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PubMed1993

Effect of fibronectin on permeability of normal and TNF-treated lung endothelial cell monolayers.

Fibronectin is found in a soluble form in plasma and lymph and in an insoluble form in the extracellular matrix. Plasma fibronectin can incorporate into the tissue pool of fibronectin where its adhesive properties may influence cell-cell interaction, cell adhesion to a collagenous matrix, and vascular integrity. Elevation of plasma fibronectin can attenuate the increase in lung vascular permeability in sheep during postoperative gram-negative bacteremia, and plasma fibronectin deficiency can magnify the increase in lung vascular permeability with postoperative sepsis. Using pulmonary endothelial monolayers, we determined if exogenous human plasma fibronectin (pFn) would influence the protein permeability of pulmonary endothelial monolayers as determined by transendothelial clearance (microliters/min) of 125I-albumin after they were exposed to human recombinant tumor necrosis factor-alpha. Treatment of endothelial monolayers with tumor necrosis factor (TNF) (200 U/ml) for 18 h resulted in a significant (P < 0.05) increase in protein permeability. Addition of intact purified human plasma fibronectin to normal confluent endothelial monolayers to yield a medium concentration of 300, 600, and 900 micrograms/ml for 18 h had no effect on baseline protein permeability. In contrast, whereas addition of lower amounts of human plasma fibronectin (300 micrograms/ml) did not attenuate the TNF-induced increase in monolayer permeability, the higher concentrations of 600 or 900 micrograms pFn/ml significantly decreased (P < 0.05) protein permeability. The ability of soluble plasma fibronectin to attenuate the TNF-induced increase in endothelial protein permeability required an incubation time of at least 2-3 h, perhaps due to a lag time required for its incorporation into the extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

Total fibronectin in maternal plasma as a predictor for preeclampsia.

OBJECTIVE: To assess the ability of measuring total maternal plasma fibronectin in predicting the risk of developing preeclampsia. METHOD: In a cohort of 657 initially normotensive pregnant nulliparous women blood samples were collected at 26, 30 and 34 weeks of gestation. After delivery 3 groups were selected for measurement of total plasma fibronectin. The first group comprised 24 women who developed preeclampsia; the second 21 women who developed gestational hypertension, and the third 89 age-matched controls who remained normotensive. RESULTS: In women who developed preeclampsia the fibronectin levels were higher at 26 (p < 0.01), 30 (p < 0.001) and 34 weeks of gestation (p < 0.001) than in pregnant controls. In the general population of pregnant nulliparous women the estimated risk of subsequent preeclampsia correlated with the fibronectin level at each measurement. In women with fibronectin levels within the 25th percentile at the 26th week of gestation, the risk was </=1.9% and within the 90th percentile >/=6.2%. The change in fibronectin levels between 26 and 34 weeks of gestation was the only variable that remained statistically associated with preeclampsia (p = 0.0002) in multivariate analysis. In this model the risk figures for the corresponding percentiles were </=1.0 and >/=5.8%, respectively. CONCLUSION: Longitudinal assessment of the change in total fibronectin levels predicted preeclampsia slightly better than cross-sectional analysis of fibronectin at any gestational age. Nevertheless, measurement of total fibronectin in maternal plasma could not be proven to be a useful clinical predictor for preeclampsia in a general obstetric population of nulliparous women.

Adult↗

PubMed1992

Effects of the SV40 large T antigen and EJ ras oncogene on fibronectin localization in human endometrial cells as viewed by confocal laser scanning microscopy.

