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PubMed2001

Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells.

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.

Animals↗
PubMed1998

The retention of abnormal type I procollagen and correlated expression of HSP 47 in fibroblasts from a patient with lethal osteogenesis imperfecta.

Various mutations of genes encoding type I procollagen chains have been linked to osteogenesis imperfecta (OI). The mutations yield abnormal procollagen molecules that fold improperly. HSP 47, a stress-inducible protein localized to the endoplasmic reticulum (ER) of collagen-producing cells, may participate in collagen processing as a procollagen-specific molecular chaperone. The intracellular transport of abnormal procollagen molecules and the expression of HSP 47 have been studied in fibroblasts from a patient with OI. Normal and OI fibroblasts cultured with or without ascorbate were analysed by immunofluorescent double labelling with monoclonal antibodies to C-propeptide of type I procollagen and HSP 47, as observed by confocal microscopy. Procollagen and HSP 47 were also quantified by immunoprecipitation of normal and OI fibroblasts radiolabelled with 35S-methionine. By confocal microscopy, procollagen molecules were retained in the ER of both fibroblast types cultured in the absence of ascorbate, and were co-localized with HSP 47. In normal fibroblasts, 2 h after the addition of ascorbate, most of the procollagen had disappeared from the cells, while in OI fibroblasts, abnormal procollagen molecules and HSP 47 were still retained in the ER. By immunoprecipitation, procollagen was negligible in normal fibroblasts cultured with ascorbate; much larger amounts of procollagen were immunoprecipitated from OI fibroblasts despite ascorbate. Increased HSP 47 in OI fibroblasts was demonstrated by immunoprecipitation with a specific monoclonal antibody. These results suggest the increase in HSP 47 in the ER of OI fibroblasts is related to its collagen-specific chaperone function.

Ascorbic Acid↗
PubMed1986

Glucocorticoids decrease the synthesis of type I procollagen mRNAs.

Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression.

Actins↗
PubMed1996

Interleukin-1 beta and phenytoin reduce alpha 1 (I) procollagen mRNA expression in human gingival fibroblasts.

Effects of and interactions between interleukin-1 beta (IL-1 beta) and phenytoin (PHT) on alpha 1 (I) procollagen gene and protein expression in human gingival fibroblasts and its relation to prostaglandin E2 (PGE2) formation were studied. IL-1 beta (300 pg/ ml) reduced the steady-state level of alpha 1 (I) procollagen mRNA by 50% and decreased the amount of procollagen I by 35%. PHT (10 micrograms/ml) reduced the level of alpha 1 (I) procollagen mRNA by 40% but the amount of procollagen I in the medium was unchanged. In combination with IL-1 beta, PHT potentiated the inhibitory effect of IL-1 beta on alpha 1 (I) procollagen mRNA level that was accompanied by an increased PGE2 formation. Preincubation with indomethacin (10(-6) M) partially reduced the inhibitory effect of IL-1 beta as well as of IL-1 beta in combination with PHT on the mRNA level of alpha 1 (I) procollagen. The inhibitory effect of PHT was unaffected by indomethacin treatment. Addition of exogenous PGE2 (> or = 10 nM) dose-dependently reduced steady-state level of alpha 1 (I) procollagen mRNA as well as the amount of procollagen 1. The study indicates that IL-1 reduces the expression of alpha 1 (I) procollagen mRNA in human gingival fibroblasts partly by a prostaglandin endoperoxide (PGH) synthase-mediated pathway and partly by a PGH-synthase independent pathway, whereas PHT reduces alpha 1 (I) procollagen gene expression by a PGH-synthase independent pathway. The potentiation of the inhibitory effect of IL-1 induced by PHT was mediated mainly by a PGH-synthase dependent pathway.

Anticonvulsants↗
PubMed1999

Amino acid availability regulates type I procollagen accumulation in human lung fibroblasts.

