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1.
Collagen phenotypes were determined for rabbit articular chondrocytes in cartilage slices and first through fifth monolayer cultures. During the first 24 hr of slice culture, chondrocytes exhibited the following collagen phenotype: 96% type II, 3% X2Y and 1% type III. In primary monolayer culture, no other types of collagen were added to this differentiated chondrocyte phenotype; however, the synthesis per cell of each of the expressed collagens was stimulated. By the fifth day of primary culture, X2Y synthesis increased 10 fold, and by the eighth day, a further 4 fold. In contrast, the synthesis of collagen types II and III showed no change by the fifth day, but increased 7 fold by the eighth day. These results suggest independent regulation of X2Y in this situation. In a separate experiment, first through fifth cultures were studied. The synthesis per cell of type II collagen declined steadily and essentially ceased by the fifth culture, indicating the loss of differentiated function by these chondrocyte progeny. The loss of type II synthesis was not quantitatively replaced by the synthesis of type I trimer and type I collagen which was first detected in the third culture. While these qualitative changes in phenotype occurred, the stimulated rate of type III collagen synthesis did not change and that of X2Y declined only slightly. Thus the termination of type II synthesis did not significantly alter the synthesis of the other collagens produced by differentiated chondrocytes. The final “de-differentiated” phenotype was 41% type I, 25% X2Y, 20% type I trimer, 13% type III and 1% type II. 相似文献
2.
J R Lichtenstein E A Bauer J Uitto 《Biochemical and biophysical research communications》1976,73(3):665-672
Human skin fibroblasts in monolayer culture synthesize and secrete precursor forms of collagen into the culture medium. The type I collagen precursor, the major precursor in the culture medium, was isolated on DEAE cellulose chromatography and subjected to mammalian collagenase cleavage. The amino terminal cleavage fragments had a higher molecular weight than α1A and α2A, but did not contain interchain disulfide bonds. The carboxy-terminal cleavage fragments formed high molecular weight aggregates which contained interchain disulfide bonds. These results indicate that human type I procollagen contains noncollagenous amino and carboxy-terminal extension peptides and that all of the interchain disulfide bonds are on the carboxy-terminal portion of the molecule. 相似文献
3.
Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [3H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X2Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis. 相似文献
4.
Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro. 总被引:6,自引:0,他引:6
Analysis of pepsin-resistant proteins produced in culture by monkey aortic smooth muscle cells (SMC) indicates the synthesis of types I and III collagen. As determined by carboxymethylcellulose chromatography and disc gel electrophoresis, SMC cultures synthesize more type III collagen than monkey skin fibroblast cultures; aortic adventitial cell cultures (a mixture of SMC and fibroblasts) synthesize an intermediate amount of type III collagen. Both types I and III procollagens can also be isolated from the culture medium of SMC and skin fibroblasts. The procollagens were separated by diethylaminoethylcellulose (DEAE-cellulose) chromatography in identified by electrophoresis and after cleavage with pepsin and cyanogen bromide. Quantitation of the procollagen by DEAE-cellulose chromatography suggests that 68% of the SMC procollagens and less than 10% of the skin fibroblast procollagens are type III. On the other hand, estimation of the proportions of collagen types secreted by cells, employing pepsin digestion of cell culture medium at 15 degrees C, leads to an underestimation of the amount of type III collagen relative to type I. SMC and fibroblasts may differ in their ability to convert type I procollagen to collagen ad indicated by the observation that skin fibroblast culture medium contains both pN and pC collagen intermediates after 24 h, while cultures of SMC essentially lack the pC collagen intermediates. 相似文献
5.
We previously reported that Kirsten sarcoma virus transformed BALB 3T3 (Ki-3T3) cell cultures contained mainly type I collagen and about 30% of another type designated by us as Y and which appears to be type III collagen, [α1 (III)]3. Clones of BALB 3T3 which exhibited contact-inhibition were found to contain mainly type I collagen [α1(I)]2α2, and about 25% of another type (X) which was composed of three α1 chains differing from those of type III (Hata, R. and B. Peterkofsky, 1977 Proc. Nat. Acad. Sci. (U.S.A.), 74: 2933—2937). Since dibutyryl 3′:5′ cyclic adenosine monophosphate (dbcAMP) increases collagen synthesis and alters other transformation specific properties of Ki-3T3 cells, we determined whether treatment of Ki-3T3 cells with this compound restored the normal collagen phenotype. We also analyzed the collagen of a revertant of Ki-3T3 which exhibits properties similar to those of the dbcAMP treated transformant. Procollagen labeled with radioactive proline was isolated from the medium or cells of cultures and was converted to collagen with pepsin; the collagen was analyzed by carboxymethyl cellulose (CMC) chromatography or gel electrophoresis under denaturing conditions. Ki-3T3 cells treated with 0.5 mM dbcAMP continued to accumulate type III collagen but there was an increase in the number of α1 chains eluting from CMC columns in the same position as α1 (I) suggesting increased accumulation of type X collagen. Although the revertant was similar to dbcAMP treated cells in that it exhibited a flattened morphology and a high relative rate of collagen synthesis, the collagen profile was similar to that of the transformant, consisting mainly of types I and III. These results indicate that accumulation of type III collagen is unaffected by dbcAMP but suggest that cAMP may be involved in the regulation of type X collagen. The failure of dbcAMP or reversion to affect the occurrence of type III collagen supports the mechanism of cell selection as a means of explaining the specific occurrence of type III collagen in sarcoma virus transformed 3T3 cells. 相似文献
6.
