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PubMed1993

Further characterization of the binding of fibronectin to gelatin reveals the presence of different binding interactions.

The specific interaction between fibronectin and collagen has permitted the isolation of fibronectin from plasma using gelatin-Sepharose affinity matrices. In this study we obtained evidence suggesting the presence of an additional high affinity binding interaction between fibronectin and collagen. The following information supports this idea: (i) Successive isolations of fibronectin using the same regenerated gelatin matrix resulted in progressively increased yields of fibronectin, thus indicating the progressive occupation of high affinity fibronectin-binding sites within collagen. (ii) This tightly bound fibronectin could not be eluted from gelatin-Sepharose matrices with strong denaturing agents, such as 8.0 M urea or 6.0 M guanidium chloride. (iii) The fibronectin-occupied matrices supported fibroblast adhesion and spreading, indicating that the amount of tightly bound fibronectin was quantitatively sufficient for this function, and that the fibronectin that bound with high affinity retained its biologically active conformation. (iv) Two fibronectin-derived fragments, CB52kDa and T55 kDa, selectively bound to fresh gelatin-Sepharose but not to gelatin-Sepharose previously employed to purify fibronectin, suggesting that these fragments recognize only the high affinity binding sites in gelatin. N-terminal amino acid sequence analysis of T55 kDa showed that it contained the previously described collagen-binding domain and included the repeat III-1 of fibronectin. These results support the existence of multiple fibronectin-binding sites in collagen and implicate a high affinity site(s) with a propensity for tenacious binding.

Binding Sites↗
PubMed1983

The localization of fibronectin in rheumatoid arthritis synovium by light and electron microscopic immunohistochemistry.

The distribution of fibronectin in rheumatoid arthritis (RA) synovium has been investigated by light and electron microscopic immunohistochemistry using an antihuman fibronectin antibody. Heavy accumulation of fibronectin was observed in the lining layer and the areas of proliferation of fibroblasts. Rough endoplasmic reticulum (RER) and peripheral vesicles of proliferated type B lining cells and fibroblasts contained large amounts of fibronectin. Thus these cells seem to participate actively in the local synthesis and secretion of this glycoprotein. Type A lining cells and migrated mononuclear phagocytes contained many phagolysosomes in some of which dense accumulation of fibronectin was observed. Some of the materials in the phagolysosomes, with dense accumulation of fibronectin, resembled the fibrinous material-fibronectin complexes frequently seen in the pericellular spaces. Accordingly fibronectin seems to play a role in the clearance of fibrinous materials by these phagocytes. The proliferated capillaries and small vessels possessed multilamellated basement membranes with heavy accumulation of fibronectin. However, RER or Golgi apparatus of the endothelial cells contained no detectable amounts of fibronectin. This indicates that these cells do not actively participate in the synthesis of fibronectin and that the majority of this glycoprotein in the basement membranes originates in fibronectin from blood vessel exudate. Fibrinous material-fibronectin complexes were frequently seen on the endothelial cell surfaces. Circulating platelets and mononuclear cells occasionally came in contact with these complexes, suggesting an association of fibronectin with the formation and clearance of thrombi in the vascular lumina at the inflammatory sites of RA synovium.

Arthritis, Rheumatoid↗
PubMed1983

Exogenous fibronectin is not required for organogenesis in vitro.

