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1.
To investigate whether peripheral blood granulocytes can synthesize the adhesive glycoprotein, fibronectin, we sought to demonstrate the presence of messenger RNA coding for fibronectin within mature circulating granulocytes. Polyadenylated-enriched RNA was isolated from human peripheral blood granulocytes, human skin fibroblasts (synthesize fibronectin) and HeLa cells (lack fibronectin) and probed with a cDNA clone coding for the cell attachment domain of fibronectin. Hybridization of a fibronectin cDNA fragment occurred with fibroblast RNA but did not occur with granulocyte RNA despite a 100 fold excess granulocyte RNA. Incubation of granulocytes with n-formyl methionyl leucyl phenylalanine, a chemotactic peptide known to augment the release of fibronectin from granulocytes, failed to induce detectable levels of mRNA for fibronectin in granulocytes. There was no difference in the quantity of fibronectin released from chemotactic peptide-stimulated granulocytes pre-incubated in the presence or absence of the protein synthesis inhibitor, cycloheximide, suggesting that fibronectin exists in a stored form in granulocytes. These data suggest that fibronectin in mature granulocytes is the product of synthesis during early myeloid maturation.  相似文献   

2.
1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-3H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated 3H was present as [3H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [3H]fucose-labelled material before and after trypsin digestion. 3. The [3H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [3H]fucose and 14C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [35S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.  相似文献   

3.
P J McCormick  B J Keys  C Pucci  A J Millis 《Cell》1979,18(1):173-182
This report describes the identification and partial characterization of a 100K dalton “glucose-regulated” cell surface protein of human diploid fibroblasts (HDF). This protein is released into and can be recovered virtually intact from the surrounding culture medium. At the present level of analysis, the protein recovered from the culture medium (“conditioned medium”) is indistinguishable from the protein extracted directly from the cell surface by 1 M urea treatment. Both proteins have molecular weights of 100K daltons when analyzed by gel electrophoresis. The protein is readily labeled at the cell surface via lactoperoxidase-catalyzed iodination, and the label can be chased into the released form of this protein in conditioned medium. Antiserum raised against the medium form of the protein reacts with the surface form of the protein but does not react with fibronectin, the major cell surface protein of HDF. Conditioned medium from SV40-transformed human fibroblasts does not contain the 100K protein, but instead contains a component that has a slightly lower molecular weight (97K daltons). The lower molecular weight band does not iodinate at the cell surface and is apparently an underglycosylated form of the 100K protein. Its molecular weight is shifted back to 100K by growing transformed cells in medium containing excess glucose. After the shift, the component becomes accessible to the radioiodine label. We suggest that the 100K protein is a glucose-regulated protein (Shiu, Pouyssegur and Pastan, 1977; Pouyssegur and Yamada, 1978) that is released into the culture medium. An underglycosylated form of the same glycoprotein is released from transformed cells.  相似文献   

4.
Thrombospondin (TS), a 450,000 molecular weight glycoprotein, is released from α-granules of thrombin-activated platelets and is secreted and incorporated into the extracellular matrix by several cell types in culture. We have examined the effects of cell density and transformation on the production of TS in cell culture. The levels of TS, per cell, in the culture medium of endothelial cells, smooth muscle cells, and fibroblasts were greater at lower cell densities; in fibroblasts the levels of two other extracellular matrix proteins, fibronectin and collagen, were unaffected by cell density. Our evidence indicates that the higher levels of TS in the culture medium, determined for lower-density cells, were achieved by an increased secretion of the protein rather than by a reduction in degradation or incorporation into the extracellular matrix. TS production by normal and transformed Wl-38 fibroblasts was the same, although the fibronectin level in the culture medium of the transformed cells was substantially decreased. These findings suggest that the production of TS by cells in culture is regulated in a different fashion from that of fibronectin or collagen.  相似文献   

5.
The major protein released into the medium by human skin fibroblasts in culture has been shown to be a fucosylated glycoprotein (designated MFGP). Analysis by gel filtration chromatography and polyacrylamide gel electrophoresis demonstrated that under reducing conditions MFGP has a molecular weight of approx. 250,000, but occurs as a disulphide-linked aggregate in the medium. Three lines of evidence are presented to establish that MFGP is a non-collagenous molecule.  相似文献   

