Fibronectin expression in human mesangial cell cultures and its alterations by adriamycin

Human mesangial cell (HMC) cultures synthesize cellular fibronectin (FN), which is secreted and incorporated into a fibrillar extracellular matrix (ECM). The anticancer drug adriamycin (ADR) induces changes in extracellular FN deposition. As revealed by immunofluorescence staining, a 24 h incubation of the cells with 2 g ADR/ml resulted in a marked expansion of the pericellular FN fibers, which may be due to either an increased synthesis or a decreased FN degradation. The effects of ADR on FN mRNA were analysed by northern hybridization andin vitro translation. Steady-state FN mRNA levels were significantly increased by 60% following ADR administration. However, yields of radioactivity incorporated into FN by cell-free translation remained constant (2.3±0.7%,n=24, vs controls 2.2±0.8% of total radioactivity,n=23). The quality of translation products was not affected by the drug, whereas translation efficiency of total RNA from ADR-treated HMC was only 75% of controls. The data presented suggest a negative feedback control of FN expression on the level of translation. Extracellular FN accumulation in the experimental model of ADR-induced progressive glomerulopathy therefore cannot be explained by an increased FN synthesis, but is rather regarded a consequence of proteinase inhibition. This assumption is compatible with a diminished number of FN fragments recently demonstrated in the culture medium of ADR-treated HMC, and is further corroborated by the loss of urinary FN degradation products accompanying the onset of proteinuria in ADR-treated rats.Abbreviations ADR adriamycin - BSA boyine serum albumin - ECM extracellular matrix - EDTA ethylenediamine tetraacetic acid - FITC fluorescein isothiocyanate - FN fibronectin - HMC human mesangial cell - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - SSC standard saline citrate - SSPE standard saline phosphate ethylenediamine tetraacetic acid disodium salt - TGF- transforming growth factor      

Sustained changes in lung expansion alter tropoelastin mRNA levels and elastin content in fetal sheep lungs

Our objective was to determine the effects of sustained alterations in fetal lung expansion on pulmonary elastin synthesis. In fetal sheep, lung expansion was either decreased between 111 and 131 days' gestation (term approximately 147 days) by tracheal drainage or increased for 2, 4, 7, or 10 days by tracheal obstruction, ending at 128 days' gestation. Lung tropoelastin mRNA levels were assessed by Northern blot analysis, total elastin content was measured biochemically, and staining of lung sections was used to assess the localization and form of elastic fibers. Tracheal obstruction significantly elevated pulmonary tropoelastin mRNA levels 2.5-fold at 2 days, but values were not different from controls at 4, 7, and 10 days; elastin content tended to be increased at all time points. A sustained decrease in lung expansion by tracheal drainage reduced pulmonary tropoelastin mRNA levels 2.5-fold; elastin content was also decreased compared with controls, and tissue localization was altered. Our results indicate that the degree of lung expansion in the fetus influences elastin synthesis, content, and tissue deposition.     

Plasma fibronectin therapy and lung protein clearance with bacteremia after surgery

Plasma fibronectin modulates macrophage phagocytic function and can also incorporate into the insoluble tissue pool of fibronectin where it influences endothelial cell adhesion and tissue integrity. We studied the effect of postoperative bacteremia on lung protein clearance in relation to plasma fibronectin levels using the unanesthetized sheep lung lymph fistula model and the effect of infusion of purified human plasma fibronectin on lung protein clearance. Sheep received live Pseudomonas aeruginosa (5 X 10(8) iv) at a time of normal plasma fibronectin (590 +/- 37 micrograms/ml) or 5 days later at a time corresponding to elevation of plasma fibronectin (921 +/- 114 micrograms/ml). After the first bacterial challenge, there was a 22% decrease (P less than 0.05) in plasma fibronectin. Lung lymph flow (QL) initially increased 308% (P less than 0.05) by 2 h (0 h = 4.7 +/- 1.1 ml/h; 2 h = 14.4 +/- 3.5 ml/h), and the total protein lymph-to-plasma concentration ratio (L/P) declined. This was followed by a sustained second phase response over 3-12 h which was characterized by a 202-393% elevation in QL (P less than 0.05), an increase in the L/P ratio, and a 240-480% (P less than 0.05) increase in lung transvascular protein clearance (TVPC = QL X L/P). Sheep with elevated fibronectin levels also manifested the early (2 h) elevation in QL (P less than 0.05) coupled with a decline in L/P ratio after the second bacterial challenge, but the second-phase increase in TVPC was markedly attenuated. Intravenous infusion of 500 mg of human plasma fibronectin into normal sheep to elevate the fibronectin level comparable to that in the hyperfibronectinemic sheep also attenuated (P less than 0.05) the second-phase (3-12 h) increase in lung protein clearance with sepsis. Thus elevation of plasma fibronectin during postoperative Gram-negative bacteremia may protect the lung vascular barrier. This response may be mediated by either fibronectin's opsonic support of phagocytic function or its influence on lung endothelial cell adhesion.     

