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1.
Plasminogen activators of the urokinase- and tissue-type and fetal calf serum (u-PA, t-PA, FCS) exert their mitogenic effect on quiescent human dermal fibroblasts and modulate the mRNA expression of cell-cycle related genes. The present study deals with the effects of PAs on the expression of fibronectin (FN), a heterodimeric extracellular matrix (ECM) protein that can be modulated in different ways by various mitogens. The kinetics of FN gene response was examined in quiescent fibroblasts upon PA stimulation (30 min -24 h). The results obtained evidenced that: (i) all mitogens tested (u-PA, t-PA and FCS) led to an increase of FN mRNA expression in early G1, as shown by the analysis of two sequences, III-9, common to all FN mRNAs, and EDA+, present only in the EDA+FN isoform; (ii) the kinetic profiles of FN mRNA stimulation were comparable for the three mitogens, although the effects on the FN-ECM assembly were distinct; (iii) t-PA and FCS led to FN assembly in the ECM, which was absent or decreased in u-PA-treated cultures. Immunobiochemical analysis of total FN and EDA+ FN showed that FN induced by t-PA was mainly dimeric (450-500 kDa), whereas FN induced by u-PA was mainly monomeric (230-250 kDa). These differences are probably due to the differential enzymatic action of t-PA and u-PA on FN, which might be related to a differential role of the two PAs in several physiopathological conditions. 相似文献
2.
Preparation of gellan sulfate as an artificial ligand for removal of extra domain A containing fibronectin 总被引:1,自引:0,他引:1
Miyamoto K Asakawa Y Arai Y Shimizu T Tokita M Komai T 《International journal of biological macromolecules》2001,28(5):381-385
The extra domain A containing fibronectin (EDA(+)FN) concentration in plasma of rheumatoid arthritis (RA) is abnormally higher than the normal level. We synthesized various gellan-sulfate (GS) candidates as artificial ligands for removing EDA(+)FN from plasma. The interaction between these artificial ligands and EDA(+)FN was evaluated using affinity constants (KA), which were determined by surface plasmon resonance measurement. The KA (3.6×108 per M) of GS-25 [degree of substitution for sulfonation (DS)=25%] with EDA(+)FN was higher than those of other molecules: GS-16 (DS=16%) at 8.3×107 per M, and GS-35 (DS=35%) at 1.7×108 per M. Furthermore, GSs displayed selectivity of EDA(+)FN for binding with plasma FN (KAEDA(+)FN/KAplasma FN>2). The removal ratio in plasma was measured by using GS-immobilized gel. Removals of 66, 11, 7.7, 6.2, 6.9, and 12% for EDA(+)FN, plasma FN, fibrinogen, albumin, immunoglobulin G (IgG) and antithrombin III from the patient-model plasma were, respectively, achieved with GS-25-immobilized gel. These results suggest that GS may be used as a selective artificial ligand for EDA(+)FN removal from plasma in RA treatment. 相似文献
3.
Induction of Fibronectin (FN) gene expression by platelet-derived growth factor (PDGF) isoforms in rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances FN levels in SMC cultures in a time- and concentration-response fashion. PDGF-AA and PDGF-AB show no effect on FN levels. The effects of insulin and insulin-like growth factor-I (IGF-I) on PDGF-BB-induced FN levels were examined. No additivity of FN levels is observed between PDGF-BB and insulin and/or IGF-I. Experiments also show that PDGF-BB enhances FN mRNA levels, implying that acquisition of additional FN mRNA units accounts for the increase in FN levels. Induction of FN and FN mRNA levels by PDGF-BB could be one of the initial events in vascular SMC proliferation and extracellular matrix expansion, leading to atherosclerosis and hypertension. 相似文献
4.