6.
Phosphatidylinositol (PI) specific phospholipase C (PIase C) treatment of human platelets caused release of a surface glycoprotein in the medium. Human blood platelets were isolated by low speed centrifugation and surface glycoproteins were labelled with periodate/[3H]borohydride procedure. Intact surface-labelled platelets were treated with PIase C purified from culture filtrates of Staphylococcus aureus (SA) or Bacillus thuringiensis (BT). After PIase C treatments platelets were spun at low speed, pellet and supernatant were separated. The supernatant was further centrifuged at high speed (140,000 x g) for 30 min. The resulting supernatant and the pellet from low speed were subjected to SDS-PAGE analysis. Protein patterns were obtained by fluorography. Release of a specific glycoprotein of approx. 150 kDa in the medium was observed due to the PIase C treatment. Prolonged incubation of platelets in 0.25 M sucrose and depletion of NaCl concentrations also affected the release of this glycoprotein. BT-PIase C released more approx. 150 kDa protein than SA-PIase C. Western blot experiment with a monoclonal antibody (mAB), epitope SZ2, reactive to human platelet surface glycoprotein Ib (GPIb) complex, confirmed that released 150 kDa glycoprotein reacted with mAB of GPIb. The release of this protein by PIase C was not inhibited by proteinase inhibitors (EDTA, PMSF and leupeptin). Treatment of human platelet membranes with PIase C also caused release of this glycoprotein as evidenced by reactivity to GPIb-mAB. These studies demonstrate that PIase C treatment causes release of 150 kDa glycoprotein from human platelet membrane surface. It is suggested that 150 kDa glycoprotein is anchored to PI in human platelets and that this glycoprotein represents the GPIb complex.  相似文献   

7.
The distribution in the cellular monolayer of the de novo synthetized pre-labeled glycoproteins and fibronectin upon culturing of fibroblasts in the medium with low serum content was analyzed. It was found that in rheumatoid arthritis (RA) the amount of total glycoproteins on the surface and within fibroblasts is higher and in the extracellular matrix is lower than in skin fibroblasts of healthy donors (HD). However, the amount of pre-labeled fibronectin on the surface of skin fibroblasts from patients with RA was considerably lower than in those from HD This finding as well as a rapid decrease in the amount of pre-labeled fibronectin in the extracellular matrix of RA fibroblasts is indicative of a more rapid metabolism of this protein in RA. In the skin fibroblasts from HD there was a practically uniform decrease in the amount of pre-labeled fibronectin in the cellular monolayer. The presence of caseinolytic activity in the culture medium even upon the first day of cell culturing in the serum-free medium, as well as the effect of various proteinase inhibitors on glycoprotein content in the cellular monolayer provide evidence that the rate of glycoprotein and fibronectin metabolism, especially in connective tissue cells of patients with RA, might possibly be determined not only by the level of their synthesis but also by the level of proteolytic activity in the connective tissue cells.  相似文献   

8.
Virus-transformed fibroblasts show an increased production of proteases as well as loss of extracellular adhesive proteins. To determine whether these transformation-associated events are related, we investigated the capacity of Rous sarcoma virus-transformed cells (embryonic chick fibroblasts and mouse BALB/c 3T3) to degrade fibronectin by using a novel cross-linked protein substratum: fluorescence-labeled or radiolabeled fibronectin covalently linked to the surface of a fixed gelatin film. In serum-containing medium, the coupled fibronectin was not released when incubated without cells, and only a small amount was released when incubated with nontransformed cells. However, when transformed cells were seeded on the radiolabeled fibronectin-coupled substratum, there was a threefold increase in the time-dependent release of radioactivity into the medium. The released material was characterized as peptides with molecular sizes of less than 30,000 daltons. Correspondingly, growth of transformed cells on the rhodamine-fibronectin substratum resulted in the appearance of discrete negative fluorescent spots beneath the cells and along their migratory paths, whereas a uniform fluorescent carpet was detected with nontransformed cells. The release of radioactivity was partially inhibited by protease inhibitors, including alpha 2-macroglobulin, leupeptin, and benzamidine, but the negative fluorescent spots appeared unaffected by any of these inhibitors. However, both the release of radiolabeled peptides and the appearance of fluorescence-negative spots were inhibited by 1,10-phenanthroline at concentrations that did not affect cellular attachment and protein synthesis, thus supporting a role for proteases in localized degradation of fibronectin substratum. These fluorescence-negative spots coincided with sites of fibronectin disappearance as judged by indirect labeling with antibodies to cellular fibronectin. In addition, immunofluorescent analyses showed a correlation between vinculin localization and the negative fibronectin spots found under transformed cells, indicating that degradation occurs at cell substratum contact sites. These results can be correlated with other transformation-associated phenotypic changes, and are discussed in terms of the invasion of tumor cells into the extracellular matrix.  相似文献   