Fibrotic lung diseases are characterized by excessive deposition of type I collagen. Amino acid availability regulates type I collagen mRNA levels in quiescent human lung fibroblasts. In these studies, the effect of amino acid availability on type I collagen protein accumulation in quiescent human lung fibroblasts was examined. Following amino acid deprivation, alpha1(I) procollagen protein levels were not detected by Western blot analysis in either the intracellular or the extracellular compartments. Fibronectin levels and total protein levels were not affected. Amino acid deprivation resulted in a more pronounced decrease in alpha1(I) procollagen protein levels than in alpha1(I) procollagen mRNA levels, suggesting that post-transcriptional events were responsible for the further decrease inalpha1(I) procollagen protein levels. The addition of transforming growth factor-beta to amino acid deprived fibroblasts increased alpha1(I) procollagen mRNA levels without affecting alpha1(I) procollagen protein levels, confirming a post-transcriptional site for regulatory control by amino acid deprivation. In the absence of ascorbic acid, alpha1(I) procollagen protein levels increased in amino acid deprived fibroblasts, but alpha1(I) procollagen mRNA levels were not affected. The absence of ascorbic acid likely resulted in the accumulation of nonhelical procollagen in the endoplasmic reticulum, indicating that translational mechanisms for alpha1(I) procollagen were intact. The addition of chloroquine, an inhibitor of lysosomal degradation of proteins, increased alpha1(I) procollagen protein levels in amino acid deprived fibroblasts. These data suggest that following amino acid deprivation of quiescent fibroblasts, newly synthesized type I collagen was degraded intracellularly, primarily by a process that involved lysosomal proteinases.

Amino Acids↗
PubMed1988

Remodeling of the rough endoplasmic reticulum during stimulation of procollagen secretion by ascorbic acid in cultured chondrocytes. A biochemical and morphological study.

The cellular and molecular mechanisms regulating the reversible accumulation of nonhelical, underhydroxylated procollagen in the rough endoplasmic reticulum (RER) remain obscure. To clarify these mechanisms, we isolated chondrocytes from chick vertebral cartilage and kept them in scorbutic monolayer cultures. By Day 9 of culture, the chondrocytes had accumulated a large amount of underhydroxylated Type II procollagen in their RER. Within 1 h of ascorbate treatment, the accumulated procollagen was hydroxylated; this was accompanied by a slight stimulation of procollagen secretion and was followed by a marked stimulation starting between 2 and 3 h of treatment. Secretion of the accumulated procollagen was completed by about 24 h of treatment. Strikingly, the marked stimulation of procollagen secretion at 2-3 h of treatment was associated with marked remodeling of the RER. This organelle came to consist of a few, unusually large cisternae ("sacs") and many flat cisternae while the RER in untreated cells consisted of uniform, oval cisternae. The RER remodeling was accompanied by a comparable redistribution of the accumulated Type II procollagen stored in it. The RER sacs and flat cisternae invariably communicated directly and were still detectable by 8 h but not by 24 h of treatment. RER remodeling and procollagen redistribution also occurred in untreated chondrocytes that had been shifted to 23 degrees C for 2-3 h. Together, the data indicate that folding of the accumulated procollagen molecules into their normal helical configuration is followed by procollagen redistribution within, and remodeling of, the RER. These processes may have a role in stimulating procollagen export from the RER and secretion.

Animals↗
PubMed1983

Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs. Reversal of the bleomycin effect by dexamethasone.

Bleomycin treatment of primary chick skin fibroblasts and chick lung fibroblasts resulted in a selective dose-dependent increase of cell layer procollagen synthesis. Solid support hybridization of total cellular RNA to 32P-labeled pro-alpha 1(I) and pro-alpha 2(I) cDNAs did not indicate an increase of total cellular procollagen type I mRNAs in bleomycin-treated cells. However, bleomycin treatment of chick skin fibroblasts causes a redistribution of procollagen type I mRNAs within the nuclear, cytoplasmic, and polysomal subcellular fractions. Both the nuclear and cytoplasmic procollagen type I mRNAs are significantly decreased in concentration after bleomycin administration. In contrast, the polysomal procollagen type I mRNAs are significantly increased in both chick skin and lung fibroblasts treated with bleomycin. Administration of dexamethasone to bleomycin-treated fibroblasts resulted in a reversal of the bleomycin-induced increase in cell layer procollagen synthesis. The increased amounts of polysomal procollagen type I mRNAs in bleomycin-treated cells were also reduced by subsequent administration of dexamethasone. These data indicate that bleomycin treatment of chick skin and chick lung fibroblasts results in a specific increase in procollagen synthesis in the cell layer which is mediated by elevated levels of polysomal type I procollagen mRNAs via a repartitioning of these mRNAs within the fibroblast. Furthermore, dexamethasone reverses the bleomycin-induced elevations of both cell layer procollagen synthesis and polysomal type I procollagen mRNAs.