Altered rate of fibronectin matrix assembly by deletion of the first type III repeats 总被引:9,自引:4,他引:5
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《The Journal of cell biology》1996,134(2):573-583
The assembly of fibronectin (FN) into a fibrillar matrix is a complex stepwise process that involves binding to integrin receptors as well as interactions between FN molecules. To follow the progression of matrix formation and determine the stages during which specific domains function, we have developed cell lines that lack an endogenous FN matrix but will form fibrils when provided with exogenous FN. Recombinant FNs (recFN) containing deletions of either the RGD cell- binding sequence (RGD-) or the first type III repeats (FN delta III1-7) including the III1 FN binding site were generated with the baculovirus insect cell expression system. After addition to cells, recFN matrix assembly was monitored by indirect immunofluorescence and by insolubility in the detergent deoxycholate (DOC). In the absence of any native FN, FN delta III1-7 was assembled into fibrils and was converted into DOC-insoluble matrix. This process could be inhibited by the amino- terminal 70 kD fragment of FN, showing that FN delta III1-7 follows an assembly pathway similar to FN. The progression of FN delta III1-7 assembly differed from native FN in that the recFN became DOC-insoluble more quickly. In contrast, RGD- recFNs were not formed into fibrils except when added in combination with native FN. These results show that the RGD sequence is essential for the initiation step but fibrils can form independently of the III1-7 modules. The altered rate of FN delta III1-7 assembly suggests that one function of the missing repeats might be to modulate an early stage of matrix formation. 相似文献
5.
The role of endogenously synthesized fibronectin (FN) in assembly was studied with cells lacking or expressing FN. Cells were cultured as homogeneous or mixed populations on surfaces coated with different matrix proteins. Compared with FN-expressing cells, FN-null cells poorly assembled exogenous plasma FN (pFN) when adhered to vitronectin or the recombinant cell-binding domain (III(7-10)) of FN. Vitronectin had a suppressive effect that was overcome by co-adsorbed pFN or laminin-1 but not by soluble FN. In co-cultures of FN-expressing cells and FN-null cells, endogenous FN was preferentially assembled around FN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was vitronectin, exogenous pFN assembled preferentially around cells expressing cellular FN or recombinant EDa- or EDa+ FN. In co-cultures on vitronectin of FN-null cells and beta(1) integrin subunit-null cells, fibrils of cellular FN and pFN were preferentially deposited by FN-null (beta(1)-expressing) cells immediately adjacent to (FN-secreting) beta(1)-null cells. In co-cultures on vitronectin of FN-null cells and beta(1)-null cells expressing a chimera with the extracellular domain of beta(1) and the cytoplasmic domain of beta(3), preferential assembly was by the chimera-expressing cells. These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN (suppressive if vitronectin, non-suppressive but non-supportive if III(7-10), supportive if pFN or laminin-1) and suggest that efficient interaction of freshly secreted cellular FN with a beta(1) integrin, presumably alpha(5)beta(1), substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or pFN to support assembly of FN. 相似文献
6.
Bjorn Steffensen Zhihua ChenSanjay Pal Margarita MikhailovaJianrong Su Yao WangXiaoping Xu 《Matrix biology》2011,30(1):34-42
Fibronectin (FN) purified by gelatin affinity chromatography is unstable and undergoes fragmentation. The cleavage has been ascribed to inherent autolytic protease activities as well as co-purified matrix metalloproteinases (MMP). Understanding the mechanism by which the proteolysis of FN occurs is important, because the FN fragments have biological activities that differ from those of intact FN. Having excluded contributions of other plasma-derived proteases, the present experiments demonstrated that cleavage of FN by MMP-2 to distinct fragments occurred in synergy with inherent FN activities. Limited heat treatment of FN at 56 °C for 30 min inactivated the inherent protease activities sharply reducing autolysis of FN in a manner similar to that seen in the presence of serine proteinase inhibitors. Heat treatment did not alter cell attachment to FN, but significantly increased the susceptibility of FN to enzymatic cleavage by MMP-2. The carboxyl-terminal hemopexin-like domain (PEX) of MMP-2 was shown to possess critical exodomain properties required for the interactions of MMP-2 with FN, and FN was cleaved at a significantly reduced rate by an MMP-2 variant with deletion of PEX. Verifying the specificity of interactions, isolated PEX competed FN cleavage by MMP-2 in a concentration-dependent manner. These results have further elucidated the synergistic contributions of inherent autolytic serine protease-like activities and MMP-2 to fragmentation of FN and provide the rationale and basis for modified preparation and handling of FN used in biological research. 相似文献
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9.