9.
Summary In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.  相似文献   

10.
F Grinnell  M K Feld 《Cell》1979,17(1):117-129
Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.  相似文献   

11.
The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) did not alter the binding and release kinetics of externally added 125I-labeled plasma fibronectin to human lung fibroblasts in culture. Cell layer-bound plasma fibronectin was found to be chased into the medium at the same rate in tumor-promoter-treated as in non-treated cells. Unlabeled fibronectin accumulated to a much higher degree in the medium when tumor promoter was present. We conclude that TPA does not interfere with the fibronectin receptor on substrate-attached fibroblasts, but may influence intracellular fibronectin before it is bound to the extracellular matrix.  相似文献   

12.
Eicosanoids are lipid-signaling mediators released by many cells in response to various stimuli. Increasing evidence suggests that eicosanoids such as leukotrienes and prostaglandins (PGs) may directly mediate remodeling. In this study, we assessed whether these substances could alter extracellular matrix (ECM) proteins and the inflammatory profiles of primary human airway smooth muscle cells (ASM) and fibroblasts. PGE(2) decreased both fibronectin and tenascin C in fibroblasts but only fibronectin in ASM. PGD(2) decreased both fibronectin and tenascin C in both ASM and fibroblasts, whereas PGF(2α) had no effect on ECM deposition. The selective PGI(2) analog, MRE-269, decreased fibronectin but not tenascin C in both cell types. All the PGs increased IL-6 and IL-8 release in a dose-dependent manner in ASM and fibroblasts. Changes in ECM deposition and cytokine release induced by prostaglandins in both ASM and fibroblasts were independent of an effect on cell number. Neither the acute nor repeated stimulation with leukotrienes had an effect on the deposition of ECM proteins or cytokine release from ASM or fibroblasts. We concluded that, collectively, these results provide evidence that PGs may contribute to ECM remodeling to a greater extent than leukotrienes in airway cells.  相似文献   

13.
K Olden  K M Yamada 《Cell》1977,11(4):957-969
The major cell surface glycoprotein of cultured chick embryo fibroblasts (CSP, a LETS protein) is substantially decreased after neoplastic transformation. We investigated the regulation of this glycoprotein by determining the kinetics of CSP biosynthesis, transit to the cell surface, and degradation before and after transformation by Rous sarcoma virus. CSP synthesis, as measured by immunoprecipitation after pulse-labeling with 14C-leucine, is decreased 3–6 fold after transformation by the Bryan high titer, Schmidt-Ruppin and temperature-sensitive ts68 and T5 strains of Rous sarcoma virus. Steady state quantities of CSP in intracellular pools are also decreased 4–5 fold after transformation. However, the rate at which newly synthesized CSP is processed and exported to the cell surface is similar before and after transformation.Degradation and release of CSP from cells were measured after labeling for 24 hr. The half-life of CSP on normal cells is 36 hr and is decreased to 16–26 hr after transformation. The absolute amount of intact CSP released into the culture medium is decreased 3 fold after transformation; these amounts, however, represent losses of approximately 20 and 40% of the total CSP synthesized by normal and transformed cells, respectively. These results indicate that the major mechanism for the decrease in CSP after transformation is reduction in its biosynthesis, although increased degradation and loss from the cell surface also contribute significantly. These changes can account for the observed 5–6 fold decreases in cell-associated CSP after transformation of chick embryo fibroblasts.  相似文献   

14.
In testing the hypothesis that the stimulation of the release of fibronectin (FN) by 12-O-tetradecanoylphorbol 13-acetate (TPA) from human lung fibroblasts in culture is the result of activation of protein kinase C (PKC), we found that the PKC inhibitor sphingosine strongly inhibited FN release in presence and even in absence of TPA. However, a different PKC inhibitor, calphostin C, despite almost complete inhibition of PKC, had no effect on FN release. We concluded that sphingosine is a potent inhibitor of FN release from the cell surface, independent of its inhibition of PKC; and that TPA stimulates release of FN by a pathway other than activation of PKC. We found that the activation of PKC by TPA was accompanied by inhibition of the cAMP-dependent protein kinase (PKA). When PKA was inhibited by an antagonist (H8, a cAMP analogue) at a concentration specific for PKA inhibition, the release of FN was stimulated similar to the stimulation with TPA. Activation of PKA with forskolin resulted in decreased FN release. In conclusion, we have shown that: (1) sphingosine had a robust effect inhibiting the release of FN from fibroblasts, independent of its action on PKC; (2) TPA treatment of these cells resulted in inhibition of PKA; (3) inhibition of PKA stimulated FN release whereas its activation decreased this release. It is possible that PKA, by phosphorylating a protein, may function, directly or indirectly, in keeping FN attached to the cell surface of fibroblasts.  相似文献   