Animals↗
PubMed1995

Prolyl hydroxylation regulates intracellular procollagen degradation in cultured rat cardiac fibroblasts.

To determine the regulatory role of prolyl hydroxylation in intracellular cardiac procollagen turnover, we examined the effects of prolyl 4-hydroxylase inhibitors (alpha, alpha-dipyridil, 3,4-dihydroxybenzoic acid ethyl ester, pyridine 2,4-dicarboxylic acid ethyl ester) and ascorbic acid on procollagen metabolism by cultured, neonatal rat cardiac fibroblasts. Ascorbate-deficient fibroblasts showed decreased rates of prolyl hydroxylation and total collagen accumulation without a significant reduction in alpha 1(I) and alpha 1(III) mRNA levels. The fraction of newly synthesized procollagens degraded intracellularly was also substantially increased in ascorbate-deficient cells (50 +/- 7 v 30 +/- 3% in ascorbate-deficient v control fibroblasts; P < 0.05). These findings were associated with increased intracellular accumulation of Type I procollagen, enhanced secretion of "underhydroxylated" pro alpha 1 (I) polypeptide into the cell culture medium, and decreased extracellular Type I collagen deposition. Similar results were obtained by treating cells with alpha, alpha-dipyridil (300 microns), and 3,4-dihydroxybenzoic acid ethyl ester (400 microM) in the presence of ascorbate. A major portion of the enhanced degradation of newly synthesized procollagens occurred within acidic intracellular compartments as indicated by the inhibition of procollagen degradation by chloroquine (25 microM). Inhibition of procollagen secretion by colchicine (0.5 micrograms/ml) enhanced the diversion to, and subsequent intracellular degradation of underhydroxylated procollagens in cardiac fibroblast lysosomes. We conclude that inactivation of prolyl 4-hydroxylase increases intracellular accumulation and intralysosomal degradation of newly synthesized cardiac procollagen polypeptides. These observations suggest that procollagen prolyl hydroxylation may be important in the regulation of collagen accumulation by cardiac interstitial cells during fibrotic processes in vivo

2,2'-Dipyridyl↗
PubMed1980

Collagen biosynthesis by human skin fibroblasts. II. Isolation and further characterization of type I and type III procollagens synthesized in culture.

Human skin fibroblasts in culture have previously been shown to synthesize genetically distinct procollagens type I and type III. In the present study, cultured human skin fibroblasts were incubated under conditions optimized for synthesis of these procollagens in medium containing [3H]proline. The newly synthesized type I and type III 3H-labeled procollagens in the culture medium were then isolated as native proteins by DEAE-cellulose chromatography, or by gel filtration and SDS-polyacrylamide slab gel electrophoresis under denaturing conditons after limited pepsin proteolysis. The chromatographic procedures were optimized to yield reliable and reporducible results with good recoveries. The isolated procollagens were identified by cyanogen bromide peptide mapping and characterized by cleavage with highly purified collagenase synthesized by human skin fibroblasts. Assay of the relative synthesis of type I/III procollagens by normal human skin fibroblasts using DEAE-cellulose chromatography indicated that 80% of the procollagen in the medium was type I while the remaining 20% consisted of type III. When the ratio of newly-synthesized type I/III collagens was estimated by gel filtration or using SDS-polyacrylamide slab gel electrophoresis after limited pepsin proteolysis, relatively fewer type III collagen alpha-chains were recovered. This observation suggests that some of the type of the type III collagen molecules are in a conformation which is less resistant to digestion by pepsin than the triple-helix of type I procollagen. The coefficient of variation for the relative synthesis of type I and type III procollagens by control cultures was relatively small (16%), indicating that the phenotypic expression of type I and type III procollagen genes, under optimized culture conditions, is under a relatively tight control. The results further suggest that the optimized methodology developed for assay of the relative synthesis of type I and type III procollagens and collagens by cultured human skin fibroblasts can be utilized in studies on collagen aberrations in acquired and inherited diseases of connective tissue.