P. Kern L. Robert Y. Courtois M. Laurent 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1985,826(4):174-179
Quantification and biosynthesis of type I and type III collagens were determined in skin of control and Fraser mice (CatFraser mutation), which exhibit a genetically determined cataract. Skin organ cultures were labelled with [3H]proline. Pepsin-solubilized collagens were studied using three different approaches: (a) differential salt precipitation at neutral pH, followed by SDS-polyacrylamide gel electrophoresis; (b) differential salt precipitation at acid pH followed by SDS-polyacrylamide gel electrophoresis. (c) CNBr peptide analysis. These methods gave consistent and reproducible results, indicating a selective decrease of type I collagen in Fraser mouse skin as compared to control mouse skin. Metabolic labelling of skin organ cultures showed a decreased specific radioactivity of hydroxy[3H]proline in type I collagen of Fraser mouse skin. The concordant results of these experiments suggest a genetically determined alteration of interstitial collagen metabolism in the Fraser mutation apparently specifically concerning the expression of type I collagen gene(s). 相似文献
7.
Fibroblasts cultured from affected skin areas of five patients with cutaneous scleroderma were found to produce increased amounts of collagen when compared with nonaffected control cells. Total RNA was isolated from the cultures and analyzed for its level of pro alpha 1 (I)collagen mRNA by hybridization of RNA blots with a cloned cDNA probe. The levels of pro alpha 1 (I)collagen mRNAs relative to total RNA were two- to sixfold higher in the samples from affected cells, accounting for the increased synthesis of type I collagen. Cytoplasmic dot hybridizations were performed to measure the cellular content of pro alpha 1 (I)collagen mRNA: up to ninefold increases in the level of this mRNA per cell were found. Upon subculturing, scleroderma fibroblasts were found to reduce gradually the increased synthesis of collagen to the level of nonaffected controls by the tenth passage. The levels of type I collagen mRNAs were also reduced, but more slowly. The results suggest that in scleroderma fibroblasts the genes for type I collagen are activated at procollagen mRNA level or that they are more stable and that the activating factors are lost during prolonged cell culture because cells from affected areas lose their activated state. 相似文献
8.
MIMI TAMAMORI HIROSHI ITO MICHIAKI HIROE FUMIAKI MARUMO RYU-ICHIRO HATA 《Cell biology international》1997,21(3):175-180
Synthesis of type I and type III collagens by rat cardiac fibroblasts was stimulated when the cells were cultured under 95% N2/5% CO2for one hour followed by incubation under normoxic conditions for 24 hours. The stimulative effect was attenutated by the presence of atrial natriuretic peptide (ANP, 10−6m) or brain natriuretic peptide (BNP, 10−6m) in the culture medium. Northern blot analysis indicated that α1(I) and α1(III) collagen mRNA levels were also increased by hypoxia, and decreased with the addition of ANP or BNP in a dose-dependent manner. These results indicate interaction between intracellular signals of a physical stimulus (hypoxic stress) and those of a chemical one (ANP or BNP) and demonstrate that both signals regulate collagen synthesis by cardiac fibroblasts at the levels of the mRNAs. The results also suggest that natriuretic peptides produced by cardiomyocytesin vivomay function as paracrine factors that play a role in the prevention of cardiac fibrosis in ischaemic heart diseases. 相似文献
9.
C E Schwartz C G Hellerqvist L W Cunningham 《Biochemical and biophysical research communications》1979,90(1):240-246
The predominant collagenous protein secreted during the attachment of freshly trypsinized human foreskin fibroblasts was found to be Type I procollagen. Evidence is presented that both the α1 and α2 chains exhibit a 3-hydroxyproline/4-hydroxyproline ratio 4–5 fold higher than that of normal Type I collagen. These findings suggest that caution should be exercised in assigning an observed increase in the 3-hydroxyproline/4-hydroxyproline ratio to the synthesis of a basement membrane type collagen. 相似文献
10.
