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1.
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
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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
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Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I. 总被引:1,自引:0,他引:1
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H Rohde E Wachter W J Richter P Bruckner O Helles R Timpl 《The Biochemical journal》1979,179(3):631-642
The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule. 相似文献
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Understanding the relationship between cell shape and cellular function is important for study of cell biology in general and for regulation of cell phenotype in tissue engineering in particular. In this study, microcontact printing technique was used to create cell-adhesive rectangular and circular islands. The rectangular islands had three aspect ratios: 19.6, 4.9, and 2.2, respectively, whereas circular islands had a diameter of 50 microm. Both rectangular and circular islands had the same area of 1960 microm(2). In culture, we found that human tendon fibroblasts (HTFs) assumed the shapes of these islands. Quantitative immunofluorescence measurement showed that more elongated cells expressed higher collagen type I than did less stretched cells even though cell spreading area was the same. This suggests that HTFs, which assume an elongated shape in vivo, have optimal morphology in terms of expression of collagen type I, which is a major component of normal tendons. Using immunohistochemistry along with cell traction force microscopy (CTFM), we further found that these HTFs with different shapes exhibited variations in actin cytoskeletal structure, spatial arrangement of focal adhesions, and spatial distribution and magnitude of cell traction forces. The changes in the actin cytoskeletal structure, focal adhesion distributions, and traction forces in cells with different shapes may be responsible for altered collagen expression, as they are known to be involved in cellular mechanotransduction. 相似文献
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SPARC regulates processing of procollagen I and collagen fibrillogenesis in dermal fibroblasts 总被引:3,自引:0,他引:3
Rentz TJ Poobalarahi F Bornstein P Sage EH Bradshaw AD 《The Journal of biological chemistry》2007,282(30):22062-22071
A characterization of the factors that control collagen fibril formation is critical for an understanding of tissue organization and the mechanisms that lead to fibrosis. SPARC (secreted protein acidic and rich in cysteine) is a counter-adhesive protein that binds collagens. Herein we show that collagen fibrils in SPARC-null skin from mice 1 month of age were inefficient in fibril aggregation and accumulated in the diameter range of 60-70 nm, a proposed intermediate in collagen fibril growth. In vitro, procollagen I produced by SPARC-null dermal fibroblasts demonstrated an initial preferential association with cell layers, in comparison to that produced by wild-type fibroblasts. However, the collagen I produced by SPARC-null cells was not efficiently incorporated into detergent-insoluble fractions. Coincident with an initial increase in cell association, greater amounts of total collagen I were present as processed forms in SPARC-null versus wild-type cells. Addition of recombinant SPARC reversed collagen I association with cell layers and decreased the processing of procollagen I in SPARC-null cells. Although collagen fibers formed on the surface of SPARC-null fibroblasts earlier than those on wild-type cells, fibers on SPARC-null fibroblasts did not persist. We conclude that SPARC mediates the association of procollagen I with cells, as well as its processing and incorporation into the extracellular matrix. 相似文献
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An investigation was conducted on cadmium accumulation and its molecular distribution in growing cultures of human fetal lung fibroblasts (IMR-90). For the first 24–48 h post-exposure, the amount of cadmium per cell remained low and relatively constant; more than 50% of the intracellular Cd was associated with molecular weight components less than 2000 daltons. The presence of a Cd-binding component with a molecular weight of 11 800 daltons was also detected during this initial period. Based upon its molecular size, sensitivity to trypsin, and ability to coincorporate 35S along with 115Cd, we have concluded that this component is a protein. Its similarity to metallothionein was suggested by its molecular size, low level in cells never exposed to Cd, spectral properties, and heat stability. During the late log phase of growth, accumulation of Cd by fibroblasts occurred at a near-linear rate and the intracellular Cd level was proportional to the exogenous concentration. There was a corresponding increase in the amount of the fibroblast Cd-binding protein present, accompanied by a reduction in the amount of Cd associated with low molecular weight components. Synthesis of the Cd-binding protein appeared to occur at a more rapid rate than accumulation suggesting that its presence may be necessary for Cd transport and/or accumulation, an interpretation strengthened by the finding that cells previously passaged in Cd exhibited no lag in accumulation and accumulated 8–10-fold more Cd. By 168 h post-exposure, a plateau occurred in the intracellular Cd level as well as the amount of Cd-binding protein present. After this period, a redistribution of Cd from the metallothionein-like protein to high molecular weight proteins occurred. It is possible that this redistribution might be the cellular event that triggers the pathological changes known to occur after Cd-grown cultures have reached confluency. 相似文献