We utilized confocal laser scanning microscopy to examine the localization of fibronectin deposition in cultures of human endometrial stromal cells. We found that fibronectin in normal human endometrial stromal cell cultures was both intracellular, occurring in rough endoplasmic reticulum and in perinuclear regions, and extracellular, occurring diffusely over the entire cell surface. Endometrial stromal cells were transfected with a plasmid containing an origin-defective Simian Virus 40 (SV40) which codes for a temperature-sensitive large T antigen. When these cells were placed under temperature-restrictive conditions for large T-antigen function, they exhibited staining patterns similar to normal endometrial cells. Fibronectin deposition in cultures of partially or fully transformed endometrial cells was not intracellular as in normal cells, but was localized primarily between cells. Cells expressing the SV40 large T antigen deposited fibronectin mainly in parallel clumps between cells. Cells expressing both the SV40 large T antigen and the EJ ras oncogene, at high cell density, displayed networks of fibronectin arranged in matrix-like patterns between cells. The malignant cell line examined, sarcoma cells, also exhibited fibronectin networks between cells. Cell density affected fibronectin deposition in endometrial stromal cells expressing the EJ ras oncogene. At low density, cells expressing the SV40 large T antigen and the EJ ras oncogene displayed diffuse fibronectin patterns and, at high density, these cells formed colonies with networks of fibronectin between cells.

Antigens, Polyomavirus Transforming↗

PubMed1987

Fibronectin in pemphigus.

In the skin organ culture model of pemphigus, fibronectin concentrations of 300 and 500 micrograms/ml inhibited pemphigus plasma-induced acantholysis and intraepidermal binding of the pemphigus antibodies examined by direct immunofluorescence. A direct interaction of fibronectin with pemphigus antibodies could not be demonstrated by chromatography of pemphigus plasma on immobilized fibronectin or by incubation with fibronectin and subsequent precipitation with antifibronectin. We then measured fibronectin concentrations in the plasma of 15 patients suffering from various types of pemphigus and presenting different activities of their disease. Immunoreactive fibronectin levels in untreated patients with active disease were generally in the low normal or even clearly in the subnormal range. They had the tendency to decrease when the disease subsided during therapy with high doses of glucocorticoids, sometimes in combination with azathioprine. The fibronectin concentration in the blister fluid of a patient with acute, untreated pemphigus vulgaris was similar to that in a plasma sample taken at the same time. Skin biopsies of pemphigus patients exhibited an essentially normal fibronectin pattern in direct immunofluorescence. We discuss a possible protective role of fibronectin in pemphigus e.g., by reducing the permeability for pemphigus antibodies in the dermal-epidermal junction zone.

Acantholysis↗

PubMedDate not supplied

Fibronectin in platelet adhesion to human collagen types I and III. Use of nonfibrillar and fibrillar collagen in flowing blood studies.

Platelet deposition at high wall shear rates on collagen type I and type III purified from human umbilical arteries is dependent on the presence of fibronectin and von Willebrand factor (VWF). The role of fibronectin at low wall shear rates (less than or equal to 500 s-1), where platelet deposition was independent of VWF, was studied with purified collagen I and III. Platelet deposition on nonfibrillar collagen I was fibronectin-dependent at all wall shear rates. Platelet deposition on nonfibrillar collagen type III was fibronectin-dependent at 300 s-1 and higher shear rates. By using a mixture of nonfibrillar type I and III, platelet deposition was found to be fibronectin-dependent at the tested wall shear rates (20, 100, and 300 s-1). This dependency was less than with nonfibrillar type I only, but more than with nonfibrillar type III. For platelet deposition on reconstituted type I or type III collagen fibrils, no fibronectin dependency was observed up to the highest wall shear rate tested (1800 s-1). The same results were obtained with a mixture of native type I and III fibrils. Thus, the dependence of platelet deposition on fibronectin is determined by the collagen type and the wall shear rate. The dependence on the fibronectin concentration was tested with nonfibrillar collagen type I at a wall shear rate of 300 s-1. Platelet deposition increased with increased fibronectin concentration up to a level of 700 micrograms/ml and leveled off above this concentration.

Collagen↗

PubMed1999

Segmental antigen challenge increases fibronectin in bronchoalveolar lavage fluid.

