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PubMed1982

Opsonic fibronectin is necessary for optimal serum-mediated phagocytosis of Staphylococcus aureus by human neutrophils.

Opsonic fibronectin is known to mediate reticuloendothelial (RE) cell and neutrophil uptake of nonbacterial particulates. In a recent study opsonic fibronectin deficiency following burn preceded the onset of sepsis, leading us to hypothesize a role for this protein in antibacterial defense. To test this hypothesis we compared pooled normal human serum to fibronectin-depleted serum in its ability to opsonize and promote phagocytosis of Staphylococcus aureus by human neutrophil monolayers. Phagocytosis and intracellular killing were evaluated using acridine orange staining and ultraviolet (UV) microscopy. Human serum depleted of opsonic fibronectin by gelatin-sepharose affinity chromatography manifested a marked reduction in its ability to support phagocytosis of S aureus by human neutrophils. Reconstitution of fibronectin-deficient human serum with purified human plasma fibronectin restored its opsonic activity. The direct interaction of fibronectin with the bacteria was shown by mixing and/or incubation of the bacteria with normal serum followed by centrifugation and removal of the bacteria. This resulted in a marked (P less than 0.05) depletion (adsorption) of the fibronectin from the serum. Fibronectin appears not to act independently, but was an important cofactor in the ability for serum to stimulate phagocytosis. Thus, plasma fibronectin may be an important protein essential for maximal opsonic activity of serum. Its depletion following trauma and burn may undermine RE cell and neutrophil defense against infection and bacteremia, thus contributing to organ failure during septic shock.

Fibronectins↗
PubMed1995

Cyclic adenosine monophosphate and protein kinase C modulate fibronectin production in cultured human mesangial cells.

To identify potential intracellular mediators of mesangial cell fibronectin production, we examined the effect of cyclic adenosine monophosphate and protein kinase C on fibronectin biosynthesis in cultured human mesangial cells. We found that increasing intracellular cyclic adenosine monophosphate resulted in a 2.6-fold increase in fibronectin mRNA expression within 15 minutes; this reached a plateau after 1 hour and fell below control levels by 24 hours. Cyclic adenosine monophosphate significantly increased both immunoreactive fibronectin levels and the incorporation of [35S]-labeled methionine into fibronectin after 1 and 2 hours (251% +/- 3% and 157% +/- 18% of controls, respectively). The induction of protein kinase C with phorbol ester also increased fibronectin mRNA expression. The effect was time and dose dependent. Phorbol ester significantly increased both immunoreactive fibronectin levels and the incorporation of [35S]-labeled methionine after 1 and 2 hours (174% +/- 2% and 224% +/- 6% of controls, respectively). Inhibition of protein kinase C with calphostin C attenuated constitutive and phorbol ester induced fibronectin biosynthesis. These studies indicate that cyclic adenosine monophosphate and protein kinase C regulate mesangial cell fibronectin biosynthesis and that protein kinase C may contribute to control of constitutive fibronectin production.

Cells, Cultured↗
PubMed1994

[Effects of angiotensin II and pressure on cellular fibronectin in the vascular wall in organotypic culture].

Fibronectin is a dimeric glycoprotein found in the extracellular matrix of most tissues, which can influence processes, including cell growth, adhesion and migration. Fibronectin synthesis has been shown to be overexpressed in hypertension. However, the respective effects of humoral factors, including angiotensin II, versus mechanical factors in vascular remodeling have not yet been clarified. To study fibronectin de novo synthesis in the arterial wall, we have developed a new model for organ culture of rabbit thoracic aorta. Arteries held at their in vivo length were incubated and perfused (40 ml/min) in DME medium containing antibiotics, supplemented with 20% fetal calf serum or with 5% bovine serum albumin. In a series of experiments, angiotensin II (10(-6) M) and indomethacin (10(-5) M) were added to culture media. Vessels were pressurized at 0, 80 or 150 mmHg, and kept for 3 days in incubator at 37 degrees C under 5% CO2. De novo synthesis of fibronectin was detected by immunofluorescence using anti-cellular fibronectin antibodies (1/200). In the absence of angiotensin II and serum, fibronectin was expressed in the sub-endothelium at 80 mmHg, and in the inner media at 150 mmHg. In the presence of serum, fibronectin expression was increased by the high pressure. When angiotensin II was added, a gradient of fibronectin became apparent in the inner media at 80 mmHg with a marked expression at the luminal side. Angiotensin II markedly enhanced fibronectin expression at 150 mmHg, the protein being detected in almost the whole media. Our results indicate that both angiotensin II and transmural pressure can induce fibronectin expression in the arterial wall, and both act synergically.