Fibronectin turnover in human mesangial cell cultures as affected by Adriamycin

Fibronectin (FN) turnover and turnover changes induced by the anticancer drug Adriamycin (ADR) were measured in human mesangial cells (HMC) in vitro. HMC cultures synthesize cellular FN (2.2+-0.3% of totalprotein synthesis; n = 12) which is secreted and incorporated into a fibrillar extracellular matrix (ECM). A 24 hr incubation of HMC with ADR (0.5–5 g/ml) resulted in an accumulation of FN in the culture medium, with a maximum increase following 5 pglml(7.3+-2.3pg/cell vs. controls: 4.4+-1.9pg/cell; n= 10). Correspondingly, radioactively labeled immunoprecipitable FN was increased in a dosage-dependent manner in the culture medium up to 50% vs. controls. The incorporation of radioactively labeled FN into ECM was significantly increased following 2 g ADR/ml. In accordance, immunofZuorescence staining revealed an expansion ofpericellular FNfibers in cultures exposed to 2 g ADR/ml. Concomitant with the accumulation of extracelhlar FN, radioactively labeled FN in the cells was reduced by 22%. Qualitative characterization of FN patterns revealed a diminished number of degradation products in the culture medium ofADR-treated HMC. These data suggest thatADR interferes with the turnover of FN secreted by HMC in vitro in such a way that FN accumulates extracellularly. This in turn leads to a reduced FN synthesis. These findings are compatible with a loss of urinary FN degradation products accompanying the onset ofproteinuria in ADR-treated rats.Abbreviations ADR adriamycin - BSA bovine serum albumin - DTT dithiothreitol - ECM extracellular matrix - EDTA ethylenediamine tetraacetic acid disodium salt - ELISA enzyme-linked immunosorbent assay - FCS fetal calf serum - FITC fluorescein isothiocyanate - FN fibronectin - HMC human mesangial cell - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

The kinetics of fibronectin synthesis and release in normal and tumor promoter-treated human lung fibroblasts

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     

Proteolytically stabilizing fibronectin without compromising cell and gelatin binding activity

Excessive proteolytic degradation of fibronectin (FN) has been implicated in impaired tissue repair in chronic wounds. We previously reported two strategies for stabilizing FN against proteolytic degradation; the first conjugated polyethylene glycol (PEG) through cysteine residues and the second conjugated PEG chains of varying molecular weight on lysine residues. PEGylation of FN via lysine residues resulted in increased resistance to proteolysis with increasing PEG size, but an overall decrease in biological activity, as characterized by cell and gelatin binding. Our latest method to stabilize FN against proteolysis masks functional regions in the protein during lysine PEGylation. FN is PEGylated while it is bound to gelatin Sepharose beads with 2, 5, and 10 kDa PEG precursors. This results in partially PEGylated FN that is more stable than native FN and whose proteolytic stability increases with PEG molecular weight. Unlike completely PEGylated FN, partially PEGylated FN has cell adhesion, gelatin binding, and matrix assembly responses that are comparable to native FN. This is new evidence of how PEGylation variables can be used to stabilize FN while retaining its activity. The conjugates developed herein can be used to dissect molecular mechanisms mediated by FN stability and functionality, and address the problem of FN degradation in chronic wounds. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:277–288, 2015     

Synthesis and glycosylation of fibronectin in hepatocytes from vitamin A-deficient rats

