrated greatly enhanced production and more rapid reduction of intracellular procollagen molecules in the 1,25-(OH)2 D3-treated cells as compared to the nontreated case. After a chase for 1 h, labeled procollagen was reduced by nine-tenths in 1,25-(OH)2 D3-treated cells, while half of the radioactivity still remained in nontreated cells. The expression rate of type I collagen, which was examined by pulse label experiment, was elevated in association with an increase in the mRNA coding for the type I collagen alpha 1 chain by 1,25-(OH)2 D3 treatment. However, the amount of intracellular procollagen present after 4 h continuous labeling was almost the same, independent of the 1,25-(OH)2 D3 treatment. Thus, we conclude that strage of the molecule was not affected. The results therefore suggest an increase in both the synthesis and secretion of type I collagen. The 1,25-(OH)2 D3 treatment was also found to induce the alpha subunit of prolyl 4-hydroxylase and to be associated with an elevated level of hydroxyproline in the procollagen. Moreover, gelatinase B-resistant procollagen molecules, indicative of intracellular procollagen molecules in the stable triple helical form, were detected only in the 1,25-(OH)2 D3-treated cells. These data suggest more efficient proline hydroxylation is involved in rapid secretion of procollagen after hormone administration. The present evidence points to posttranslational control of procollagen synthesis.

Animals↗
PubMedDate not supplied

Serum type III procollagen peptide concentrations in severe chronic active hepatitis: relationship to cirrhosis and disease activity.

To analyze the correlations between the presence of cirrhosis and hepatocellular inflammation and the serum concentrations of the amino-terminal peptide of procollagen type III in chronic liver disease, we measured procollagen type III concentrations in paired serum samples from 46 patients (17 had cirrhosis) with severe chronic active hepatitis during a therapeutic treatment trial. Coded sera were analyzed for procollagen type III concentrations using both a standard and a recently described Fab radioimmunoassay to compare their relative diagnostic accuracy. Mean procollagen type III levels were elevated to the same extent in the cirrhotic and noncirrhotic groups at entry into the study. In response to immunosuppressive therapy, the initially elevated procollagen type III levels improved to normal values at remission in both groups. Qualitatively, the results were similar using either assay, but the standard assay was more sensitive for identifying the clinical stage of disease (i.e., active disease vs. disease in remission) than the Fab assay. Since both procollagen type III levels and standard liver function tests correlated well individually with the presence or absence of active disease, they also correlated with each other when both entry and remission values were considered. However, procollagen type III levels correlated poorly with indicators of inflammation (histologic grade and serum transaminase levels) during active disease. It is concluded that procollagen type III levels change in concert with standard liver function tests but do not quantitatively reflect inflammation or static measurements of hepatic fibrosis in severe chronic active hepatitis. However, these preliminary results suggest that procollagen type III can distinguish active disease from chronic active hepatitis in remission.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Antinuclear↗
PubMed1989

In situ hybridization for procollagen types I, III and IV mRNA in normal and fibrotic rat liver: evidence for predominant expression in nonparenchymal liver cells.

The expression of alpha 2(I), alpha 1(III) and alpha 1(IV) procollagen mRNA was analyzed in normal and CCl4-induced fibrotic rat liver by in situ hybridization using RNA probes. In normal liver, moderate amounts of alpha 2(I) and alpha 1(III) procollagen transcripts were found in sinusoidal cells, in stromal cells of the portal tracts and in the vicinity of central veins, whereas a1(IV) procollagen gene expression was below the threshold of detection. After 2 weeks of CCl4 treatment, increased transcription of alpha 2(I) and alpha 1(III) procollagen genes was observed in sinusoidal cells. At this stage, alpha 1(IV) procollagen mRNA was detectable in the same cell types and localization as alpha 2(I) and alpha 1(III) procollagen transcripts, although with a weaker signal. After 4 weeks, newly formed fibrous septa showed many cells intensely labeled by alpha 2(I), alpha 1(III) and alpha 1(IV) procollagen probes. Neither in normal liver nor at any stage of fibrosis was any hybridization signal above background observed in hepatocytes. These patterns suggest that in the liver Type I, Type III and Type IV procollagen expression takes place predominantly in nonparenchymal cells. Therefore, hepatocytes do not appear to be significantly involved in procollagen production in this experimental model of liver fibrosis.

Animals↗
PubMed1991

Type I procollagen gene expression in normal and early healing of the medial collateral and anterior cruciate ligaments in rabbits: an in situ hybridization study.