Fibronectin may contribute to asthma pathogenesis by recruitment and activation of inflammatory cells, and by promotion of subepithelial fibrosis. Fibronectin is produced by several types of airway cells, including epithelial cells, fibroblasts, and alveolar macrophages. To test the hypothesis that antigen-induced airway inflammation is associated with increased local generation of fibronectin, segmental bronchoprovocation (SBP) with antigen and saline was performed in 17 atopic patients. Bronchoalveolar lavage (BAL) was performed at 5 min and 48 h after segmental challenge with saline or antigen. Fibronectin concentrations in BAL fluid, measured by enzyme-linked immunosorbent assay (ELISA), increased more than 5-fold 48 h after antigen challenge (65 [47 to 110] versus 407 [240 to 697] ng/ml, median and 25 to 75% interquartiles, p < 0.05). Fibronectin concentrations 48 h after antigen challenge correlated with histamine concentrations 5 min after antigen challenge and numbers of eosinophils, neutrophils, macrophages, and total cells in BAL fluid 48 h after antigen challenge. BAL was more enriched in fibronectin 48 h after challenge than would be predicted solely from increased permeability of plasma proteins. Western blot analysis showed that fibronectin in BAL fluid was largely intact and contained the extra domain-A (ED-A) splice variant of cellular fibronectin, indicative of local production. We conclude that antigen challenge in atopic subjects causes increased production of fibronectin by airway cells and speculate that this response may contribute to airway remodeling in allergic inflammation.

Adult↗

PubMed1993

Immunofluorescent analysis of plasma fibronectin incorporation into the lung during acute inflammatory vascular injury.

Incorporation of plasma fibronectin into tissues is believed to influence endothelial cell-cell interaction, as well as endothelial cell adhesion to matrix. We used immunofluorescent microscopy coupled with tissue extraction of noncovalently incorporated fibronectin to delineate the time course for matrix incorporation of soluble plasma-derived fibronectin into the lung of sheep during postoperative bacteremia. Adult sheep were surgically prepared with both lung and peripheral lymph fistulas. Sheep were anesthetized 2 days following surgery and injected intravenously with a sublethal dose of live Pseudomonas aeruginosa, which consisted of 5 x 10(8) live organisms suspended in 0.9% saline. Bacterial infusion elicited a 300% increase in lung transvascular protein clearance but no increase in peripheral transvascular protein clearance. Purified dimeric human plasma fibronectin (hFn), used as an "immunologic marker," was then infused intravenously (100 mg/sheep) into two additional groups of sheep (nonbacteremic control group and bacteremic experimental group) and allowed to mix with the plasma pool of endogenous soluble sheep fibronectin (sFn). Incorporation of the plasma-derived hFn into the lung matrix and its distribution in relation to endogenous sheep fibronectin in the matrix was assessed by dual-label immunofluorescence using antibodies specific to either sFn or hFn. Human fibronectin from the vascular compartment codistributed with endogenous sheep fibronectin in the lung matrix. Moreover, its deposition into the lung was markedly increased in postoperative bacteremic sheep compared with nonbacteremic control sheep. Increased hFn deposition in the lung with bacteremia was clearly apparent within 2 h. The hFn deposited in the lung was nonextractable using a heparin-urea tissue extraction buffer, suggesting its rapid covalent cross-linking and incorporation into the lung matrix. Microscopic analysis of serial lung biopsies revealed focal areas of inflammation with an intense mononuclear infiltrate into the lungs by 2 h in the bacteremic sheep. Interstitial edema and vascular endothelial injury were observed by 4 h, with alveolar edema apparent over 6 to 8 h. Thus, postoperative bacteremia results in a rapid incorporation of plasma fibronectin into the lung matrix. This may be a physiologic mechanisms to stabilize the integrity of the lung vascular barrier.

Animals↗

PubMed2003

Permissive effect of fibronectin on collagen gel contraction mediated by bovine trabecular meshwork cells.

PURPOSE: The effect of fibronectin on the contractility of trabecular meshwork (TM) cells was investigated. METHODS: The contractility of bovine TM cells was evaluated by culture of the cells in a collagen gel and measurement of the change in the diameter of the gel under various conditions. The formation of stress fibers and the localization of integrin alpha5 and beta1 chains (which together form a fibronectin receptor) in bovine TM cells were investigated by laser confocal microscopy of cells stained with phalloidin and antibodies to the integrin subunits. RESULTS: The addition of fibronectin to collagen gels containing bovine TM cells induced marked gel contraction in a time- and concentration-dependent manner. Cytochalasin D (an inhibitor of microfilament formation) and the peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), a fibronectin receptor antagonist, each inhibited this effect of fibronectin, whereas nocodazole (an inhibitor of microtubule polymerization) and the control peptide GRGESP (Gly-Arg-Gly-Glu-Ser-Pro) did not. Furthermore, fibronectin induced the spreading of cells, the formation of actin stress fibers, and the expression of integrin alpha5 in the collagen gel-embedded TM cells. CONCLUSIONS: Fibronectin promotes collagen gel contraction mediated by bovine TM cells. Moreover, the formation of actin stress fibers and upregulation of integrin alpha5 appear to contribute to this permissive effect of fibronectin. The interaction of fibronectin with TM cells may thus be a determinant of the contractility of TM tissue.