The biological effect of plasma fibronectin on the differentiation of embryonic mouse kidney and tooth was studied in organ cultures. Transferrin (50 micrograms/ml) was a strong mitogen for kidney cells, whereas the addition of soluble fibronectin (50 to 250 micrograms/ml) had no detectable effect on differentiation or proliferation. The same serum-free, transferrin-containing medium did not support tooth differentiation. However, fibronectin was not a necessary serum component because fibronectin-free serum supported tooth development. It was demonstrated with antibodies specific for human fibronectin that the exogenously added human fibronectin at 50 micrograms/ml did not become incorporated to the cultured organs. Only minimal incorporation to the kidney basement membrane area was observed when fibronectin concentration was 250 micrograms/ml. The mesenchymal stroma and the basement membranes of the kidney and tooth rudiments cultured in fibronectin-free media stained intensely with conventional fibronectin antibodies, indicating endogenous production of fibronectin. Outgrowing epithelial cells from isolated kidney tubules produced fibronectin as well as laminin. The results suggest that the fibronectin found in the stroma and basement membranes is an endogenous product of the developing tissues and that plasma fibronectin is not required for in vitro organogenesis. The results also indicate that it is difficult to study the effect of fibronectin on morphogenetic processes because it may not penetrate the organ explants in vitro.

Animals↗
PubMed1986

Reversal of opsonic deficiency in surgical, trauma, and burn patients by infusion of purified human plasma fibronectin. Correlation with experimental observations.

Plasma fibronectin deficiency has been documented in critically ill surgical, trauma, and burn patients. Human plasma fibronectin was isolated by gelatin-Sepharose affinity chromatography and evaluated with respect to its opsonic activity following pasteurization, its in vivo clearance kinetics, and its short-term influence on cardiovascular hemodynamics in postoperative septic sheep. Six patients with low plasma fibronectin levels were also evaluated with respect to temporal changes of immunoreactive fibronectin and opsonic activity following infusion of fibronectin at a dose calculated to elevate the plasma fibronectin level to 400 micrograms/ml. With utilization of three different in vitro radioisotopic phagocytic assays, i.e., liver slice assay, peritoneal macrophage monolayer assay, and Kupffer cell monolayer assay, retention of opsonic activity by fibronectin following pasteurization was documented. The normal biphasic kinetics associated with plasma clearance of fibronectin were also not altered by pasteurization. In postoperative septic sheep with hemodynamic instability, intravenous infusion of 500 mg of purified human fibronectin initiated no abnormal hemodynamic response. Indeed, as compared with placebo, the infusion of fibronectin into the postoperative septic sheep resulted in a more stable systemic vascular resistance and pulmonary vascular resistance with a higher arterial pressure. It also elevated immunoreactive fibronectin levels (p less than 0.05) and increased opsonic activity (p less than 0.05). Surgical, trauma, and burn patients (ages 18 to 80 years) with low plasma fibronectin levels (160 to 236 micrograms/ml) manifested no disturbance in cardiovascular, respiratory, or hematologic parameters following fibronectin infusion (590 to 988 mg per patient), but did display an early increase of opsonic activity. This standardized, pasteurized, and opsonically active preparation of purified human plasma fibronectin (5.0 mg/ml after reconstitution) has utility for future randomized clinical trials in injured patients with sepsis.

Adolescent↗
PubMed1993

Influence of follicular maturation on the deposition of fibronectin by chicken granulosa cells in vitro--effect of 8-bromo cyclic AMP.

Fibronectin deposition by avian granulosa cells and the effect of 8-bromo cAMP (8-br-cAMP) on this process was investigated in vitro. Granulosa cells obtained from the first (F1) and third (F3) largest preovulatory follicles and from small yellow follicles (SYF) were incubated in serum free medium 199 for 12 or 24 hr with and without 8-br-cAMP. Fibronectin deposited in incubation wells secreted in medium or associated with cells was measured by ELISA. Basal fibronectin deposition increased with time and was greatest for F1 cells. Within 12 hr, 8-br-cAMP enhanced fibronectin deposition dose-dependently by F3 and SYF cells, but not by F1 cells. The amount of fibronectin deposition caused by 8-br-cAMP (which was greater after 24 hr incubation) was 0.10- to 0.16-fold for F1 cells; 0.20- to 0.81-fold for F3 cells and more than 30-fold for SYF cells. Fibronectin secreted in the medium by unstimulated cells was also greatest in F1 cells. 8-bromo cAMP stimulated fibronectin secretion in medium dose-dependently by F3 and SYF cells, however, it had only a marginal stimulatory effect on this process in F1 cells. Cell-associated fibronectin was not increased significantly by 8-br-cAMP in F1, F3 or SYF cells. Total (deposited plus medium plus cell associated) fibronectin production was elevated in dose- and time-dependent manner by 8-br-cAMP in F3 and SYF cells, but only at high concentrations in F1 cells. Like deposited fibronectin, the relative effect of 8-br-cAMP on fibronectin secreted in the medium and on total fibronectin production was greatest in immature SYF cells. These results demonstrate that fibronectin synthesized and deposited by unstimulated chicken granulosa cells in vitro increase concomitantly with follicular maturation and cellular differentiation, and the relative change in fibronectin production caused by 8-br-cAMP was greatest in immature SYF cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