Cells, Cultured↗
PubMed1977

Genetics of the connective tissue proteins: assignment of the gene for human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids.

Somatic cell hybrids between mouse and human cell lines have been used to identify the specific chromosome that governs the synthesis of type I procollagen. Fourteen hybrid clones and subclones were derived independently from crosses between mouse parents [LM (thymidine kinase-negative) or A9 (hypoxanthine phosphoribosyltransferase-negative)] and human cells (human diploid lung fibroblasts WI-38 or diploid skin fibroblasts GM5, GM17, and GM9). The cultures were labeled with [(3)H]proline in modified Eagle's medium without serum. Radioactive procollagens were purified from the medium by the method of Church et al. [(1974) J. Mol. Biol. 86, 785-799]. DEAE-cellulose chromatography was used to separate collagen and type I and type III procollagen. Human type I procollagen was assayed by double immunodiffusion analysis with type I procollagen antibodies prepared by immunizing rabbits with purified human type I procollagen. These analyses combined with karyology and isozyme analyses of each hybrid line have produced evidence for the assignment of the gene for human type I procollagen to chromosome 17. A human microcell-mouse hybrid cell line containing only human chromosome 17 was positive for human type I procollagen, lending further support to the assignment of the human type I procollagen gene to chromosome 17. Finally, by using a hybrid line containing only the long arm of human chromosome 17 translocated onto a mouse chromosome, the type I procollagen gene can be assigned more specifically to the long arm of chromosome 17.

Animals↗
PubMed2002

Interaction properties of the procollagen C-proteinase enhancer protein shed light on the mechanism of stimulation of BMP-1.

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.

Binding Sites↗
PubMed1991

Alternatively spliced type II procollagen mRNAs define distinct populations of cells during vertebral development: differential expression of the amino-propeptide.

Type II collagen is a major component of cartilage providing structural integrity to the tissue. Type II procollagen can be expressed in two forms by differential splicing of the primary gene transcript. The two mRNAs either include (type IIA) or exclude (type IIB) an exon (exon 2) encoding the major portion of the amino (NH2)-propeptide (Ryan, M. C., and L. J. Sandell. 1990. J. Biol. Chem. 265:10334-10339). The expression of the two procollagens was examined in order to establish a potential functional significance for the two type II procollagen mRNAs. First, to establish whether the two mRNAs are functional, we showed that both mRNAs can be translated and the proteins secreted into the extracellular environment. Both proteins were identified as type II procollagens. Secondly, to test the hypothesis that differential expression of type II procollagens may be a marker for a distinct population of cells, specific procollagen mRNAs were localized in tissue by in situ hybridization to oligonucleotides spanning the exon junctions. Embryonic vertebral column was chosen as a source of tissue undergoing rapid chondrogenesis, allowing the examination of a variety of cell types related to cartilage. In this issue, each procollagen mRNA had a distinct tissue distribution during chondrogenesis with type IIB expressed in chondrocytes and type IIA expressed in cells surrounding cartilage in prechondrocytes. The morphology of the cells expressing the two collagen types was distinct: the cells expressing type IIA are narrow, elongated, and "fibroblastic" in appearance while the cells expressing type IIB are large and round. The expression of type IIB appears to be correlated with abundant synthesis and accumulation of cartilagenous extracellular matrix. The expression of type IIB is spatially correlated with the high level expression of the cartilage proteoglycan, aggrecan, establishing type IIB procollagen and aggrecan as markers for the chondrocyte phenotype. Transcripts of type II collagen, primarily type IIA, are also expressed in embryonic spinal ganglion. While small amounts of type II collagen have been previously detected in noncartilagenous tissues, the detection of this new form of the collagen in relatively high abundance in embryonic nerve tissue is unique. Taken together, these findings imply a potential functional difference between type IIA and type IIB procollagens and indicate that the removal of exon 2 from the pre-mRNA, and consequently the NH2-propeptide from the collagen molecule, may be an important step in chondrogenesis. In addition, type II procollagen, specifically type IIA, may function in noncartilage tissues, particularly during development.