The purpose of this study was to compare the levels of procollagen type I messenger RNA (mRNA) in normal and healing medial collateral ligament (MCL) and anterior cruciate ligament (ACL) in a rabbit model. Our method of injury involved a surgical model with identical partial lacerations in the midsubstance of the MCL and ACL. Paraffin sections of normal ligaments, and ligaments 3, 7, 14, and 28 days postlaceration were studied by in situ hybridization to compare and follow the level of type I procollagen mRNA in the two ligaments. A complementary DNA (cDNA) probe corresponding to alpha 1(I) procollagen mRNA was labeled with [32P]d-CTP. After hybridization, autoradiography, and staining of the sections, the level of procollagen mRNA was assessed by microscopic examination. A higher level of procollagen mRNA was consistently detected in normal MCL than in normal ACL, suggesting higher collagen synthetic activity in the MCL. At the injury sites of the MCL and ACL, the levels of type I procollagen mRNA increased at all post-laceration periods, reaching its highest level at 14 days postsurgery. The MCL healing site had a considerably higher level of procollagen mRNA than the ACL healing site (i.e., injury site) at all postoperative intervals. The results demonstrate that procollagen mRNA levels in MCL tissue are higher than those in ACL tissue under normal conditions, as well as in response to injury. The differences in the procollagen mRNA levels of MCL and ACL may reflect the synthesis of collagen in these tissues, and may contribute to the differences in their healing capacities.

Animals↗
PubMed1994

Inhibition of procollagen I degradation by colligin: a collagen-binding serpin.

Colligin is a collagen-binding glycoprotein localized to the endoplasmic reticulum (ER) and has been proposed to play a role in collagen biosynthesis. Its membership in the serpin family prompted us to examine its effect on procollagen degradation. We first showed that procollagen degradation can take place in the ER of L6 myoblasts by using brefeldin A to block transit from the ER. This degradation could be prevented by the serine protease inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). To examine procollagen degradation in vitro, isolated liver microsomes were incubated with procollagen. Intact microsomes were unable to degrade labeled procollagen I, fibronectin, or the cytoplasmic proteins, phosphorylase b and the RI subunit of the cAMP-dependent protein kinase. However, when the microsomes were permeabilized by treatment with detergent, they became capable of degrading procollagen and fibronectin, but not the cytoplasmic proteins. The degrading activity was not due to cross-contamination by lysosomal or cytoplasmic, multicatalytic proteases. The proteolysis of procollagen chains in the treated microsomes was partially inhibited by TPCK, TLCK, and leupeptin. The most effective inhibitor was, however, colligin. In its presence, the breakdown of procollagen I, but not of fibronectin, was specifically inhibited. Colligin itself was not degraded by the microsomal preparations. The protein degrading activity was localized to the microsomal membranes, and showed a pH optimum of about 8.0. From these studies it is inferred that one of the roles of colligin may be to protect the procollagen I chains in the ER from degradation prior to their transport to the cis-Golgi compartment.

Amino Acid Sequence↗
PubMed1999

Quantitative analysis of alpha 1(I) and alpha 1(III) procollagen mRNA expression in systemic sclerosis skin tissue--an in situ hybridization study.

Human alpha 1(I) and alpha 1(III) procollagen mRNA expression in skin tissue from 15 systemic sclerosis (SSc) patients and from 7 normal control subjects was quantitatively analyzed using in situ hybridization. The grains accumulating in each area, representing procollagen mRNA expression per cell, were counted. To normalize the results from each subject, the number of cells and the number of grains per cell were divided by the area of the skin specimen (in square millimeters). The number of cells per square millimeter expressing alpha 1(I) and alpha 1(III) procollagen mRNA in SSc skin was significantly elevated compared with normal control skin (both P < 0.01). The number of grains per cell per square millimeter expressing alpha 1(III) procollagen mRNA in SSc skin was also significantly elevated compared with normal control skin (P < 0.01). The relationship between procollagen mRNA expression and the histological findings in SSc was also studied. The numbers of cells and grains per cell per square millimeter expressing alpha 1(I) procollagen mRNA in fibrotic zone SSc skin were significantly elevated compared with normal control skin (both P < 0.01). The numbers of cells and grains per cell per square millimeter expressing alpha 1(III) procollagen mRNA in SSc skin were significantly elevated compared with normal control skin (both P < 0.01) and with border zone SSc skin (number of cells P < 0.01, number of grains P < 0.05). These results indicate an increase in the number of cells showing elevated expression of alpha 1(I) and alpha 1(III) procollagen mRNA, and a close relationship between alpha 1(I) and alpha 1(III) procollagen mRNA expression and the histological findings in SSc.