Bleomycin-induced synthesis of type I procollagen by human lung and skin fibroblasts in culture 总被引:9,自引:0,他引:9
Bleomycin is a chemotherapeutic agent sometimes associated with pulmonary fibrosis and skin lesions in patients undergoing treatment. We examined the mechanisms of increased collagen deposition on bleomycin-induced fibrosis by incubating human lung and skin fibroblast cultures with [14C]proline; the synthesis of [14C]hydroxyproline relative to DNA or cell protein was taken as an index of procollagen formation. Procollagen synthesis by lung cells in the presence of 0.1 and 1.0 microgram/ml bleomycin was significantly increased and similar results were obtained with skin fibroblasts. The relative synthesis of genetically distinct types of collagen was measured by isolating the newly synthesized type I and type III procollagens by DEAE-cellulose chromatography. The proportion of type III procollagen of total newly synthesized procollagen in control lung fibroblast cultures was 17.4 +/0 0.6% (mean +/- S.E.) while the corresponding value in cells incubated in 1 microgram/ml bleomycin was 12.5 +/- 0.6% (n = 6, P < 0.01). Similar results were obtained when the ratios of newly synthesized type I and type III collagens were estimated by interrupted polyacrylamide disc gel electrophoresis in sodium dodecyl sulfate after a limited proteolytic digestion with pepsin. The results indicate that the increased procollagen synthesis induced by bleomycin in fibroblast cultures is predominantly directed towards the synthesis of type I procollagen. 相似文献
11.
Background
Hypertrophic scars are pathologic proliferations of the dermal skin layer resulting from excessive collagen deposition during the healing process of cutaneous wounds. Current research suggests that the TGF-β/Smad signaling pathway is closely associated with normal scar and hypertrophic scar formation. TRAP-1-like protein (TLP), a cytoplasmic protein, has been reported to efficiently regulate Smad2- and Smad3-dependent signal expression in the TGF-β pathway. The relationship between TLP and Type I/III collagen (Col I/III) synthesis explored in the present study provides an effective target for wound healing and gene therapy of hypertrophic scarring.Objective
To investigate the effects of TLP on collagen synthesis in human dermal fibroblasts.Methods
Lentiviral vectors encoding TLP was constructed to transfect fibroblasts derived from normal human skin. The expression of Col I/III and phosphorylation of Smad2 and Smad3 in fibroblasts were examined after TLP treatment. In addition, the comparison of TLP expression in normal skin tissues and in hypertrophic scar tissues was performed, and the effect of TLP on cell viability was analyzed by MTT assay.Results
TLP expression in hypertrophic scar tissue was markedly higher than in normal skin tissue. The Real Time PCR and Western blot test results both revealed that the synthesis of Col I/III was positively correlated with the expression of TLP. TLP also facilitate Smad2 phosphorylation while, conversely, inhibiting Smad3 phosphorylation. TLP may play a cooperative role, along with the cytokine TGF-β1, in improving the overall cell viability of skin fibroblasts.Conclusions
TLP likely acts as a molecular modulator capable of altering the balance of Smad3- and Smad2-dependent signaling through regulation of phosphorylation, thus facilitating collagen synthesis in fibroblasts. Based on genetic variation in TLP levels in different tissues, these results suggest that TLP plays a key role in the process of TGF-β1/Smad3 signaling that contributes to wound healing and genesis of pathologic scars. 相似文献12.
Yuki Kishimoto Norikatsu Saito Katsumi Kurita Kentaro Shimokado Naoki Maruyama Akihito Ishigami 《Biochemical and biophysical research communications》2013,430(2):579-584
Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24 h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis. 相似文献
13.
Samuel Taddese Michael C. JungChristian Ihling Andrea HeinzReinhard H.H. Neubert Christian E.H. Schmelzer 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(4):731-739
Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly775-Leu776- in α-2 type I collagen and -Gly775-Ile776- in α-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III. 相似文献
14.
Human recombinant gamma-interferon stimulates proliferation and inhibits collagen and fibronectin production by human dental pulp fibroblasts 总被引:2,自引:0,他引:2
M Melin D J Hartmann H Magloire E Falcoff C Auriault J A Grimaud 《Cellular and molecular biology, including cyto-enzymology》1989,35(1):97-110
Human recombinant-gamma-interferon was tested on human dental pulp fibroblast activity in vitro. Fibroblast proliferation was estimated by a colorimetric test. Type I and type III collagens and fibronectin were quantified by radioimmunoassay in culture supernatant from confluent fibroblasts. A dose dependent stimulation of the proliferation was observed when fibroblasts were treated with recombinant-gamma-interferon. In contrast, an inhibition of the synthesis of soluble types I and III collagen and fibronectin by confluent cell cultures treated with recombinant-gamma-interferon occurred without apparent modification of the insoluble collagen level in the cell layer. Quantimetric analysis of type I collagen immunoperoxidase labelling have demonstrated that there was no intracellular storage of type I collagen in these cultured fibroblasts. These data support the view that human recombinant-gamma-interferon can affect human dental pulp fibroblast functions and thus may play an important part in the regulation of fibrosis. 相似文献
15.