A quantitative analysis of the incorporation of fibulin-1 into extracellular matrix indicates that fibronectin assembly is required.

Fibulin-1 is an extracellular matrix glycoprotein found in both loose and dense connective tissues, elastic fibers and some basement membranes. Cultured cells such as fibroblasts assemble endogenously synthesized or exogenously added fibulin-1 into matrix fibrils that also contain fibronectin. Since we have previously shown that fibulin-1 binds to fibronectin (Balbona, K., Tran, H., Godyna, S., Ingham, K. C., Strickland, D. K. and Argraves, W. S. J. Biol. Chem. 267: 20120-20125, 1992), we sought to investigate fibulin-1 incorporation into fibroblast extracellular matrix with an emphasis on evaluating the potential role of fibronectin in the process. In this study, we have used quantitative assays to measure the binding of 125I-fibulin to monolayers of cultured fibroblasts. Our results show that the kinetics of fibulin-1 incorporation into the cell layer and its partitioning into detergent-soluble and -insoluble fractions were similar to those of fibronectin. It was found that antibodies to fibronectin or to the fibulin-1-binding domain of fibronectin-inhibited fibulin-1 incorporation. Cell lines that fail to assemble fibronectin into the matrix, such as HT1080 or PFHR-9, do not incorporate fibulin-1 into their cell layers. However, when HT1080 cells were induced to assemble fibronectin by treatment with dexamethasone, they subsequently acquired the ability to incorporate fibulin-1. Moreover, treatment of cultured fibroblasts with antibodies that inhibit fibronectin assembly significantly inhibit fibulin-1 incorporation into the matrix. When increased amounts of fibronectin were incorporated into cells layers by incubating the cells for varying lengths of time with exogenous fibronectin, a corresponding increase in fibulin-1 incorporation was also observed. Taken together, the data indicate that the incorporation of fibulin-1 requires fibronectin assembly and suggests a dependence on the amount of fibronectin in a matrix. These results highlight the potential of fibronectin to control the deposition of fibulin-1 into those extracellular matrices where both proteins coincide and may have implications in the formation of fibulin-1-containing matrix structures such as basement membranes or elastic fibers.

Antibodies↗
PubMed2000

The effect of fetal fibronectin testing on admissions to a tertiary maternal-fetal medicine unit and cost savings.