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Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not beta-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled alpha 2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment. 相似文献
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Hypoxia and amino acid deprivation downregulate expression of extracellular matrix genes in lung fibroblasts. We examined the effect of hypoxia on amino acid uptake and protein formation in human lung fibroblasts. Low O(2) tension (0% O(2)) suppressed incorporation of [(3)H]proline into type I collagen without affecting [(35)S]methionine labeling of other proteins. Initial decreases in intracellular [(3)H]proline incorporation occurred after 2 h of exposure to 0% O(2), with maximal suppression of intracellular [(3)H]proline levels at 6 h of treatment. Hypoxia significantly inhibited the uptake of radiolabeled proline, 2-aminoisobutyric acid (AIB), and 2-(methylamino)isobutyric acid (methyl-AIB) while inducing minor decreases in leucine transport. Neither cycloheximide nor indomethacin abrogated hypoxia-related suppression of methyl-AIB uptake. Efflux studies demonstrated that hypoxia inhibited methyl-AIB transport in a bidirectional fashion. The downregulation of amino acid transport was not due to a toxic effect; function recovered on return to standard O(2) conditions. Kinetic analysis of AIB transport revealed a 10-fold increase in K(m) accompanied by a small increase in maximal transport velocity among cells exposed to 0% O(2). These data indicate that low O(2) tension regulates the system A transporter by decreasing transporter substrate affinity. 相似文献
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Shingo Tajima Sheldon R. Pinnell 《Biochemical and biophysical research communications》1982,106(2):632-637
Ascorbic acid specifically stimulates collagen production in cultured human skin fibroblasts, an effect that appears to be independent of its cofactor role in prolyl and lysyl hydroxylation. In order to investigate the level of regulation of ascorbic acid on collagen synthesis, we have translated mRNA in a cell-free system derived from rabbit reticulocytes. Total RNA was prepared from normal human skin fibroblasts and similar fibroblasts which had been exposed to 100 uM ascorbic acid for four days. Ascorbic acid treatment resulted in a twofold stimulation of procollagen mRNA whereas non-collagenous mRNA was unchanged. These results reveal that ascorbic acid has a preferential stimulating effect on type I procollagen mRNA. 相似文献
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Coated vesicles were purified from embryonic chick tendon fibroblasts pulsed with [3H]proline. They were morphologically and biochemically similar to coated vesicles purified from other sources. Furthermore, they contained newly synthesized Type I procollagen which was protected from bacterial collagenase digestion unless detergent was present. The procollagen remained associated with coated vesicles during immune precipitation and agarose gel electrophoresis. Data from pulse-chase experiments demonstrated that the specific activity of the coated vesicle preparations was approx. 5-fold higher at the 10 min chase point than at either the 0 or 40 min chase points. These data are consistent with the hypothesis that coated vesicles are intermediates in the intracellular transport of newly synthesized Type I procollagen in chick tendon fibroblasts. 相似文献
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Transformation of the human embryonic lung fibroblast line, WI-38, with simian virus 40 (SV40) results in inactivation of the type I procollagen genes. No type I collagen or procollagen mRNA is detected in these transformed cells, as determined by polyacrylamide gel electrophoresis. Analysis of the methylation patterns of these genes showed the type I procollagen genes to be hypermethylated at certain cytosine residues in the transformed cells. However, several of the cytosine residues were methylated in the normal cells where these genes are expressed. These methylation patterns can be altered by treatment of the cells with 5-azacytidine or 5-azadeoxycytidine, but without a resultant activation of the type I procollagen genes. These results show that demethylation alone is not sufficient for gene activation, but that other signals are also required. 相似文献
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H M Garnett 《Archives internationales de physiologie et de biochimie》1979,87(3):443-454
Human embryonic fibroblasts accumulate Ca2+ in the presence of extracellular ATP and Mg2+, the uptake being maximal at 3 mM ATP. Iodoacetic acid, oligomycin and temperatures of 2 degrees C all inhibit the ATP-potentiated uptake suggesting that an active process may be involved in the transport of Ca2+ into these cells under certain conditions. 相似文献
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