Actins↗

PubMed1983

Reduced surface expression and binding of fibronectin by thrombin-stimulated thrombasthenic platelets.

Thrombin stimulation results in increased surface expression of endogeneous fibronectin and binding of plasma fibronectin to human platelets. Platelets of patients with Glanzmann's thrombasthenia, a bleeding disorder, exhibit reduced thrombin-induced platelet aggregation, little or no clot retraction, and abnormal platelet spreading on glass surfaces. Thrombin stimulation of patient platelets from four thrombasthenic kindreds resulted in little fibronectin binding. Nevertheless, thrombin did induce serotonin secretion from these cells, indicating that stimulation was occurring. Thrombasthenic platelets did not inhibit thrombin-stimulated fibronectin binding to coincubated normal cells, suggesting that their defect was not due to the presence of a soluble inhibitor of fibronectin binding. Thrombin-stimulated afibrinogenemic platelets bound similar quantities of fibronectin to normal cells, indicating that the thrombasthenic deficit is not secondary to reduced fibrinogen content or binding. The thrombasthenic cells had an endogenous fibronectin content of 2.9 +/- 0.7 micrograms/10(9) platelets, whereas cells simultaneously prepared from five normal individuals contained 1.8 +/- 0.7 micrograms/10(9) platelets, a statistically insignificant difference. Nevertheless, thrombin stimulation did not increase expression of endogeneous fibronectin antigen on the surface of the thrombasthenic platelets as judged by immunofluorescence. These defects in platelet fibronectin binding and surface expression may account for some of the manifestations of Glanzmann's thrombasthenia.

Binding Sites↗

PubMed1990

Fibronectin fragments containing the RGDS cell-binding domain mediate monocyte migration into the rabbit lung. A potential mechanism for C5 fragment-induced monocyte lung accumulation.

Many inflammatory processes are characterized by an early phase of neutrophil migration and a later phase of monocyte migration into the inflammatory site. Mechanisms that govern the transition between phases are the subject of these investigations. Acute lung inflammation induced by C5 fragments in the rabbit leads to an initial neutrophil influx and plasma leakage into the alveolar space, followed by monocyte influx that we have previously shown to be dependent on prior emigration of neutrophils. Neutrophil enzymes are known to cleave intact fibronectin into fragments that are monocyte chemotaxins in vitro. Accordingly, generation of appropriate fibronectin fragments in situ by proteolytic enzymes from infiltrating neutrophils might represent a potential mechanism for attraction of monocytes into the lung. The studies reported herein demonstrate that a 120-kD fragment of fibronectin containing the RGDS fibroblast cell-binding domain induced monocyte migration into the rabbit lung in vivo. Intact fibronectin was inactive. A significant proportion of the monocyte migration was neutrophil independent. Intact fibronectin was present in bronchoalveolar lavage fluid from C5 fragment-treated animals rendered neutropenic, but absent in lavage from normal C5 fragment-treated animals. Fibronectin fragments were present in bronchoalveolar lavage fluid from both C5 fragment-treated and control rabbits. In addition, the amount of fibronectin was significantly increased in lavage of C5 fragment-treated normal but not neutropenic animals. Monoclonal antibodies directed against an epitope of fibronectin containing the RGDS cell-binding domain significantly inhibited the C5 fragment-induced monocyte migration, but not neutrophil migration. These studies suggest that chemotactic fibronectin fragments may in part be responsible for the recruitment of monocytes into areas of acute lung inflammation.