OBJECTIVE: Fetal fibronectin bedside testing has been proposed as a diagnostic tool for the accurate diagnosis of preterm labor. The study objective was to determine whether the introduction of routine fetal fibronectin bedside testing affected costs and transfer rates from referral district hospitals to a tertiary obstetric hospital, as well as direct admissions to a tertiary referral hospital. STUDY DESIGN: We performed an 18-month prospective audit of fetal fibronectin use in 9 referral hospitals and one university maternal-fetal medicine unit. Data collected were delivery details and cervical dilatation at admission. Cost savings in terms of transport costs for patients with a negative fetal fibronectin result who were not transferred or admitted to the tertiary center were calculated for interhospital transfer (road ambulance or fixed-wing retrieval). RESULTS: One hundred fifty-one patients had a presumptive diagnosis of threatened preterm labor. Forty-five patients had a positive fetal fibronectin result and 106 had a negative fetal fibronectin result (3 with cervical dilatation >/=3 cm). Eleven (24%) patients with a positive fetal fibronectin result were delivered within 7 days, and 5 (5%) with a negative fetal fibronectin result were delivered within 7 days. One patient was delivered at 34 weeks, and the remaining patients were delivered at or after 36 weeks' gestation. All 3 patients with negative fetal fibronectin results with cervical dilatation of >/=3 cm were delivered within 5 days, leaving 2 (1.9%) patients (with closed cervices and negative fetal fibronectin results) being delivered 5 days after the fetal fibronectin testing. Ninety percent of the patients admitted to a referral hospital with threatened preterm labor who had a negative fetal fibronectin result were not transferred; thus an unnecessary transfer was avoided, with cost savings ranging from $30,297 for road and fixed-wing transport. CONCLUSION: A negative fetal fibronectin result is not helpful if cervical dilatation is present, and these patients should be treated as having a high risk of preterm delivery. The use of a fetal fibronectin test was associated with a 90% reduction in maternal transfer and can substantially reduce the costs and inconvenience associated with unnecessary transfer.

Antibodies, Monoclonal↗
PubMed1997

Expression, localization and alternative splicing pattern of fibronectin messenger RNA in fibrotic human liver and hepatocellular carcinoma.

BACKGROUND/AIMS: Fibronectin is a multifunctional glycoprotein and plays important roles in cell-to-cell or cell-to-matrix interaction. The molecular and functional diversity of fibronectin arises from alternative splicing of pre-mRNA at three variable regions, termed ED-A, ED-B and IIICS. Cellular fibronectin with ED-A and ED-B regions has different biological activities from plasma fibronectin lacking these regions. This study was aimed at investigating the type-specific expression of fibronectin in human liver diseases. METHODS: Immunohistochemistry with anti-total and anti-cellular fibronectin monoclonal antibodies, in situ hybridization with cDNA probes detecting common and ED-A regions and RT-PCR to amplify each variable region were performed in 35 specimens, including 4 control, 16 chronic hepatitis, 7 liver cirrhosis and 8 hepatocelular carcinoma. RESULTS: In control liver, there were slight deposits of cellular fibronectin [ED-A(+)fibronectin] in portal areas. In chronic hepatitis, it was strongly deposited at the margin of the fibrously enlarged portal areas where new collagen fibers were formed. Cellular fibronectin was evenly and abundantly accumulated in fibrotic septa in liver cirrhosis, and in fibrotic septa and capsules of tumor nodules in hepatocellular carcinoma. In control liver, cellular fibronectin mRNA was localized in a few hepatocytes and non-parenchymal cells around central veins, and was increased in the same cell populations near fibrously enlarged portal areas as hepatic fibrosis progressed. In hepatocellular carcinoma, it was expressed in most hepatoma cells. Fibronectin mRNA with three variable regions was detectable by RT-PCR in control liver as well as in each disease group. CONCLUSIONS: The expression of cellular fibronectin was increased in fibrotic human liver and hepatocellular carcinoma. In human liver, both non-parenchymal cells and hepatocytes participated together in cellular fibronectin production. In hepatocellular carcinoma, hepatoma cells were the main producer. Our results indicate that, in human liver, cellular fibronectin may participate in the hepatic fibrogenesis and in the malignant phenotypes of hepatocellular carcinoma.

Alternative Splicing↗
PubMed1987

Decreased interaction of fibronectin, type IV collagen, and heparin due to nonenzymatic glycation. Implications for diabetes mellitus.