Adult↗
PubMed1980

Collagen biosynthesis by human skin fibroblasts. I. Optimization of the culture conditions for synthesis of type I and type III procollagens.

Skin fibroblasts in culture can provide a convenient means to study aberrations of collagen metabolism in a variety of clinical conditions. In the present study, the culture conditions for the synthesis of procollagen by cultured human skin fibroblasts were optimized by independently varying parameters in the cell culture environment. To study the synthesis of procollagen the cell cultures were labeled with [3H]proline and the collagenous polypeptides were determined either by measuring the synthesis of hydroxy[3H]proline or by assaying the 3H-labeled proteins digested into dialyzable 3H-labeled peptides by bacterial collagenase. On the basis of the experimental results, the following culture conditions are suggested for optimal synthesis of procollagen: (a) cell culture medium should be supplemented with ascorbic acid (25--50 micrograms/ml) and fetal calf serum (20%); (b) the pH of the culture medium should be kept above 7.2 and preferably in the pH range 7.5--7.8; (c) the cell cultures should be used one to two days after reaching visual confluency. Under these conditions the synthesis and secretion of [3H]procollagen was found to be linear through a 24 h incubation period, and procollagen was demonstrated to be a major gene product of the fibroblasts. The relative synthesis of type I and type III procollagens was also monitored by isolating these genetically distinct procollagens by DEAE-cellulose chromatography or by measuring type I and III collagens by sodium dodecyl sulfate polyacrylamide gel electrophoresis after limited pepsin proteolysis. No marked changes were observed in type I/III procollagen ratios in situations where the total formation of hydroxy[3H]proline was significantly affected. The average coefficient of variance for procollagen synthesis between replicate cultures was found to be relatively small (14%), and the optimization of the culture conditions for the control cells has, therefore, created a reliable and reproducible basis for employing human skin fibroblasts to study collagen metabolism in acquired and inherited diseases.

Ascorbic Acid↗
PubMed1978

Collagen synthesis by human amniotic fluid cells in culture: characterization of a procollagen with three identical proalpha1(I) chains.

Second trimester human amniotic fluid cells synthesize and secrete a variety of collagenous proteins in culture. F cells (amniotic fluid fibroblasts) are the most active biosynthetically and synthesize predominantly type I with smaller amounts of type III procollagen. Epithelioid AF cells (the predominating clonable cell type) synthesize a type IV-like procollagen and a procollagen with three identical proalpha chains, structurally and immunologically related to the proalpha1 chains of type I procollagen. The latter procollagen, when cleaved with pepsin and denatured, yields a single non-disulfide-bonded alpha chain that migrates more slowly than F cell or human skin alpha1(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but coelutes with these chains from carboxymethyl-cellulose. The major cyanogen bromide produced peptides demonstrate a similar behavior relative to peptides derived from alpha1(I). The collagen is characterized by an increased solubility at neutral pH and high ionic strength, relative to type I collagen. The amino acid composition of the pepsin-resistant alpha chain is essentially identical with that of human alpha1(I), except for marked increases in the content of 3- and 4-hydroxyproline and hydroxylysine. Preliminary experiments suggest that these increased posttranslational modifications are responsible for the unusually slow migration of this collagen and its cyanogen bromide peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The procollagen has, therefore, been assigned the chain composition [proalpha1(I)]3. Like type I procollagen, [proalpha1(I)]3 undergoes a time-dependent conversion, in the medium and cell layer, to procollagen intermediates and alpha chains. The production of [proalpha1(I)]3 probably reflects the state of differentiation and/or embryologic derivation of AF cells rather than a characteristic of the fetal phenotype, since F cells do not synthesize significant amounts of the procollagen.

Amino Acids↗
PubMed1992

Early increased renal procollagen alpha 1(IV) mRNA levels in streptozotocin induced diabetes.