Animals↗

PubMed1991

Fibronectin biosynthesis in the rat aorta in vitro. Changes due to experimental hypertension.

This study was undertaken to determine if changes in fibronectin biosynthesis accompany the phenotypic changes that occur in aortic tissue following experimental hypertension. An in vitro procedure was developed to measure fibronectin synthesis in aortic rings obtained from normotensive or hypertensive rats. There was a three to sixfold increase in fibronectin biosynthesis by aortic rings taken from rats treated with deoxycorticosterone/salt for 7 and 21 d, the change being more pronounced at 21 d. In contrast, there was no major change at either time point in net incorporation into total protein. Studies comparing fibronectin biosynthesis in aortic rings from Wistar rats and spontaneously hypertensive rats at ages between 10 and 40 wk showed increased fibronectin biosynthesis in older animals of both strains, but only slight differences between strains. Studies using rats infused with angiotensin II showed a correlation between blood pressure elevation and increased aortic fibronectin biosynthesis. Western blot analysis of aortic extracts showed that the fibronectin content was increased in the hypertensive models. The in vitro procedure for measuring fibronectin biosynthesis appears to provide a reliable reflection of in vivo changes in fibronectin expression, and the methodology could prove useful for studying the factors influencing protein expression in vascular tissue.

Age Factors↗

PubMed1997

Microtubule-associated protein 1 light chain 3 is a fibronectin mRNA-binding protein linked to mRNA translation in lamb vascular smooth muscle cells.

Intimal cushions form in the fetal ductus arteriosus by fibronectin-dependent smooth muscle cell migration which is associated with greater efficiency of fibronectin mRNA translation. We investigated whether the AU-rich element (ARE), UUAUUUAU, in the 3'-untranslated region (3'UTR) of fibronectin mRNA is involved in this mechanism by transfecting smooth muscle cells with plasmids containing the chloramphenicol acetyltransferase coding region with its 3'UTR replaced by fibronectin 3'UTR bearing intact or mutated ARE. More efficient translation of fusion mRNA with intact versus mutated ARE was observed. This effect was amplified in ductus (10.9-fold) compared with nonmigratory, lower fibronectin-producing aorta cells (6.5-fold). Ductus cells transfected with wild-type but not ARE-mutated plasmid reverted to the stellate phenotype of aorta cells associated with reduced fibronectin production. This suggested that plasmid ARE sequesters RNA-binding factors, thereby reducing endogenous fibronectin mRNA translation. We next purified a 15-kD fibronectin ARE-dependent RNA-binding protein and identified it as microtubule-associated protein 1 light chain 3 (LC3). LC3 is present in greater amounts in ductus compared with aorta cells, and overexpression of LC3 in aortic cells by transfection enhances fibronectin mRNA translation to levels observed in ductus cells.

Amino Acid Sequence↗

PubMed1987

Dental papilla cells synthesize but do not deposit fibronectin in culture.

The dental papilla cells play a major regulatory role during tooth morphogenesis, and they are the only mesenchymal cells capable of differentiating into odontoblasts secreting dentin. In this paper, we have extended our studies on the behavior of cultured dental papilla cells which have been disaggregated from 17-day mouse embryo teeth. Quite unexpectedly, we observed that these cells, which in vivo are embedded in a fibronectin-rich extracellular matrix, lose all surface-associated fibronectin when cultured as monolayers. Fibronectin was, however, detected intracellularly, and metabolic labeling and immunoprecipitation studies indicated that the dental papilla cells continued to synthesize fibronectin in culture. Furthermore, when purified plasma fibronectin was added at 50 micrograms/mL to the culture medium, it became incorporated as fibrillar matrix on the surfaces of dental papilla cells. This indicates that the cells are not deficient in cell-surface receptors or other surface-associated molecules which bind fibronectin. When pieces of dental papillae were cultured as explants, an abundant matrix containing fibronectin was deposited on their surfaces. This matrix was gradually lost as the cells migrated from the explants. Furthermore, when the cells were disaggregated and cultured at high cell density, the cells in the central area of the pellet were covered by fibronectin containing fibrillar structures which were lost as the cells spread out. This indicates that the maintenance of close contacts between the dental papilla cells is required for the assembly of fibronectin into the extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Decreased plasma fibronectin during starvation in man.