Summary Recent work from our laboratory (Kim and Wolf, J Biol Chem 262: 365–371, 1987) has shown increased uptake of labeled amino acids into fibronectin (FN), increased net synthesis of FN and increased levels of FN-mRNA in primary cultures of hepatocytes from vitamin A-deficient rats compared to controls. We now find, surprisingly, decreased uptake of labeled sugars into the oligosaccharide chains of FN from vitamin A-deficient hepatocytes. This decrease could be reversed by added retinoic acid at physiological concentration. At the same time, FN from deficient hepatocytes (–A.FN) was more susceptible to proteolytic degradation. Decreased uptake of the core sugar mannose into –A.FN was similar to that of glucosamine, yet the percent of label in sialic acid was the same as in +A.FN, suggesting a smaller number of oligosaccharide chains per molecule of –A.FN. Upon enzymatic removal of oligosaccharide and labeling with sodium borotritide, it was found that both –A.FN and +A.FN had biantennary oligosaccharide structures. Selective enzymatic removal of sialic acid showed that +A.FN had both sialic acids in an 23 linkage, whereas –A.FN apparently had one 23 and one 26-linked sialic acid. The borotritide experiments allowed us to calculate that +A.FN appeared to have 5 oligosaccharide chains per FN monomer, whereas the –A. FN showed only 4 chains. These results would account for the decreased glycosylation and increased susceptibility to proteolysis of the –A. FN. We conclude that vitamin A controls both the rate of synthesis of the polypeptide chain of FN via its mRNA, as well as the rate of its glycosylation.Abbreviations FN Fibronectin - ELISA Enzyme-linked Immunosorbent Assay - DOC Deoxycholate - TCA Trichloroacetic Acid - PMSF Phenylmethylsulfonyl Fluoride - PBS Phosphate-buffered Saline - BSA Bovine Serum Albumin - AGP Alpha-1 acid Glycoprotein - SDS-PAGE Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis     

Constitutive and UV-induced fibronectin degradation is a ubiquitination-dependent process controlled by beta-TrCP

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.     

The Involvement of Tissue-Type Plasminogen Activator in Parietal Endoderm Outgrowth

When F9 stem cells are treated in suspension with retinoic acid, they differentiate into embryoid bodies (EBs) consisting of an inner core of undifferentiated stem cells surrounded by an outer layer of visceral endoderm (VE). When these EBs are plated onto a fibronectin (FN)-coated substrate, VE-derived parietal endoderm (PE) cells migrate onto the substrate. It has been suggested that increased levels of tPA associated with the emerging PE cells may help mediate PE outgrowth. We now show that goat anti-human tPA, an anticatalytic antibody that crossreacts with mouse tPA, and a panel of serine protease inhibitors partially inhibit PE outgrowth. Extracellular matrix (ECM) degradation analysis demonstrates that PE cell-mediated degradation of [³H]proline-labeled ECM is time- and cell concentration-dependent. A serine protease inhibitor reduced the extent of degradation, suggesting that tPA might play a role in PE outgrowth by cleaving the ECM. In support of this contention, we demonstrate that incubation of purified FN with conditioned medium plus plasminogen results in FN proteolysis. The degradation of FN is blocked by either serine protease inhibitors or goat anti-human tPA. Our data suggest that enhanced production of tPA during PE outgrowth may facilitate the migratory behavior of PE cells by mediating the degradation of ECM components such as FN.     

Effects of transforming growth factor-beta 1 and fibronectin on SPARC expression in cultures of human periodontal ligament cells

Transforming growth factor-beta(1) (TGF-beta(1)) increases synthesis of secreted protein, acidic and rich in cysteine (SPARC), as well as fibronectin (FN) and type I collagen. However, little is known about the regulatory mechanism of SPARC expression. We examined the effect of FN on SPARC expression by TGF-beta(1) in cultures of human periodontal ligament cells (HPL cells). TGF-beta(1) increased the SPARC and SPARC mRNA levels in HPL cells. Extracellular matrix (ECM) produced by HPL cells in the presence of TGF-beta(1) also increased the SPARC levels. Contents of FN and type I collagen in the ECM were increased by TGF-beta(1). HPL cells cultured on FN-coated plates secreted more SPARC than those on non-coated plates. However, type I collagen had little effect on SPARC levels. The addition of anti-alpha5 antibody to the cultures abolished the increase in SPARC mRNA expression by TGF-beta(1). This study demonstrated that FN may be partly involved in the increase in SPARC expression by TGF-beta(1) in HPL cells.     

A major fraction of fibronectin present in the extracellular matrix of tissues is plasma-derived

The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA(+)FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA(+)FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues.     