Changes in renal procollagen mRNA levels were measured shortly after the induction of streptozotocin induced diabetes in the rat. "Medullary" procollagen alpha 1(IV) levels seven days after diabetes induction was significantly higher in untreated diabetic rats (DM, N = 12; 244 +/- 57% of the mean control value), than in diabetic rats receiving small doses of insulin insufficient to achieve euglycemia (NPH, N = 10; 87 +/- 12%) and in diluent injected nondiabetic control rats (C, N = 15; 100 +/- 12%; P less than 0.01, DM vs. C and DM vs. NPH). "Medullary" procollagen alpha 1(I) mRNA levels were numerically increased in DM to a lesser degree (141 +/- 5%, ANOVA not significant) compared to C (100 +/- 13%), and this small increment was further normalized by insulin treatment (NPH, 120 +/- 11%). A trend for increased beta-actin mRNA levels in DM did not reach significance (P greater than 0.05). Increases in "medullary" procollagen mRNA levels did not correlate with kidney weight, glomerular tuft volume, creatinine clearance, food intake, or body weight gain, and occurred when renal morphology was normal by light microscopy. Statistically significant but weak correlations were noted between the serum glucose levels and "medullary" procollagen alpha 1(IV) mRNA levels (r = 0.43, P less than 0.05). In addition, weak correlations were noted between glycosuria and "medullary" procollagen alpha 1(I) levels (r = 0.38, P less than 0.05). In situ hybridization studies localized the increased procollagen alpha 1(IV) mRNA levels predominantly in the DM group primarily in the deep cortex and medullary outer stripe of proximal tubules. Glomerular procollagen alpha 1(IV), alpha 1(I), alpha 1(III) and beta-actin mRNA levels were not increased in untreated diabetic rats 7 or 28 days after diabetes induction. Thus, tubular procollagen alpha 1(IV) mRNA levels increased prior to any measurable change in glomerular levels and were ameliorated by insulin administration.

Animals↗
PubMed1994

Synthesis of recombinant human procollagen II in a stably transfected tumour cell line (HT1080).

Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.

Amino Acid Sequence↗
PubMed2005

Substrate-specific modulation of a multisubstrate proteinase. C-terminal processing of fibrillar procollagens is the only BMP-1-dependent activity to be enhanced by PCPE-1.

Members of the bone morphogenetic protein-1/tolloid (BMP-1/Tld) family of metalloproteinases, also known as procollagen C-proteinases (PCPs), control multiple biological events (including matrix assembly, cross-linking, cell adhesion/migration and pattern formation) through enzymatic processing of several extracellular substrates. PCP activities on fibrillar procollagens can be stimulated by another family of extracellular proteins, PCP enhancers (PCPE-1, PCPE-2), which lack intrinsic enzymatic activity. While PCPs have multiple substrates, the extent to which PCPEs is involved in the processing of proteins other than fibrillar procollagens is unknown. In the experiments reported here, PCPE-1 was found to have no effect on the in vitro BMP-1 processing of procollagen VII, the procollagen V N-propeptide, the laminin 5 gamma2 chain, osteoglycin, prolysyl oxidase, or chordin. In contrast, PCPE-1 enhanced C-terminal processing of human fibrillar procollagen III but only when this substrate was in its native, disulfide-bonded conformation. Surprisingly, processing of procollagen III continued to be enhanced when essentially all the triple-helical region was removed. These and previous results (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869; Bernocco, S., Steiglitz, B. M., Svergun, D. I., Petoukhov, M. V., Ruggiero, F., Ricard-Blum, S., Ebel, C., Geourjon, C., Deleage, G., Font, B., Eichenberger, D., Greenspan, D. S., and Hulmes, D. J. S. (2003) J. Biol. Chem. 278, 7199-7205) indicate that the mechanism of PCPE-1 action involves recognition sites in both the C-propeptide domain and in the C-telopeptide region of the procollagen molecule. PCPEs therefore define a new class of extracellular adaptor proteins that stimulate proteinase activity in a substrate-specific manner, thereby providing a new target for the selective regulation of PCP activity on fibrillar procollagen substrates.

Amino Acid Sequence↗
PubMed1993

Parallel regulation of procollagen I and colligin, a collagen-binding protein and a member of the serine protease inhibitor family.