This study evaluated the effect of 5 days of starvation followed by 5 days of refeeding on immunoreactive plasma and serum fibronectin and associated opsonic activity as studied by peritoneal macrophage monolayer bioassay in 12 healthy women volunteers. The temporal alteration of fibronectin was compared with the serum albumin, total iron-binding capacity, and retinol-binding protein levels. Fibronectin concentration and opsonic activity were also determined in two cachectic patients who were 61 and 78% of their ideal body weight. Prior to starvation, plasma fibronectin was 292 +/- 20 micrograms/ml and serum fibronectin was 182 +/- 16 in all subjects. After 5 days of starvation, immunoreactive fibronectin decreased (p less than 0.05) by 20-25%. This decrease was not great enough to impair opsonic activity as tested by the in vitro macrophage assay. Starvation caused no decrease in serum albumin or total iron-binding capacity, although retinol-binding protein decreased by 35%. During refeeding, subjects were randomized to a diet with (n = 6) and without (n = 6) carbohydrate. After 5 days of refeeding, fibronectin levels were normalized on the carbohydrate-containing diet, but were still low (82% of normal) on the carbohydrate-free diet. Retinol-binding protein did not fully normalize after 5 days of refeeding. In the two cachectic patients, fibronectin levels prior to total parenteral nutrition were 25 and 75% of normal. Thus, starvation can lower fibronectin levels and this protein is rapidly restored with adequate nutrition.

Adult↗

PubMed2005

Sturge-Weber syndrome: altered blood vessel fibronectin expression and morphology.

Sturge-Weber syndrome presents with vascular malformations of the brain, skin, and eye. Fibronectin has potent effects on angiogenesis, vessel remodeling, and vessel innervation density. To determine fibronectin expression in the blood vessels of Sturge-Weber syndrome brain and skin tissue and to quantify the density and circumference of Sturge-Weber syndrome blood vessels by type compared with controls, we performed in situ hybridization for fibronectin messenger ribonucleic acid (RNA) expression on six Sturge-Weber syndrome cortical brain samples, six epilepsy brain samples, skin from two port-wine stain skin lesions, and two normal skin samples from two subjects with Sturge-Weber syndrome. Fibronectin messenger RNA was expressed in blood vessels and endothelial cells in the parenchyma of both Sturge-Weber syndrome and control brain tissues and in skin samples. Fibronectin expression was significantly reduced by 23% in the Sturge-Weber syndrome meningeal vessels compared with the epilepsy controls (P < .01). Fibronectin expression was significantly increased by 19% in the Sturge-Weber syndrome parenchymal vessels compared with the epilepsy controls (P < .05). No difference was found in the expression of fibronectin in port-wine stain skin blood vessels. The density of leptomeningeal blood vessels in the Sturge-Weber syndrome brain tissue samples was 45% greater than in the epilepsy samples (P < .05). Blood vessel circumference was significantly decreased in the Sturge-Weber syndrome meningeal vessels compared with the controls (27%; P < .05). When blood vessels from different brain regions were compared, fibronectin expression was decreased in Sturge-Weber syndrome meningeal vessels and was increased in the parenchymal vessels. Altered blood vessel fibronectin expression in Sturge-Weber syndrome could contribute to abnormal vascular structure and function in this disorder.

Brain↗

PubMed1995

Differential targeting of recombinant fibronectins in AtT-20 cells based on their efficiency of aggregation.