Angiotensin II↗
PubMed1994

Evidence for Pneumocystis carinii binding to a cell-free substrate: role of the adhesive protein fibronectin.

Attachment of Pneumocystis carinii organisms to the alveolar epithelium is probably a prerequisite for the initiation of P. carinii infection. Previous studies have demonstrated a role for the extracellular matrix protein fibronectin in mediating attachment of P. carinii organisms to cultured alveolar cells; however, these studies could not clearly distinguish the role of fibronectin binding to P. carinii organisms and fibronectin binding to the alveolar cells. The current study demonstrates the direct binding of P. carinii organisms to substrate-bound soluble fibronectin P. carinii organisms bound specifically in a concentration-dependent manner to fibronectin-coated plates; maximum binding (19.1% +/- 1.9%) occurred at a fibronectin concentration of 50 micrograms/ml. P. carinii organisms did not bind significantly to bovine serum albumin-coated plates (5.8% +/- 1.2%). Binding of P. carinii organisms to fibronectin-coated plates was inhibited in a concentration-dependent manner by addition of the tetrapeptide arginine-glycine-aspartic acid-serine, which represents the active site of the fibronectin cell-binding domain. Similarly, attachment of P. carinii organisms was significantly inhibited by the addition of monoclonal antibodies directed against the cell-binding domain of fibronectin or by the addition of calcium ion chelators. To evaluate the role of the major P. carinii surface antigen gp120 in attachment of P. carinii organisms, purified gp120 and specific polyclonal anti-gp120 antibodies were used to inhibit attachment of P. carinii organisms to fibronectin-coated plates.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1998

Fibronectin expression in the developing human spinal cord, nerves, and ganglia.

AIM: Analysis of developmental role of fibronectin during differentiation of the human spinal cord, nerves, and ganglia. METHODS: Seven normal human embryos and fetuses between the 7th and 9th developmental week and a 9-week fetus with cervical spina bifida were histologically examined on hematoxylin and eosin stained serial paraffin sections of thoracic axial segments. Monoclonal antibody to the human cell fibronectin fragment was used for immunohistochemical detection of fibronectin. RESULTS: In the 7th and 8th week of development, fibronectin was weakly expressed in the ventricular and intermediate zones of the spinal cord. Intense fibrillar expression was found in the marginal zone of the spinal cord - first over the ventral gray horns and later over the lateral and dorsal gray horns, and along the pathways of ventral and dorsal roots of the spinal nerves and in the spinal ganglia. At 9th week, fibronectin expression disappeared in the ventricular and intermediate zones a nd became weak and granular in the marginal zone of the spinal cord. In the spinal cord of a 9-week malformed fetus with cervical spina bifida, fibronectin expression was completely absent. Fibronectin was expressed in the nerves and ganglia throughout the investigated period, both in normal and malformed human conceptuses. CONCLUSION: Transient expression of fibronectin in the human spinal cord coincided with the most intense neuronal differentiation. Temporal and spatial expression of fibronectin during normal development, and its absence in a malformed human fetus suggests developmental role of fibronectin for the normal formation of the spinal cord.

Biomarkers↗
PubMed2004

The role of fibroblasts in the modulation of integrin-dependent interactions between the gastric cell line HGT-1 and fibronectin.