A potential regulatory linkage between the biosynthesis of colligin, a collagen-binding protein of the ER, and procollagen I was examined under a variety of experimental conditions. Cell lines which did not produce a significant amount of procollagen I mRNA also lacked the capacity to produce colligin mRNA. Anchorage-dependent cell lines like L6 myoblasts and normal rat kidney fibroblasts produced both colligin and procollagen I mRNA, but the level of both was concurrently reduced considerably in their ras-transformed counterparts. Similarly, during the differentiation of L6 myoblasts, levels of both colligin and procollagen declined together. Treatment of myoblasts by dexamethasone or EGF led to a decrease in the steady-state levels of procollagen I mRNA, and this was, again, accompanied by a decrease in colligin mRNA synthesis. On the other hand, when the rate of procollagen I synthesis was stimulated by treatment of myoblasts with TGF beta, it led to the concurrent augmentation of both the mRNA and protein levels of colligin. A linkage between the regulation of synthesis of procollagen I and colligin thus seems to exist. The only exception to this generalization is provided by the heat induction behavior of the two proteins. Treatment of myoblasts for a very short period leads to an increase in the synthesis of both the mRNA and protein levels of colligin. This, however, is not accompanied by a change in the mRNA levels of procollagen I. These studies establish that colligin and procollagen are generally tightly co-regulated except after heat shock, suggesting an important functional linkage.

Animals↗
PubMed1985

Identification of type II procollagen in rabbit vitreous.

A procollagen in the soluble fraction of rabbit vitreous was isolated by dialysis against dilute acetic acid and partially purified by Bio-Gel A 5M gel filtration. The molecule was identified to be type II procollagen by comparing its segment-long-spacing (SLS) banding pattern with that of standard type II collagen isolated from rabbit articular cartilage. Electron microscopy of the SLS of this type II procollagen revealed a fuzzy propeptide extension at the N-terminal end of the molecule. Pepsin digestion of the procollagen removed this extension, thus converting the molecule into a collagen which had mobility similar to that of pepsin-soluble cartilage type II collagen in SDS-polyacrylamide gel electrophoresis. No inter-chain disulfide bond was found in the propeptide extension when the procollagen samples were electrophoresized with or without mercaptoethanol. Comparison of the cyanogen bromide peptide map of the type II procollagen with that of the pepsin-soluble type II collagen indicated that two extra peptides were present in the digest of procollagen. All of this evidence suggested that the procollagen in the soluble vitreous body of the rabbit eye was a novel type II procollagen with a propeptide extension only at the N-terminus.

Animals↗
PubMed1979

Scleroderma: increased biosynthesis of triple-helical type I and type III procollagens associated with unaltered expression of collagenase by skin fibroblasts in culture.

To assess potential abnormalities in collagen metabolism in systemic scleroderma, skin fibroblast lines from patients with this disease were established and compared to control cell lines derived from healthy subjects. For studies on the biosynthesis of procollagen, the cells were incubated with [(14)C]proline in a medium supplemented with ascorbic acid and beta-aminopropionitrile, and the synthesis of nondialyzable [(14)C]hydroxyproline, in relation to DNA or cell protein, was taken as an index of procollagen formation. Five of eight scleroderma fibroblast cell lines demonstrated procollagen biosynthesis rates significantly higher than the controls, and the mean rate of procollagen synthesis by scleroderma fibroblasts was about twice that of the control cells. Control experiments demonstrated that the specific activity of the intracellular free proline was not different in scleroderma and control fibroblasts, and the mean population doubling times of the scleroderma and the control fibroblast cell lines were the same. The relative synthesis of the genetically distinct procollagens was examined by isolating type I and type III procollagens from the cell culture medium using DEAE-cellulose chromatography. The ratios of type I/III procollagens in scleroderma cell lines did not differ from the controls. The helical stability of the collagenous portion of type I and type III procollagens, estimated by the resistance of (14)C-collagen to limited proteolytic digestion with pepsin under nondenaturing conditions, was the same in both scleroderma and control cultures. The capacity of the cells to synthesize enzymatically active and immunologically reacting collagenase was also studied; no marked differences in these parameters could be observed. The results suggest that cultured skin fibroblasts from patients with scleroderma demonstrate a metabolic abnormality expressed as increased synthesis of type I and type III procollagens in a normal ratio. This abnormality may play a role in the excessive accumulation of collagen in the skin and other organs affected in scleroderma.

Adolescent↗
PubMed1993

Regulation of procollagen metabolism in the pressure-overloaded rat heart.