Y Murata H Seo K Sekiguchi T Imai J Lee N Matsui 《Molecular endocrinology (Baltimore, Md.)》1990,4(5):693-699
10.
F Buchwald A L Tabachnik G A Ermolin N I Zhidkova V E Kotelianski? 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1987,(12):28-33
The interaction of 62 S. aureus clinical strains and, respectively, 20 and 17 isolated S. epidermidis and S. saprophyticus strains with human blood plasma fibronectin (FN) has been studied. The specific interaction of FN with bacteria has been evaluated simultaneously by the binding of 125I with FN (method 1), the FN-mediated agglutination of staphylococci (method 2) and the character of colonies formed in 0.15% agar medium containing FN (method 3). The data obtained in this investigation indicated that all S. aureus strains under study react with FN to a different extent. When evaluating the binding of FN with bacteria, the most pronounced correlation was observed between methods 1 and 3. None of the methods used in this investigation has revealed interaction between FN and S. epidermidis and S. saprophyticus strains under study. The authors suggest that a preliminary inference on the capacity of the isolated clinical strains of staphylococci for reaction with FN may be made from the character of colonies formed in 0.15% agar medium containing FN. 相似文献
11.
AimsWe investigated the protective effects of chemical stimulation of cerebellar fastigial nucleus (FN) on stress gastric mucosal injury (SGMI) and its possible neuro-regulatory mechanisms in rats.Main methodsChemical stimulation, electrical stimulation, chemical ablation, electrolytic lesion, and microinjection were used to investigate the effects of FN simulation on SGMI. The model of SGMI was established by restraint and water (21 ± 1 °C)-immersion (RWI) for 3 h in rats. The gastric mucosal injury index indicated the severity of gastric mucosal injuries.Key findingsWe showed that microinjection of L-glutamic acid into the FN or electrical stimulation of the FN markedly attenuated SGMI. Either chemical lesion of the FN or electrical ablation of the decussation of superior cerebellar peduncle (DSCP) obviously aggravated SGMI. The protective effect of FN stimulation on SGMI was reversed after chemical ablation of the lateral hypothalamic area (LHA). The protective effect of FN was prevented by pretreatment with the glutamic acid decarboxylase antagonist, 3-MPA into the FN or GABAA receptor antagonist, bicuculline into the LHA. The protective effect of FN was abolished by pretreatment with sympathectomy. The discharge frequency of greater splanchnic nerve (GSN) was decreased and gastric mucosal blood flow (GMBF) was increased after chemical stimulation of FN. These results indicate that the FN participates in regulation of SGMI, and is a specific area in the CNS for exerting protective effects on the SGMI. The DSCP, LHA and peripheral sympathetic nerve may be involved in this process.SignificanceOur findings might provide a new and improved understanding of the cerebellar function and an effective treatment strategy for stress gastric mucosal injury. 相似文献
12.
Fibronectins (FNs) are dimeric glycoproteins that adopt a globular conformation when present in plasma and solution and an extended conformation in the extracellular matrix. Factor XII (FXII) is a zymogen of the proteolytically active FXIIa that plays a role in thrombus stabilization by enhancing clot formation and in inflammation by enhancing bradykinin formation. To investigate whether the extracellular matrix could play a role in these events, we have recently shown that FXIIa, but not FXII, binds to the extracellular matrix (ECM), and suggested that FN may be the target for the binding. Immunofluorescence microscopy has in the present investigation confirmed that FXIIa added to the ECM colocalizes with FN deposited during growth of human umbilical vein endothelial cells. The aim of the present study, therefore, was to further elucidate the interaction between FXIIa and FN by the use of a solid face binding assay. This showed, like the binding to the ECM, that FXIIa, but not FXII, binds in a Zn2+-independent manner to immobilized FN. The K(D) for the binding was 8.5 +/- 0.9 nM (n = 3). The binding was specific for the immobilized FN, as the binding could not be inhibited by soluble FN. Furthermore, soluble FN did not bind to immobilized FXIIa. However, soluble FN could bind to FXII, and this binding inhibited the surface-induced autoactivation of FXII and subsequent binding of the generated FXIIa to immobilized FN. The presence of FXII in an anti-FN immunoprecipitate of plasma indicated that some FXII in plasma circulates bound to FN. The binding of FXIIa to FN was inhibited by gelatine and fibrin but not by heparin, indicating that FXIIa binds to immobilized FN through the type I repeat modules. Accordingly, FXIIa was found to bind to immobilized fragments of FN containing the type I repeat modules in the N-terminal domain to which fibrin and gelatine bind. 相似文献
13.