16.
J ChandraRajan 《Biochemical and biophysical research communications》1978,83(1):180-186
Types I and III collagens were solubilized from fetal human skin by limited digestion with pepsin and precipitated by dialysis against 0.02 M Na2HPO4. Heat denaturation of the collagens in 2 M guanidine-HCl, pH 7.5, resulted in the precipitation of the contaminant pepsin which could be removed by centrifugation. Renaturation of the denatured collagens by dialysis against deionized water at 22° for 2 hours selectively precipitated the type III collagen fibrils. Type I collagen remained in solution. The simplicity and high recovery (77%) make this a suitable approach for the rapid estimation of type III collagen in small tissue samples. 相似文献
17.
Synthesis of type III collagen by fibroblasts from the embryonic chick cornea 总被引:2,自引:2,他引:0
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Synthesis of collagen types I, II, III, and IV in cells from the embryonic chick cornea was studied using specific antibodies and immunofluorescence. Synthesis of radioactively labeled collagen types I and III was followed by fluorographic detection of cyanogen bromide peptides on polyacrylamide slab gels and by carboxymethylcellulose chromatography followed by disc gel electrophoresis. Type III collagen had been detected previously by indirect immunofluorescence in the corneal epithelial cells at Hamburger-Hamilton stages 20--30 but not in the stroma at any age. Intact corneas from embryos older than stage 30 contain and synthesize type I collagen but no detectable type III collagen. However, whole stromata subjected to collagenase treatment and scraping (to remove epithelium and endothelium) and stromal fibroblasts from such corneas inoculated in vitro begin synthesis of type III collagen within a few hours while continuing to synthesize type I collagen. As demonstrated by double-antibody staining, most corneal fibroblasts contain collagen types I and III simultaneously. Collagen type III was identified biochemically in cell layers and media after chromatography on carboxymethylcellulose be detection of disulfide-linked alpha l (III)3 by SDS gel electrophoresis. The conditions under which the corneal fibroblasts gain the ability to synthesize type III collagen are the same as those under which they lose the ability to synthesize the specific proteoglycan of the cornea: the presence of corneal-type keratan sulfate. 相似文献
18.
Yozo Gotoh Shigeru Saito Atsushige Sato 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,587(2):253-262
Studies were performed to determine whether cultured odontogenic cells from rabbit tooth germ (RP cell) could synthesize dentine-like collagen. When cells were cultured with [14C]proline, 33% of the total incorporated proteins present were collagenous. Cultured RP cells were labelled with [14C]proline in the presence of β-aminopropionitrile. The resulting fractions, on analysis by CM-cellulose chromatography, contained three radioactive protein peaks, α1(I), [α1(III)]3, α2. From the radioactive measurements, RP cells synthesized a significant amount of type III collagen, comparable to type I collagen.DEAE-cellulose chromatography was used to separate collagen molecules from collagen precursors. The results showed that 60% of total collagen precursor was type III precursor and the remainder was type I precursor.CM-cellulose chromatography of CNBr peptides of collagen from culture medium and cell extract revealed the presence of type I and type III collagen. Thus, the RP cell, which is a diploid cell, is unique in the predominance of type III collagen in culture, differing thereby from the character of collagen in vivo. 相似文献
19.
Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts 总被引:20,自引:0,他引:20
A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells. 相似文献
20.
Terusama Hatahara Jerome M. Seyer 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,716(3):431-438
Hepatocytes were obtained from rat liver and maintained in primary culture for periods up to 14 days. Collagen synthesis was maximal after 3–5 days and declined thereafter. The rate of collagen production was appox. one-tenth that observed by the rat skin fibroblasts of the same animals after 3–5 passages. Type I procollagen, the major macromolecular collagenous species, was identified as a 450 000 dalton molecule which was converted to 120 000 dalton, denatured, reduced procollagen chains. Prior pepsin digestion of the native procollagen released 95 000 dalton collagen chains identified as α1(I) and α2(I) by co-migration with carrier rat skin type I collagen chains. The production of type III procollagen was also tentatively identified by DEAE-cellulose chromatography. This material was isolated and identified with type-specific antibodies developed against the amino-terminal extension peptide of bovine skin type III procollagen. The relative distribution of type I:type III procollagen was estimated at 7:3 similar to the ratio previously found in whole rat liver. No evidence of type IV or type V procollagen biosynthesis was observed. These results suggest that rat hepatocytes in primary culture are capable of interstitial type I and type III collagen biosynthesis in a ratio similar to that found in their parent hepatic tissue in situ. They also suggest that the less abundant type IV (basement membrane-associated) or type V are nor major collagenous products of these cells. 相似文献