Animals↗
PubMed1997

Identification of the molecular recognition sequence which determines the type-specific assembly of procollagen.

A key question relating to procollagen biosynthesis is the way in which closely related procollagen chains discriminate between each other to assemble in a type-specific manner. Intracellular assembly of procollagen occurs via an initial interaction between the C-propeptides followed by vectorial propagation of the triple-helical domain in the C to N direction. Recognition signals within the C-propeptides must, therefore, determine the selective association of individual procollagen chains. We have used the pro alpha1 chain of type III procollagen [pro alpha1(III)] and the pro alpha2 chain of type I procollagen [pro alpha2(I)] as examples of procollagen chains that are either capable or incapable of self-assembly. When we exchanged the C-propeptides of the pro alpha1(III) chain and the pro alpha(I) chain we demonstrated that this domain is both necessary and sufficient to direct the assembly of homotrimers with correctly aligned triple-helices. To identify the sequences within this domain that determine selective association we constructed a series of chimeric procollagen chains in which we exchanged specific sequences from the pro alpha1(III) C-propeptide with the corresponding region within the pro alpha2(I) C-propeptide (and vice versa) and assayed for the ability of these molecules to form homotrimers. Using this approach we have identified a discontinuous sequence of 15 amino acids which directs procollagen self-association. By exchanging this sequence between different procollagen chains we can direct chain association and, potentially, assemble molecules with defined chain compositions.

Amino Acid Sequence↗
PubMed1993

Increased serum concentrations of procollagen type III peptide in severely injured patients: an indicator of fibrosing activity?

OBJECTIVES: To determine the serum concentrations of procollagen type III peptide in severely injured patients with different outcomes and to evaluate the relationship between serum procollagen type III peptide concentrations, sources of increased posttraumatic fibrotic activity (wounds, lung, liver, kidney), and decreased elimination of procollagen type III peptide (liver). DESIGN: Prospective study. SETTING: Surgical ICU, university hospital. PATIENTS: Fifty-seven patients (mean injury severity score: 38.5 points, range 13 to 75 points), between 16 and 70 yrs of age, treated in our institution within 6 hrs after the accident. MEASUREMENTS: Serial measurements were started on admission and continued on a 6-hr basis. After 48 hrs, the monitoring interval was extended to 24 hrs until recovery (but at least until day 14) or death. At each point of evaluation, pulmonary and circulatory function parameters and chest radiographs (once a day) were evaluated, the results were recorded, and blood samples were drawn to determine procollagen type III peptide, total bilirubin, creatinine, gamma-glutamyl transferase, polymorphonuclear elastase, and other parameters. Statistic evaluation was done with the Wilcoxon test, Spearman rank correlation, and a multiple regression model. RESULTS: Mean procollagen type III peptide serum concentrations (+/- SD) were significantly different in patients who died (8.0 +/- 3.8 U/mL) compared with those patients who survived with organ failure (2.7 +/- 1.3 U/mL) or without complications (1.4 +/- 0.5 U/mL), respectively. Significant correlations of procollagen type III peptide concentrations with the serum bilirubin concentrations (r = .7), days with need of mechanical ventilation (r = .64), PaO2/FIO2 ratio (r = -.6), polymorphonuclear elastase (r = .6), serum creatinine concentrations (r = .55), and injury severity score (r = .33) were observed. There was a tendency toward higher serum procollagen type III peptide concentrations in patients with severe skeletal injuries. CONCLUSIONS: Serum procollagen type III peptide concentrations in severely injured patients may be considerably increased in correlation with injury severity and outcome. Procollagen type III peptide serum concentrations seem to reflect the sum of increased collagen formation from wound healing and fibrogenesis of mediator-related organ damage (especially lung) and decreased procollagen type III peptide excretion due to impaired liver function. Further data are necessary to evaluate the role of hepatic elimination in these patients.