Loss of fibronectin (FN) assembly in the extracellular matrix has long been recognized as a feature of cellular transformation. However, such assembly is regulated not only by FN synthesis but also by its post-translational modifications. The mechanism controlling FN protein stability has remained unclear so far. Recently it was demonstrated that FN matrix turnover occurs intracellularly at the lysosome following caveolin-1-dependent endocytosis. Although FN was reported to undergo ubiquitindependent degradation, the ubiquitin ligase responsible for FN ubiquitination is unknown. In this study, we have identified beta-TrCP as the ubiquitin ligase for lysosomal degradation of FN. We found two conserved beta-TrCP recognition motif (DSGVVYS and DSGSIVVS) in the primary amino acid sequence of human, mouse, and rat FN. Down-regulation of either beta-TrCP1 or beta-TrCP2 by small interference (siRNA) caused significant accumulation of FN. Immunolocalization studies showed intracellular accumulation of FN in beta-TrCP siRNA-treated cells without showing much alteration in its matrix association. We also observed that exposure of cells to UV irradiation effectively down-regulated FN following increased ubiquitination, which was significantly inhibited either by lysosomal inhibitor or by siRNA-mediated down-regulation of beta-TrCP. Taken together, constitutive FN degradation, as well as UV-induced degradation, is ubiquitination dependent and controlled by beta-TrCP. 相似文献
14.
Khan ZA Farhangkhoee H Mahon JL Bere L Gonder JR Chan BM Uniyal S Chakrabarti S 《Experimental biology and medicine (Maywood, N.J.)》2006,231(6):1022-1029
Fibronectin (FN), a key extracellular matrix protein, is upregulated in target organs of diabetic angiopathy and in cultured cells exposed to high levels of glucose. FN has also been reported to undergo alternative splicing to produce the extra domain-B (ED-B) containing isoform, which is exclusively expressed during embryogenesis, tissue repair, and tumoral angiogenesis. The present study was aimed at elucidating the role and mechanism of endothelins (ETs) in FN and ED-B FN expression in diabetes. We investigated vitreous samples for ED-B FN expression from patients undergoing vitrectomy for proliferative diabetic retinopathy. Our results show increased FN and ED-B FN expression in the vitreous of diabetic patients in association with augmented ET-1. Using an antibody specific to the ED-B segment of FN, we show an increase in serum ED-B FN levels in patients with diabetic retinopathy and nephropathy. We further examined retinal tissues, as well as renal and cardiac tissues, from streptozotocin-induced diabetic rats. Diabetes increased FN and ED-B FN in all three organs, which was prevented by ET antagonist bosentan. To provide insight into the mechanism of glucose-induced and ET-mediated ED-B FN upregulation, we assayed endothelial cells (ECs). Inhibition of mitogen-activated protein kinase with pharmacological inhibitors and protein kinase B with dominant negative transfections prevented glucose- and ET-1-mediated FN and ED-B FN expression. Furthermore, treatment of cells exposed to high levels of glucose with ET antagonist prevented the activation of all signaling pathways studied and normalized glucose-induced ED-B FN expression. We then determined the functional significance of ED-B in ECs and show that ED-B FN is involved in vascular endothelial growth factor expression and cellular proliferation. These studies show that glucose-induced and ET-mediated FN and ED-B FN expressions involve complex interplays between signaling pathways and that ET may represent an ideal target for therapy in chronic diabetic complications. 相似文献
15.