15.
In our previous study the macromolecular complexes mostly consisting of fibronectin and procollagen were isolated from human fibroblast culture media using immobilized antibodies against fibronectin. At present an attempt was made to elucidate at what stage the formation of the fibronectin-collagen complex occurs--either in the course of incubation of the immobilized antibodies with labelled proteins secreted by fibroblasts, or in the extracellular space while labelling fibroblasts, or intracellularly. The results obtained show that the fibronectin-collagen complex: 1) pre-exists even before incubation with the immobilized antibodies and 2) it is of intracellular origin. Thus, the considerable amount of the fibroblast-secreted fibronectin (no less than 20%) is released from the cell not in the free form but in the complex with procollagen. It was suggested that the fibronectin-collagen complex presents a stage in the formation of the insoluble extracellular matrix.  相似文献   

16.
We investigated the actions of human placental lactogen (HPL) and human growth hormone (HGH) on [3H]thymidine incorporation and the release of immunoassayable somatomedin-C (SM-C) by isolated myoblasts, dermal fibroblasts, and costal cartilage explants taken from human fetuses at 11-21 weeks of gestation. The incorporation of [3H]thymidine by myoblasts and fibroblasts was significantly increased after incubation for 20 hr or 44 hr, and cell number after incubation for 7 days, in the presence of 50-250 ng/ml HPL. Incubation with HPL did not increase [3H]thymidine incorporation into cartilage explants, whereas incubation with HGH failed to enhance the uptake of this isotope by any of the tissues. Following extraction with acid-ethanol, culture medium conditioned by exposure to myoblasts or fibroblasts for 44 hr, and to cartilage explants for 7 days, contained radioimmunoassayable SM-C. Myoblast-conditioned medium contained significantly more SM-C [1,609 +/- 953 mU/mg cell protein (mean +/- SD); n = 10] than did that conditioned by fibroblasts (637 +/- 323; n = 5; P less than 0.02). In 1 week of culture, cartilage explants released 4.1 +/- 1.1 mU/mg wet weight (n = 7). The release of immunoassayable SM-C from cultured cells was significantly increased in the presence of 250 ng/ml HPL in five of eight experiments with myoblasts and two of four experiments with fibroblasts. Neither fibroblasts or myoblasts showed increased SM-C release following exposure to HGH. The results suggest that HPL, but not HGH, is growth-promoting for some human fetal tissues in vitro and that this action is mediated, at least in part, by an increased release of somatomedins.  相似文献   

17.
The carbohydrates of human fibronectin released from non-transformed human fibroblasts WI-38 have been compared with those of fibronectin released from SV40 virus transformed WI-38/VA13 cells and those of fibronectin prepared from human plasma. The majority of the bi-antennary glycopeptides of fibronectin released from WI-38 fibroblasts was not sialylated at the terminal galactosyl residues, but was fucosylated at the coreN-acetylglucosaminyl residue directly linked to a peptide (structure A, below). Most of the minor sialylation detected was linked 2–3 to galactose. In contrast, the majority of the bi-antennary glycopeptides released from the transformed VA13 cells was highly sialylated at the terminal galactosyl residues with both 2–3 and 2–6 linkages, but was only partially fucosylated at the coreN-acetylglucosaminyl residue (structure B, below). This structure was similar to that of the bi-antennary glycopeptide of human plasma fibronectin which was, however, predominantly sialylated with an 2–6 linkage (structure C, below). These human fibronectins, regardless of their source, lack a high molecular weight lactosaminoglycan structure.In addition to the differences in bi-antennary structure described above, the quantity of tri- to tetra-antennary glycopeptides of fibronectin released from VA13 transformed cells was found to be much greater than the quantity of these glycopeptides of fibronectin released from normal WI-38 fibroblasts. Furthermore, there was a relatively small quantity of a glycopeptide fraction having a smaller molecular weight that did not bind to Con A-Sepharose and was separated on a Bio-Gel P-4 column. The presence of this fraction was characteristic for fibronectin released from transformed VA13 cells, and the fraction was absent in fibronectin from normal fibroblasts.  相似文献   

18.
Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV-40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation.  相似文献   

19.
Late passage fibroblasts show decreased cell-substrate adhesion. We provide evidence that the reduced adhesion is due to a defect in the adhesive glycoprotein fibronectin. Late passage cells become more adhesive in culture media that has been conditioned by the growth of early passage cells. Analysis of fibronectins purified from early and late passage cell conditioned media indicates that there are striking differences in their abilities to promote cell adhesion. Young cell fibronectin supports the maximal adhesion of both young and old cells. However, old cells require quantitatively more fibronectin. In contrast, old cell fibronectin is less effective in supporting the adhesion of either cell type. In addition, neither cell type achieves a normal morphology in the presence of old cell fibronectin. The results support the conclusion that the fibronectin released by late passage cells is defective and does not support normal cell-substrate interactions.  相似文献   

20.
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