Adolescent↗
PubMed1999

Serum type III procollagen in children with type I hereditary tyrosinemia.

BACKGROUND: Type I hereditary tyrosinemia leads to hepatic dysfunction and fibrosis and is associated with a high risk of hepatic malignancy. Serum N-terminal propeptide of type III procollagen is a sensitive marker of organ fibrosis of diverse origins. The current study was conducted to determine whether analysis of serum levels of type III procollagen in hereditary tyrosinemia would be useful in the follow-up of the progressive liver disease and eventually in detecting hepatic malignancy. METHODS: Serum N-terminal propeptide of type III procollagen was sequentially studied in 10 children with type I hereditary tyrosinemia. RESULTS: At diagnosis of type I hereditary tyrosinemia, serum N-terminal propeptide of type III procollagen ranged from 0.6 to 2.9 multiples of age-related median. During follow-up, serum N-terminal propeptide of type III procollagen decreased, yet remained elevated 0.2 to 2.6 years after diagnosis. Children with the acute type of the disease tended to have higher serum N-terminal propeptide of type III procollagen than did those with the chronic type. Porphyria crises were associated with elevated serum type III procollagen. The one patient receiving 2-(2-nitro-4-trifluoromethyl-benzoyl)-1,3-cyclohexanedione (NTBC) did not differ from the other ones in serum type III procollagen levels. Serum N-terminal propeptide of type III procollagen did not increase with developing hepatocellular carcinoma. CONCLUSIONS: Serum N-terminal propeptide of type III procollagen may be useful in monitoring the hepatopathy in type I hereditary tyrosinemia but is not useful in detecting malignant transformation in the liver.

Adolescent↗
PubMed2002

Differential interaction of molecular chaperones with procollagen I and type IV collagen in corneal endothelial cells.

PURPOSE: Procollagen I is synthesized and intracellularly degraded in corneal endothelial cells (CEC), whereas type IV and VIII collagens are secreted into Descemet's membrane. In our previous study, we demonstrated that procollagen I synthesized by CEC is improperly folded and that the molecule was largely colocalized with protein disulfide isomerase (PDI) within the endoplasmic reticulum (ER). In the present study, we further investigated whether the alpha-subunit of prolyl 4-hydroxylase (P4Halpha) and glucose regulatory protein/immunoglobulin heavy chain binding protein (Grp78/BiP) were also involved in ER retention of procollagen I in CEC. METHODS: Immunocytochemical analysis was performed to determine the colocalization of procollagen I with molecular chaprones. Protein synthesis was measured by immunoblot analysis and the association between proteins was determined by coimmunoprecipitation followed by immunoblot analysis. mRNA was quantitated using RT-PCR. RESULTS: To study the interaction of procollagen I with certain molecular chaperones involved in the collagen biosynthetic pathway, we determined whether procollagen I colocalized with P4Halpha and Grp78/Bip, and then compared this molecular chaperone colocalization with their association with type IV collagen. Procollagen I was colocalized with either P4Halpha or Grp78/Bip to a much lesser degree than type IV collagen was colocalized with these same ER proteins. Colocalization between the molecular chaperones demonstrated that P4Halpha and Grp78/Bip were largely colocalized in the peripheral region of the ER, whereas colocalization of P4Halpha and PDI was mostly limited to a small region of the ER. When cells were treated with alpha,alpha-dipyridyl, the inhibitor did not affect the colocalization profiles of collagens with the molecular chaperones. However, the inhibitor markedly increased colocalization of P4Halpha and PDI, but it significantly decreased colocalization between P4Halpha and Grp78/Bip. When synthesis of the molecular chaperones was compared between CEC and corneal stromal fibroblasts (CSF), more Grp78/Bip and PDI were produced by CEC than by CSF. On the other hand, expression of Hsp47 was lower in CEC than it was in CSF. Coimmunoprecipitation was used to compare the association of P4Halpha or Grp78/Bip with collagens in CEC and CSF. The association of collagens (regardless of type) with P4Halpha or Grp78/Bip was much higher in CEC than in CSF. When the association of collagen molecules with respective molecular chaperones was compared in CEC, the degree of association between Grp78/Bip and procollagen I was similar to that between the molecular chaperone and type IV collagen. On the other hand, the degree of association between P4Halpha and type IV collagen was much higher than that between P4Halpha and procollagen I. CONCLUSIONS: These data suggest that procollagen I and type IV collagen may use different molecular chaperones in the ER, thus targeting their distinctive destinations.