There is uncertainty about the potential reduction of soil nitrous oxide (N(2)O) emission when fertilizer nitrogen (FN) is partially or completely replaced by biological N fixation (BNF) in temperate grassland. The objectives of this study were to 1) investigate the changes in N(2)O emissions when BNF is used to replace FN in permanent grassland, and 2) evaluate the applicability of the process-based model DNDC to simulate N(2)O emissions from Irish grasslands. Three grazing treatments were: (i) ryegrass (Lolium perenne) grasslands receiving 226 kg FN ha(-1) yr(-1) (GG+FN), (ii) ryegrass/white clover (Trifolium repens) grasslands receiving 58 kg FN ha(-1) yr(-1) (GWC+FN) applied in spring, and (iii) ryegrass/white clover grasslands receiving no FN (GWC-FN). Two background treatments, un-grazed swards with ryegrass only (G-B) or ryegrass/white clover (WC-B), did not receive slurry or FN and the herbage was harvested by mowing. There was no significant difference in annual N(2)O emissions between G-B (2.38±0.12 kg N ha(-1) yr(-1) (mean±SE)) and WC-B (2.45±0.85 kg N ha(-1) yr(-1)), indicating that N(2)O emission due to BNF itself and clover residual decomposition from permanent ryegrass/clover grassland was negligible. N(2)O emissions were 7.82±1.67, 6.35±1.14 and 6.54±1.70 kg N ha(-1) yr(-1), respectively, from GG+FN, GWC+FN and GWC-FN. N(2)O fluxes simulated by DNDC agreed well with the measured values with significant correlation between simulated and measured daily fluxes for the three grazing treatments, but the simulation did not agree very well for the background treatments. DNDC overestimated annual emission by 61% for GG+FN, and underestimated by 45% for GWC-FN, but simulated very well for GWC+FN. Both the measured and simulated results supported that there was a clear reduction of N(2)O emissions when FN was replaced by BNF. 相似文献
16.
Regulation of fibronectin (FN) by Platelet-derived Growth Factor-BB (PDGF-BB) and Insulin-like Growth Factor-I (IGF-I) in cultured rat thoracic aortic adventitial fibroblasts were examined. PDGF-BB enhances FN mRNA levels in a time and concentration-dependent fashion. The effect of IGF-I on PDGF-BB-induced FN levels was also examined. There was a larger increase in FN mRNA levels (4-fold) in the cells treated with both IGF-I and PDGF-BB than with either IGF-I (2-fold) or PDGF-BB (2-fold) alone. The effects of PDGF-BB and IGF-I on FN levels were examined. There was a larger increase in FN levels (135%) in the cells treated with both PDGF-BB and IGF-I than with either PDGF-BB (43%) or IGF-I (45%) alone. Regulation of adventitial fibroblast FN may lead to blood vessel diseases and/or angiogenesis. 相似文献
17.
Bianca R. Tomasini-Johansson Ian A. Johnson F. Michael Hoffmann Deane F. Mosher 《Matrix biology》2012,31(6):360-367
Fibronectin (FN) is a plasma glycoprotein that circulates in the near micromolar concentration range and is deposited along with locally produced FN in the extracellular matrices of many tissues. The control of FN deposition is tightly controlled by cells. Agents that modulate FN assembly may be useful therapeutically in conditions characterized by excessive FN deposition, such as fibrosis, inflammatory diseases, and malignancies. To identify such agents by high throughput screening (HTS), we developed a microtiter assay of FN deposition by human fibroblasts. The assay provides a robust read-out of FN assembly. Alexa 488-FN (A488-FN) was added to cell monolayers, and the total fluorescence intensity of deposited A488-FN was quantified. The fluorescence intensity of deposited A488-FN correlated with the presence of FN fibrils visualized by fluorescence microscopy. The assay Z′ values were 0.67 or 0.54, respectively, when using background values of fluorescence either with no added A488-FN or with A488-FN added together with a known inhibitor of FN deposition. The assay was used to screen libraries comprising 4160 known bioactive compounds. Nine compounds were identified as non- or low-cytotoxic inhibitors of FN assembly. Four (ML-9, HA-100, tyrphostin and imatinib mesylate) are kinase inhibitors, a category of compounds known to inhibit FN assembly; two (piperlongumine and cantharidin) are promoters of cancer cell apoptosis; and three (maprotiline, CGS12066B, and aposcopolamine) are modulators of biogenic amine signaling. The latter six compounds have not been recognized heretofore as affecting FN assembly. The assay is straight-forward, adapts to 96- and 384-well formats, and should be useful for routine measurement of FN deposition and HTS. Screening of more diverse chemical libraries and identification of specific and efficient modulators of FN fibrillogenesis may result in therapeutics to control excessive connective tissue deposition. 相似文献
18.