To determine the molecular events responsible for the disproportionate accumulation of myocardial fibrillar collagens during sustained hypertension, we examined the in vivo rate of procollagen synthesis, collagen accumulation, and intracellular procollagen degradation 1-16 wk after abdominal aortic banding in young rats. These measurements were correlated with tissue mRNA levels for type I and type III procollagen polypeptides. Banded animals developed moderate, sustained hypertension and mild left ventricular hypertrophy. Increased type III procollagen mRNA levels were detected early after banding and persisted for the entire observation period. Disproportionate collagen accumulation without histological evidence of fibrosis was noted within 1 wk after hypertension induction. Fibrillar collagen accumulation at this time point resulted not from a major increase in procollagen synthesis, but rather a marked decrease in the rate of intracellular procollagen degradation. Interstitial fibrosis, however, was observed 16 wk after banding. Type I procollagen mRNA levels were increased six-fold, but only after 16 wk of hypertension. These results correlated well with the results of in vivo procollagen synthesis experiments at 16 wk, which demonstrated a threefold increase in left ventricular procollagen biosynthesis. We conclude that pretranslational as well as posttranslational mechanisms regulate fibrillar collagen deposition in the myocardial extracellular matrix during sustained hypertension.

Animals↗
PubMed1996

Serum type I and type III procollagen peptide levels in sarcoidosis.

Type I and type III are the most abundant collagens in the lung. The aim of our study was to compare type I and III procollagen peptides in sera of sarcoid patients. Sixty eight patients with sarcoidosis were studied (19 with newly recognized disease, 7 with relapsing disease, 15 with chronic disease, and 27 in stable remission). Thirty healthy volunteers served as controls. The levels of procollagen I and III peptides were determined by radioimmunoassay. Angiotensin-converting enzyme (ACE) level was evaluated by means of a colorimetric assay. In patients with newly recognized sarcoidosis, both serum procollagen I and III peptide levels were increased with respect to controls (p=0.0014 and p<0.00001, respectively). There was a poor correlation between levels of procollagen I and III (r=0.26), whereas there was a closer correlation between procollagen III and ACE (r=0.69). Procollagen I peptide level did not identify patients in roentgenological stage III. In conclusion, in patients with newly recognized sarcoidosis there is a significant increase in the serum level of procollagen I peptide. However, procollagen I peptide is not a marker of sarcoid patients with fibrosis, ie. stage III disease. Its clinical usefulness seems to be weaker than that of procollagen III peptide.

Adult↗
PubMed1997

Propeptide levels of type III and type I procollagen in the serum and bronchoalveolar lavage fluid of patients with pulmonary sarcoidosis.

No single test is available to reliably assess the activity or prognosis of pulmonary sarcoidosis. In this study, we have evaluated two procollagen markers, aminoterminal propeptide of type III procollagen (PIIINP) and carboxyterminal propeptide of type I procollagen (PICP) in serum and bronchoalveolar lavage fluid (BALF) and compared them to other disease markers of pulmonary sarcoidosis, such as serum level of angiotensin converting enzyme (S-ACE) or serum interleukin-2 receptor (S-IL-2R). Bronchoalveolar lavage was performed in 40 sarcoidosis patients without (stages 0-I) and 20 patients with lung parenchymal involvement (stages II-III), as well as in 17 controls. Serum (S)- and BALF-PIIINP and PICP, S-ACE, S-IL-2R, BALF-albumin, BALF-lymphocytes and mast cells were determined in these patients. BALF-PIIINP was clearly and S-PIIINP slightly elevated in sarcoidosis compared to the controls. Similarly BALF-PICP, but not S-PICP, was significantly higher in sarcoidosis. BALF-PIIINP, but not BALF-PICP correlated with S-ACE and S-IL-2R levels. Patients with lung parenchymal disease had higher S-ACE and BALF-PIIINP, but neither S-IL-2R, S-PIIINP nor S- or BALF-PICP were elevated. S-PIIINP and S-IL-2R but not S-ACE were higher in symptomatic than nonsymptomatic patients. Symptomatic patients with parenchymal disease had elevated BALF-PIIINP whereas in the symptomatic nonparenchymal group S-PIIINP was elevated. In conclusion, this is the first study to evaluate carboxyterminal propeptide of type I procollagen in sarcoidosis and showed elevated levels in bronchoalveolar lavage fluid. In contrast to the levels of bronchoalveolar lavage fluid aminoterminal propeptide of type III procollagen, levels of carboxyterminal propeptide of type I procollagen did not correlate with serum level of angiotensin converting enzyme and serum interleukin-2 receptor levels, suggesting that carboxyterminal propeptide of type I procollagen may be less suitable disease marker in sarcoidosis than aminoterminal propeptide of type III procollagen. However, the role of carboxyterminal propeptide of type I procollagen as an indicator of fibrogenesis must be further studied.

Adult↗