Animals↗
PubMed1986

Defects in the processing of procollagen to collagen are demonstrable in cultured fibroblasts from patients with the Ehlers-Danlos and osteogenesis imperfecta syndromes.

This is a study of the processing of procollagen to collagen in cultures of skin and tendon fibroblasts. Processing was markedly increased by growing cells for 2-4 days postconfluence and then adding ascorbate to the medium for 2 days prior to labeling with [3H] proline. With this system, more than two-thirds of the pro-alpha chains of type I procollagen in the culture medium, and more than 90% of those in the cell layer, were rapidly processed to pC-alpha, pN-alpha, or alpha chains. Purified, exogenous procollagen was also rapidly processed in cell-free culture medium. The results showed for the first time that exogenous procollagen can be processed in conditioned cell-free medium. The system was then used to compare the processing of procollagen in the medium of normal fibroblasts, cells from one bovine and four human variants of osteogenesis imperfecta, and those from eight human variants of the Ehlers-Danlos syndrome. The cells could be divided into three groups, based on their ability to process type I procollagen: normal, consistently slow, and very slow. The cause of the decreased processing was shown to be associated with either a mutation causing a shortening of an alpha chain or decreased activity of procollagen N-proteinase in cell-free culture medium. Decreased processing of procollagen to collagen occurred with cultured fibroblasts from patients with different forms of both osteogenesis imperfecta and Ehlers-Danlos syndrome. Both of these disease syndromes are associated with abnormalities in the structure or metabolism of procollagen in fibrous connective tissues, bones, and teeth. The results show that defects in the structure, synthesis, or processing of procollagen are readily demonstrated with cultured fibroblasts.

Adolescent↗
PubMed1983

Mechanism for the regulation of post-translational modifications of procollagens synthesized by matrix-free cells from chick embryos.

Three possible mechanisms are considered to account for the variations of post-translational modifications in different collagen types. 1) The cells have different amounts of post-translational modifying enzymes, 2) the rate of prolylhydroxylation of different procollagen types is varied, and 3) the rate of chain association of pro-alpha chains of different collagen types is modulated. In an attempt to examine the three possibilities, we have determined the activities of prolyl hydroxylase and lysyl hydroxylase, and we have examined the kinetics of the secretion of procollagens and the kinetics of pro-gamma chain formation of different procollagen types in matrix-free cells isolated from tissues of 17-day-old chick embryos. Type II collagen synthesized by cartilage cells contains more hydroxylysine than type I collagen synthesized by tendon and cornea cells. It was found, however, that cartilage cells contain significantly less lysyl hydroxylase than tendon and cornea cells. In contrast, we found only a small difference in the amount of prolyl hydroxylase in tendon, cornea, and cartilage cells. The secretion of type I procollagen by tendon and cornea cells can be described by two first order processes. In contrast, the secretion of type II procollagen by cartilage cells, type IV procollagen by lens cells, and type V procollagen by cornea cells can be described by single first order processes. Examination of the formation of pro-gamma components of procollagen types I and II revealed that it occurs via intermediate dimers of two pro-alpha chains. The formation or pro-gamma(I) chains in tendon and cornea cells is about three times faster than the formation of pro-gamma(II) chains in cartilage cells. These results are consistent with the hypothesis that the rate of association of pro-alpha chains regulates the synthesis of procollagens with different degrees of post-translational modifications.

Animals↗