Localization of macrophage agglutination factor activity to the gelatin-binding domain of fibronectin 总被引:2,自引:0,他引:2
H P Godfrey C V Angadi R A Wolstencroft C Bianco 《Journal of immunology (Baltimore, Md. : 1950)》1984,133(3):1417-1423
We previously proposed that macrophage agglutination factor (MAggF, a T cell-derived guinea pig lymphokine) is a fibronectin (FN). We now show MAggF binding to gelatin and to peritoneal macrophages is mediated by domains similar to corresponding domains of plasma FN. MAggF activity in lymphokine concentrates prepared by two different methods differed nearly 10-fold in m.w. on gel filtration chromatography. Despite this difference, MAggF dose-activity curves of both preparations were parallel, and MAggF in both preparations bound reversibly to gelatin and to monoclonal anti-guinea pig FN immunoadsorbents. MAggF activity in one preparation was inhibited by the addition of soluble monoclonal antibody specific for the gelatin-binding domain of human FN; inhibitory activity of this antibody was blocked by purified guinea pig plasma FN or partially purified MAggF from the other preparation. Measured MAggF activity of both preparations was reduced in a dose-dependent manner by pretreatment of indicator macrophages with monoclonal anti-human monocyte FN receptor antibody or F(ab')2 fragments or with guinea pig plasma FN. Neither anti-FN receptor antibody nor plasma FN interacted directly with MAggF. Indirect immunofluorescence studies confirmed the presence of uncomplexed plasma membrane receptors for FN on indicator macrophages in MAggF-responsive populations that were able to bind added FN. Our identification of MAggF as lymphokine FN provides a basis for future biochemical analysis of delayed hypersensitivity inflammatory reactions. 相似文献
19.
Summary The present study is a detailed kinetic analysis of the synthesis, release and multimerization of fibronectin (FN) in normal and tumor promoter-treated human lung fibroblasts. Pulse/chase and surface labeling experiments were performed to follow the fate of both newly synthesized and pre-existing cell-surface FN over time. The majority of FN (80%) left the intracellular compartment within one hour of synthesis. However, the rate of direct secretion was very low and after one hour, 70% of newly synthesized FN was still at the cell surface. This material was primarily dimeric. Dimeric and multimeric (very high molecular weight) FN was detectable at the cell surface and in the medium 4 hours after synthesis. Pulse-labeled FN multimer levels peaked at 12 hours and declined thereafter. After 24 hours, 85% of pulse-labeled FN had been shed into the medium and the labeled FN remaining at the cell surface was primarily multimeric. Surface labeling experiments confirmed that the majority of FN resides at the cell surface prior to release into the medium.One hour treatment with the phorbol ester tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), stimulated a nine-fold increase in release of preexisting, dimeric cell-surface FN (125I-labeled). The major effect of longer term TPA treatment up to nine hours was continued depletion of dimeric cell-surface FN. Increased release of cell-surface multimeric FN was also stimulated by TPA, but to a much lesser extent. Release of newly synthesized (pulse-labeled) dimeric FN was also stimulated by TPA though much less than pre-existing FN, and TPA treatment produced a small decrease in the steady-state level of multimeric FN. Thus, preexisting cell-surface IN and newly synthesized FN differ dramatically in their susceptibility to TPA treatment.Abbreviations FN
fibronectin
- HLF
human lung fibroblasts
- PMSF
phenylmethylsulfonylfluoride
- TPA
12-O-tetradecanoyl phorbol-13-acetate
- DMSO
dimethylsulfoxide
- MEM
minimal essential medium
- ELISA
enzyme linked immunosorbent assay
- PBS
phosphate-buffered saline
- BSA
bovine serum albumin
- SDS-PAGE
sodium dodecylsulfate-polyacrylamide gel electrophoresis
- DOC
deoxycholate
- DTT
dithiotreitol 相似文献