The nonenzymatic glycation of basement membrane proteins, such as fibronectin and type IV collagen, occurs in diabetes mellitus. These proteins are nonenzymatically glycated in vivo and can also be nonenzymatically glycated in vitro. After 12 days of incubation at 37 degrees C with 500 mM glucose, purified samples of human plasma fibronectin and native type IV collagen showed a 13.0- and 4.2-fold increase, respectively, in glycated amino acid levels in comparison to control samples incubated in the absence of glucose. Gelatin (denatured calfskin collagen) was glycated 22.3-fold under the same conditions. Scatchard analyses were performed on the binding of radiolabeled fibronectin to gelatin or type IV collagen. It was found that there is a 3-fold reduction in the affinity of fibronectin to type IV collagen due to the nonenzymatic glycation of fibronectin. The dissociation constant (KD) for the binding of control fibronectin to type IV collagen was 9.6 X 10(-7) M while the KD for glycated fibronectin and type IV collagen was 2.9 X 10(-6) M. This was similar to the 2.7-fold reduction in the affinity of fibronectin for gelatin found as a result of the nonenzymatic glycation of fibronectin (KD of 4.5 X 10(-7) M for the interaction of control fibronectin with gelatin vs. KD of 1.2 X 10(-6) M for the interaction of nonenzymatically glycated fibronectin with gelatin). The molecular association of control fibronectin or its glycated counterpart with [3H]heparin was also determined. Scatchard analyses of this interaction showed no difference between control fibronectin and glycated fibronectin in [3H]heparin binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Basement Membrane↗
PubMed2006

Fibronectin induces cell proliferation and inhibits apoptosis in human bronchial epithelial cells: pro-oncogenic effects mediated by PI3-kinase and NF-kappa B.

The extracellular matrix glycoprotein, fibronectin, influences a variety of cellular functions including adhesion, migration, survival, differentiation, and growth. Fibronectin has also been shown to increase the migration and proliferation of human lung carcinoma cells. However, the role of fibronectin in controlling lung airway epithelial cell phenotype remains unknown. Here, we demonstrate that fibronectin stimulates the proliferation of human bronchial epithelial cells (BEAS-2B and 16-HBE). Of note, fibronectin induced the mRNA and protein expression of c-Myc and cyclin D1, while it decreased the expressions of cyclin-dependent kinase inhibitor p21 (WAF-1/CIP1/MDA-6) (p21) and the tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten (PTEN). Fibronectin also stimulated the phosphorylation of the phosphatidylinositol 3 kinase (PI3-K) downstream signal Akt. The inhibitor of PI3-K, Wortmannin, and anti-alpha5beta1 integrin antibodies abrogated the effect of fibronectin on c-Myc, cyclin D1, p21, and PTEN expression. The stimulatory effect of fibronectin was mediated by nuclear factor kappaB (NF-kappaB) since fibronectin induced the expression of the p65 component of NF-kappaB and enhanced NF-kappaB DNA binding. Furthermore, we found that p65 small interfering RNA inhibited the effect of fibronectin on c-Myc, cyclin D1, p21, PTEN expression, and on fibronectin-induced cell proliferation. Finally, we found that fibronectin inhibits apoptosis by reducing DNA fragmentation and inhibiting the activities of caspases 3/7. Taken together, our findings demonstrate that fibronectin stimulates human bronchial epithelial cell growth and inhibits apoptosis through activation of NF-kappaB, which, in turn, increases the expression of c-Myc and cyclin D1 and decreases p21 and PTEN via alpha5beta1 integrin-dependent signals that include PI3-K/Akt. Therefore, alternations in the extracellular matrix composition of the lung, with increased fibronectin, might promote epithelial cell growth and thereby contribute to oncogenesis in certain settings.

Apoptosis↗
PubMed1985

Effect of fibronectin on adherence of Staphylococcus aureus to fibrin thrombi in vitro.