In pituitary-derived AtT-20 cells, recombinant fibronectin containing the N-terminal matrix assembly domain and the C-terminal half of fibronectin does not follow the regulated secretory pathway but instead concentrates in distinct organelles prior to secretion. These organelles are larger than the dense-core granules and localize to the cell body at sites that differ from lysosomes, endosomes and endoplasmic reticulum. Unlike the dense-core granules, their discharge is not stimulated by 8-bromo-cyclic-AMP or phorbol esters. The kinetics of intracellular transport and secretion of the recombinant fibronectin suggest that it is present in a post-Golgi pool that turns over more slowly than constitutive vesicles. Indeed, the fibronectin-containing organelles disappear with a half-time of 3 hours after inhibiting protein synthesis. Presence of the organelles correlates with intracellular aggregation of dimeric fibronectin polypeptides. The organelles are absent in cells expressing monomeric recombinant fibronectin (lacking C-terminal dimerization sites) or the C-terminal half of fibronectin (which dimerizes but lacks the N-terminal matrix assembly domain), both of which aggregate less efficiently than dimeric fibronectin. Instead, the latter polypeptides enter the dense-core granules. Thus while the formation of the fibronectin-containing organelles may require efficient aggregation, it may not require a specific structural signal. Moreover, efficient aggregation is not necessarily a prerequisite for following the regulated pathway.

Adrenocorticotropic Hormone↗

PubMed1995

Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts.

The regulation of cell shape, fibronectin mRNA level, secretion and assembly by substratum surface topography was investigated in early passage human gingival fibroblasts cultured on titanium-coated smooth or V-shaped grooved substrata produced by micromachining. Cells on grooved surfaces were significantly elongated and orientated along the grooves of the substratum, while cell height, measured using confocal scanning laser microscopy, was approximately 1.5-fold greater than that of cells on smooth surfaces. Northern hybridization analysis revealed that on a per cell basis the grooved surface increased the amounts of fibronectin mRNA/cell approximately 3.5-fold at 16 hours, approximately 1.9-fold at 40 hours and approximately 2.2-fold at 90 hours, while the mRNA levels of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD) were constant. The amounts of secreted fibronectin on the grooved surface were increased approximately 2-fold for all time points. The stability of fibronectin mRNA was also altered by substratum surface topography. The half-life of fibronectin mRNA on smooth surfaces was estimated to be approximately 5 hours, but on the grooved surfaces the half-life of fibronectin mRNA showed a two-phase response: a rapid 60% reduction in the first half-life (t1/2 approximately 2 hours) and a 2.4-fold increase in the second half-life (t1/2 approximately 12 hours) relative to that observed on the smooth surface. The GAPD mRNA half-lives were essentially unaffected by the surface topography of the substrata. The grooved surface was also found to alter the amount of fibronectin assembled into the extracellular matrix, producing a approximately 2-fold increase in the cultures at all time points. It thus appears that substratum surface topography alters cell shape and modulates fibronectin at the transcriptional and post-transcriptional levels, as well as the amount of fibronectin assembled into extracellular matrix. Micromachining, which has the ability to precisely control surface topography over a wide range of dimensions and shapes, appears to be a useful technique in investigating the relationship between cell shape and function.

Adult↗

PubMed1996

Tenascin mediates human glioma cell migration and modulates cell migration on fibronectin.

The role of tenascin in mediating tumor cell migration was studied using two cell migration models. In migration/invasion Transwell assays U251.3 glioma cells rapidly migrated through the 8 mu m pore size membranes onto tenascin- and fibronectin-coated surfaces. In this assay the number of cells migrating onto tenascin was 52.2 +/- 9.6% greater than on fibronectin within 4 hours. To assess cell migration rates and cell morphology, U251.3 migration was examined in a two-dimension spheroid outgrowth assay. The radial distance migrated by U251.3 cells from tumor spheroids was found to be 53.8 +/- 4.9% greater on tenascin than on fibronectin. Cells migrating on tenascin display a very motile appearance, while cells migrating on fibronectin spread and maintain close intercellular contacts. Cell migration in the presence of integrin blocking antibodies demonstrated that migration on tenascin and fibronectin is mediated by distinct integrins, alpha2beta1 and alphavbeta5/alphavbeta3, respectively. Since tenascin is coexpressed in malignant tumor matrices with fibronectin, we assessed the effects of tenascin on U251.3 cell migration mediated by fibronectin. Tenascin was found to provide a positive effect on fibronectin-mediated migration by altering cell morphology and enhancing cell motility. These effects of tenascin on fibronectin-mediated cell migration were inhibited by blocking beta1 and alpha2beta1 integrins. The results suggest that tenascin may play a significant role in promoting tumor cell migration and invasiveness by modulating cell responses to normal matrix components.

Base Sequence↗