Interaction between pathogenic amebas and fibronectin: substrate degradation and changes in cytoskeleton organization

Invasion of human tissues by the parasitic protozoan Entamoeba histolytica is a multistep process involving, as a first step, the recognition of surface molecules on target tissues by the amebas or trophozoites. This initial contact is followed by the release of proteolytic and other activities that lyse target cells and degrade the extracellular matrix. In other parasitic diseases, as well as in certain cancers, the interaction of invasive organisms or cells with fibronectin (FN) through specific receptors has been shown to be the initial step in target cell recognition. Interaction with FN triggers the release of proteolytic activities necessary for the effector cell migration and invasion. Here, we describe the specific interaction of Entamoeba histolytica trophozoites with FN, and identify a 37-kD membrane peptide as the putative receptor for FN. The interaction between the parasite and FN leads to a response reaction that includes the secretion of proteases that degrade the bound FN and the rearrangement of amebic actin into "adhesion plates" at sites of contact with FN-coated surfaces. The kinetics of the interaction was determined by measuring the binding of soluble 125I-FN to the trophozoites and visualization of the bound protein using specific antibodies. Degradation of FN was measured by gel electrophoresis and the release of radioactivity into the incubation medium. Focal degradation of FN was visualized as black spots under the trophozoites at contact sites with fluorescent FN. We conclude that the interaction of E. histolytica with FN occurs through a specific surface receptor. The interaction promotes amebic cytoskeleton changes and release of proteases from the parasite. The binding and degradation of extracellular matrix components may facilitate the migration and penetration of amebas into tissues, causing the lesions seen in human hosts.     

Increase in extracellular cross-linking by tissue transglutaminase and reduction in expression of MMP-9 contribute differentially to focal segmental glomerulosclerosis in rats

Tissue transglutaminase (tTG) is a Ca²⁺-dependent enzyme which stabilizes the extracellular matrix (ECM) through post-translational modification, and may play an important role in the pathogenesis of focal and segmental glomerulosclerosis (FSGS). Here, we have investigated whether tTG contributes to the glomerular ECM expansion in the puromycin aminonucleoside (PAN)-injection-induced experimental rat model of FSGS. The localization and expression of tTG, MMP-9 gelatinase, and the ECM component fibronectin (FN) in kidneys was determined by immunohistochemistry and measured by semi-quantitative analysis. Protein levels of tTG and MMP-9 were also analyzed by Western blotting.In situtransglutaminase activity was assayed by measurement of incorporated substrate and the immunofluorescence staining for the cross-linking product, ε-(γ-glutamyl) lysine. Prominent proteinuria, a typical pathological feature of FSGS, was observed in PAN injection group rats. tTG immunoreactivity was located markedly in glomeruli and the levels of this protein in whole-kidney homogenates of PAN injection group rats were significantly increased (361± 106% control, P< 0.05). Similarly, transglutaminase activity and ε-(γ-glutamyl) lysine were also predominately located within glomeruli and were much more intense in the PAN-injected group than that in control animals. MMP-9 was also located primarily within glomeruli. In PAN-injected kidneys, protein levels of active MMP-9 were significantly reduced (59± 27% control, P< 0.01), while pro-MMP-9 levels increased (148± 42% control, P< 0.05). Remarkable expression of glomerular fibronectin (FN) was found in PAN injection group rats. Semi-quantitative analysis demonstrated this increased intensity of FN staining in the PAN-injected rats was 149± 23% of the control values (P< 0.05). Enhanced cross-linking of ECM by tissue transglutaminase and decreased degradation due to reduced active MMP-9 expression may be at least partially responsible for the deposition of FN within injured glomeruli in experimental FSGS.     

Hepatocyte growth Factor/Scatter factor (HGF/SF) is a regulator of fibronectin splicing in MDCK cells: comparison between the effects of HGF/SF and TGF-beta1 on fibronectin splicing at the EDA region.