Staphylococcus aureus binds to purified fibronectin in solution and may bind to fibronectin present in wound tissue. When incorporated into a solid fibrin thrombus, however, plasma fibronectin may fail to bind S. aureus, because the S. aureus-binding sites on fibronectin may be occupied by fibrin. Both S. aureus and fibrin bind to the same 27-kilodalton amino-terminal fragment of fibronectin. To determine whether fibronectin incorporated into fibrin still promotes the adherence of S. aureus, we clotted citrated normal plasma and fibronectin-depleted plasma onto petri dishes. We then measured bacterial adherence to these in vitro fibrin thrombi. We found that the adherence of five of seven S. aureus strains decreased significantly (by 26 to 58%) when fibronectin had been depleted from the fibrin thrombi. Adding fibronectin back reversed this decrease in adherence. The reversal was dose dependent; the increase was in proportion to the amount of fibronectin added back to the plasma. Bacteria known not to bind to fibronectin (Escherichia coli and Staphylococcus epidermidis) adhered 100-fold less than S. aureus, and their adherence was unaffected by the absence of fibronectin in the fibrin thrombus. We conclude that fibronectin incorporated into solid fibrin thrombi does mediate the adherence of most S. aureus strains to fibrin thrombi. Fibronectin may be an important molecule that mediates the adherence of S. aureus to fibrin in wounds.

Adhesiveness↗
PubMed1983

Kinetics of endogenously labeled plasma fibronectin: incorporation into tissues.

Fibronectin exists in a soluble form in plasma and lymph as well as in a relatively insoluble form in tissues. The disappearance of endogenously labeled fibronectin from plasma and its subsequent extravascular localization was studied over a 38-h period in normal rats (350-400 g) utilizing plasma fibronectin labeled in vivo with 75Se-selenomethionine. For comparative analysis, 125I-albumin was utilized in these dual isotopic experiments. After the simultaneous injection of 75Se plasma fibronectin (20 nCi) and 125I-albumin (2 microCi), all measured tissues demonstrated accumulation of both radiolabeled proteins in extravascular sites. Plasma fibronectin demonstrated a rather specific and high affinity for liver and spleen, which are enriched with reticuloendothelial cells. Albumin manifested the expected washout from extravascular sites, whereas fibronectin primarily displayed retention in tissues. The plasma disappearance of 75Se-labeled fibronectin was well described by two exponentials: an early phase with a half time of 0.52 h and a later phase with a half time of 21 h. To account for tissue retention of labeled fibronectin, a two-compartment kinetics model was required that included loss from the extravascular exchangeable compartment. Analysis of the disappearance kinetics and fibronectin distribution allowed estimation of the fractional turnover rate, pool size, and mean residence time. Accordingly the total rate of loss for plasma fibronectin was 0.51-0.54 mg/h. The calculated pool size of the soluble form of fibronectin was 15.5-16.3 mg. The mean residence time for exchangeable fibronectin was 29.6-29.9 h. These findings suggest that plasma fibronectin can be incorporated within the insoluble pool of fibronectin in tissues.

Animals↗
PubMed1984

Mechanism of acute depletion of plasma fibronectin following thermal injury in rats. Appearance of a gelatinlike ligand in plasma.

Plasma fibronectin was depleted within 15 min following sublethal burn, followed by partial recovery at 8 h and complete restoration by 24 h in anesthetized rats. Radiolabeled 75Se-plasma fibronectin, injected intravenously before burn, was rapidly sequestered in burn skin as well as the liver. Fibronectin levels at 2 h postburn as detected by immunoassay vs. 75Se-plasma fibronectin indicated that more fibronectin was in the plasma than detected by electroimmunoassay. Crossed immunoelectrophoretic analysis of fibronectin in early postburn plasma demonstrated a reduced electrophoretic mobility of the fibronectin antigen. Addition of heparin or fibrin, both of which have affinity for fibronectin, to normal plasma was unable to reproduce this altered fibronectin electrophoretic pattern. In contrast, addition of gelatin or native collagen to normal plasma reproduced the abnormal electrophoretic pattern of fibronectin seen in burn plasma. Extracts of burned skin, but not extracts of normal skin, when added to normal plasma, elicited a similar altered electrophoretic pattern for fibronectin. By gel filtration, fibronectin in burn plasma had an apparent molecular weight approximately 40% greater than that observed in normal plasma. These data suggest the release into the blood of a gelatinlike ligand from burned skin, which complexes with plasma fibronectin. Thus, fibronectin deficiency acutely postburn appears mediated by (a) its accumulation at the site of burn injury; (b) its removal from the circulation by the liver; and (c) its presence in the plasma in a form that is less detectable by immunoassay.

Animals↗
PubMed1986

Cytodifferentiation of granulosa cells induced by gonadotropin-releasing hormone promotes fibronectin secretion.

An analysis of the hormonal regulation of fibronectin production by granulosa cells showed that GnRH stimulated fibronectin secretion. GnRH increased fibronectin production 2.5- to 5-fold over that of control untreated cultures and greater than 20-fold over that of FSH-treated cultures. The GnRH concentration required for a minimal response was 10(-10) M, and that required for a maximal response was 10(-8) M. In contrast to the effects of GnRH, FSH suppressed fibronectin production to low or undetectable levels. In addition, FSH abolished the actions of GnRH on fibronectin production. Treatment of cultured granulosa cells with either (Bu)2cAMP or methylisobutylxanthine to influence cellular cAMP levels mimicked the actions of FSH on fibronectin secretion. Similar to FSH, both (Bu)2cAMP and methylisobutylxanthine abolished the actions of GnRH on fibronectin production. These results indicated that an increase in cAMP levels resulted in suppression of fibronectin secretion by granulosa cells and inhibition of the actions of GnRH. Insulin treatment of granulosa cells also suppressed fibronectin secretion, but to a lesser extent than FSH. In addition, insulin inhibited the actions of GnRH on fibronectin production by approximately 40-50% of stimulated levels. GnRH promoted a state of cytodifferentiation of the granulosa cell which had a high level of fibronectin production and, as shown previously, a low level of steroidogenesis. In contrast, FSH promoted a state of cytodifferentiation which had a low level of fibronectin production and a high level of steroidogenesis Both fibronectin and steroidogenic enzymes (e.g. aromatase) provide useful markers for an analysis of the cytodifferentiation of granulosa cells between these two distinct differentiated states. Results are discussed in relation to the possible mechanisms controlling granulosa cell cytodifferentiation and the possible functions of fibronectin in the ovary.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Intracrine role of progesterone in follicle-stimulating hormone- and cyclic adenosine 3',5'-monophosphate-induced fibronectin production and deposition by chicken granulosa cells: influence of follicular development.

The role of progesterone in FSH- and 8-bromo-cAMP (8-Br-cAMP)-induced fibronectin production by chicken ovarian granulosa cells was examined. Granulosa cells isolated from the third largest (F3; developing; 15-20 mm in diameter) preovulatory follicle and a pool of immature small yellow follicles (SYF; 6-8 mm in diameter) were incubated in serum-free medium 199, and the total amount of fibronectin produced (deposited, secreted into the medium, and associated with cells) was measured by enzyme-linked immunosorbent assay. Unstimulated F3 cells deposited greater amounts of fibronectin than unstimulated SYF cells. FSH or 8-Br-cAMP significantly increased fibronectin deposition. Similarly, both agents increased the quantity of fibronectin secreted into the medium and that associated with cells. The magnitude of FSH- and 8-Br-cAMP-enhanced fibronectin deposition or secretion into medium by SYF cells was greater than that by F3 cells. Cyanoketone (an inhibitor of progesterone synthesis) significantly suppressed basal fibronectin production by F3 cells, but not that by SYF cells. Cyanoketone completely blocked FSH- or 8-Br-cAMP-induced fibronectin production by F3 cells, but caused only a modest inhibition (nonsignificant) of agonist-induced fibronectin production by SYF cells. Exogenous progesterone completely reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The nondegradable synthetic progestin R5020 also reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The antiprogestin, ZK 98.299, inhibited basal and FSH-stimulated fibronectin production. The data demonstrate that FSH- and cAMP-stimulated fibronectin production by chicken granulosa cells is dependent (at least in part) on de novo progesterone synthesis. Furthermore, they indicate that fibronectin production and deposition by these cells are stimulated by progesterone, perhaps in an intracrine/autocrine manner, and that the role of progesterone increases with advancing stages of follicular development.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Fibronectin regulates calvarial osteoblast differentiation.