EDA-containing fibronectin (EDA + FN) is selectively produced under several physiological and pathological conditions requiring tissue remodeling, where cells actively proliferate and migrate. Only a few growth factors, such as transforming growth factor (TGF)-beta1, have been reported to regulate FN splicing at the EDA region. In the present study, we showed for the first time that hepatocyte growth factor/scatter factor (HGF/SF), which is mainly produced by mesenchymal cells and functions as a motogenic and mitogenic factor for epithelial cells, modulates FN splicing at the EDA region in MDCK epithelial cells. HGF/SF treatment increased the ratio of EDA + FN mRNA to mRNA of FN that lacks EDA (EDA - FN) (EDA+/EDA- ratio) more than TGF-beta1 treatment did: at a range from 0.02 to 20 ng/ml, HGF/SF increased the ratio in a dose-dependent manner by up to 2. 1-fold compared with nontreated control, while TGF-beta1 stimulated the EDA+/EDA- ratio by 1.5-fold at the optimum dose of 10 ng/ml. However, TGF-beta1 increased total FN mRNA levels by 3-fold at 10 ng/ml, but HGF/SF did not. We previously demonstrated that fibroblasts cultured at low cell density expressed more EDA + FN than those at high cell density. The same effect of cell density was also observed in MDCK cells. Furthermore, at low cell density, HGF/SF stimulated EDA inclusion into FN mRNA more effectively than did TGF-beta1, whereas at high cell density, TGF-beta1 was more potent than HGF/SF. Simultaneous treatment of cells with HGF/SF and TGF-beta1 synergistically stimulated EDA inclusion into FN mRNA. This stimulation of EDA inclusion into FN mRNA by HGF/SF led to increased EDA + FN protein production and secretion by cells, which was demonstrated by immunoblotting. Thus, our studies have shown that HGF/SF is an enhancer of EDA inclusion into FN mRNA as is TGF-beta1. However, these two factors were different in their effects at low and high cell densities and also in their effects on total FN mRNA levels.     

Interferon enhances fibronectin expression in various cell types.

Fibronectin (FN), a normal plasma and extracellular matrix glycoprotein, plays a significant role in various phases of wound healing. At wound site FN is synthesized locally by various cell types involved in the healing process (viz. epithelial, endothelial, fibroblast and macrophage cells) or deposited from the plasma. The present study was undertaken to investigate the in vitro effect of IFN on FN synthesis as well as release in the culture medium by various cell types. Indirect immunofluorescence and immunoelectron microscopy studies, using specific antibodies, revealed that IFN treatment resulted in significantly more staining for FN as compared to untreated control cells. Metabolic labeling with 35S-methionine, immunoprecipitation and SDS-page studies showed an increase in FN synthesis and release by IFN treated cells. In addition, to determine whether this increased synthesis was reflected at mRNA levels, poly (A)+ RNA was isolated from human lung epithelial cells (A549) and probed with FN specific cDNA. We found that IFN treatment increased the level of FN mRNA.     

Fibronectin, aging and related pathologies]

It could be demonstrated that plasma and tissue fibronectin (FN) increase with age. Some age dependent diseases as diabetes, osteoarthritis and Werner syndrome produce also an increase of tissue fibronectin biosynthesis. Plasma fibronectin decreases in diabetes and in breast cancer. Alternative splicing of the FN gene appears also to vary with age and in some related pathologies. Nutritional status and UV light also influence FN biosynthesis. It appears therefore that the determination of plasma FN and its isoforms as well as the study of tissue FN may be of interest for the study of chronological aging and related pathologies.     

Fibronectin matrix turnover occurs through a caveolin-1-dependent process

Extracellular matrix remodeling occurs during development, tissue repair, and in a number of pathologies, including fibrotic disorders, hypertension, and atherosclerosis. Extracellular matrix remodeling involves the complex interplay between extracellular matrix synthesis, deposition, and degradation. Factors that control these processes are likely to play key roles in regulating physiological and pathological extracellular matrix remodeling. Our data show that fibronectin polymerization into the extracellular matrix regulates the deposition and stability of other extracellular matrix proteins, including collagen I and thrombospondin-1 (Sottile and Hocking, 2002. Mol. Biol. Cell 13, 3546). In the absence of continual fibronectin polymerization, there is a loss of fibronectin matrix fibrils, and increased levels of fibronectin degradation. Fibronectin degradation occurs intracellularly after endocytosis and can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and by caveolae-disrupting agents. Down-regulation of caveolin-1 by RNAi inhibits loss of fibronectin matrix fibrils, fibronectin internalization, and fibronectin degradation; these processes can be restored by reexpression of caveolin-1. These data show that fibronectin matrix turnover occurs through a caveolin-1-dependent process. Caveolin-1 regulation of fibronectin matrix turnover is a novel mechanism regulating extracellular matrix remodeling.