The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.

Amino Acid Sequence↗
PubMed1991

Fibronectin mRNA and protein accumulation, distribution, and breakdown in rabbit anti-glomerular basement membrane disease.

Fibronectin is a multifunctional matrix protein which by immunofluorescence appears to be present in increased amounts during glomerular injury. To examine fibronectin metabolism in glomerular injury, an anti-glomerular basement membrane model that progresses to severe glomerular crescent formation, glomerulosclerosis, and interstitial fibrosis was used. Fibronectin was purified from rabbit plasma, and a monoclonal antibody raised against rabbit fibronectin was used for immunolocalization and quantitation of fibronectin protein. RNA and protein were extracted from isolated glomeruli and whole renal cortex at various times during progression of disease. At day 4, there was a 2.5-fold increase in fibronectin protein which by immunofluorescence appeared to be in the glomerular mesangial area. There was no increase in glomerular fibronectin mRNA at this time. This discrepancy is consistent with the conclusion that, at this early time point, the increased glomerular fibronectin comes predominantly from plasma. By day 7, glomerular fibronectin mRNA and extractable fibronectin protein were increased in association with bright immunofluorescence along the inner aspect of Bowman's capsule where early crescents were forming. Similarly, at day 14, crescents stained very brightly for fibronectin. These results are consistent with the conclusion that, at later time points, fibronectin is synthesized in glomeruli in association with cell division and crescent formation. Degradation of fibronectin in glomerular and cortical extracts was demonstrated under normal and nephritic conditions by finding fibronectin proteolytic fragmentation by Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Nonenzymatic glycation of fibronectin and alterations in the molecular association of cell matrix and basement membrane components in diabetes mellitus.

This study reports the nonenzymatic glycation of plasma fibronectin in vivo in diabetic dogs and also in vitro by incubation of human plasma fibronectin with excess glucose. Although no difference is observed in the total plasma fibronectin level, the nonenzymatic glycation of fibronectin is increased 2.3-fold in inbred male beagle dogs made diabetic with alloxan in comparison with age-matched controls. The extent of non-enzymatic glycation of fibronectin is shown to be proportional to blood glucose levels. HPLC reverse-phase analysis of the hydrolyzed amino acids and glyco-amino acids from plasma fibronectin samples of normal and diabetic dogs show that nonenzymatic glycation occurs only on lysine residues. When purified human plasma fibronectin was incubated in vitro with 500 mM glucose, the extent of nonenzymatic glycation of fibronectin was observed to increase proportionately with time. Ligand binding assays conducted in solution with varying concentrations of 3H-heparin in the presence of a constant amount of normal or nonenzymatically glycated human plasma fibronectin gave virtually identical binding curves. However, the binding of 3H-heparin to normal fibronectin could be increased fourfold by the concomitant addition of normal gelatin (denatured calfskin collagen). If in vitro glycated fibronectin and/or in vitro glycated gelatin are added under this latter condition with 3H-heparin, there is a tremendous decrease in the expected heparin binding seen with normal levels of nonenzymatic glycation. Other experiments were performed to quantitate the binding of 3H-labeled fibronectin to gelatin-coated nitrocellulose filters. Nonenzymatic glycation of fibronectin in vitro resulted in markedly decreased binding of 3H-fibronectin to collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