Fibronectin plays an important role in gastric cancer progression. However, little is known about the microenvironmental factors modulating integrin-dependent interactions between gastric cancer cells and fibronectin. We therefore studied the regulation by fibroblasts of the integrin-dependent adhesion and migration of the gastric cancer cell line HGT-1 onto fibronectin. We first determined, by immunofluorescence, immunoblotting and flow cytometry, that HGT-1 cells expressed alpha3, alpha5, alpha6, alphaV and beta1 integrin chains, and the alphaVbeta3 and alphaVbeta5 dimers. We verified that HGT-1 cells xenografted to the immunosuppressed newborn rat retained the integrin repertoire detected in vitro and were able to induce the formation of tumors rich in fibronectin. By using an in vitro assay in the presence of neutralizing antibodies, we verified that HGT-1 adhesion and migration onto fibronectin involved beta1, alphaV and alpha5 integrin chains; we verified, by using an in situ adhesion test to rat gastric wall frozen sections, that in situ HGT-1 adhesion to fibronectin was integrin dependent. In coculture experiments, we showed that organ-specific fibroblasts from stomach, lung and dermis were able to induce, in a site-specific manner, the expression of beta1, alpha5 and alphaV integrin chains in HGT-1 cells, their integrin-dependent adhesion and migration on fibronectin and their capacity to secrete oncofetal fibronectin. In conclusion, our results show the capacity for tissue-derived fibroblasts to modulate the integrin-dependent interactions between the gastric cell line HGT-1 and fibronectin. They strongly suggest that, in gastric cancer, stromal fibroblasts contribute to promote fibronectin-mediated local invasion by tumor cells.

Animals↗
PubMed1993

Expression of fibronectin messenger RNA in hypertrophic and normal dermal tissues and in vitro regulation by interferon alfa-2b.

After severe thermal injury, hypertrophic scarring which is associated with accumulation of extracellular matrix proteins including fibronectin, frequently develops. We have recently demonstrated that interferon alfa-2b significantly reduces the level of type 1 procollagen messenger RNA expressed by both hypertrophic and normal dermal fibroblasts. In this report, we provide evidence that this cytokine also significantly decreases the expression of fibronectin messenger RNA in human hypertrophic scar and normal dermal fibroblasts. Four dermal fibroblast cell strains were established in cell culture from four human postburn hypertrophic scar tissues with the use of normal dermal fibroblasts from the same patients as controls. These cells were then treated with 2000 U/ml interferon alfa-2b in culture medium at various times. The results of Northern analysis of interferon-treated dermal fibroblasts indicate that this cytokine reduced the expression of fibronectin messenger RNA as early as 12 hours after treatment and reached its lowest level (24% relative to untreated fibroblasts) after 96 hours. When the expression of fibronectin messenger RNA was quantified by densitometry for each individual paired cell strain, a differential response to interferon treatment was found among cell strains. The level of fibronectin messenger RNA expression decreased from 17.2% to 69% in hypertrophic scar fibroblasts and 47% to 83.7% in normal fibroblasts relative to that of untreated control values. Although this decrease was less pronounced in normal fibroblasts than in hypertrophic scar fibroblasts, this reduction was significant in both interferon alfa-2b treated hypertrophic scar fibroblasts (6.39 +/- 0.71 versus 2.88 +/- 0.9, n = 4, p < 0.05) and normal cells compared with untreated controls (5.47 +/- 0.89 versus 3.64 +/- 0.99, n = 4, p < 0.05) as assessed with Student's paired t test. Rehybridization of the RNA blot prepared from interferon alfa-2b treated and untreated hypertrophic scar fibroblasts with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 gelatinase inhibitor showed no significant changes in abundance of this transcript. This result suggests that this cytokine selectively suppresses the expression of fibronectin messenger RNA and that this reduction is not due to RNA loading. A dot blot analysis of total RNA extracted from these tissues was carried out to compare the expression of fibronectin messenger RNA between human hypertrophic scar tissues and normal dermis obtained from the same patients. The blot was initially hybridized with fibronectin complementary DNA and subsequently with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 to correct for RNA loading. When the ratio of fibronectin to tissue inhibitor or metalloproteinase type 2 messenger RNA expression for each hypertrophic scar tissue was compared with its normal control, this ratio was fourfold higher in human hypertrophic scar tissues relative to normal controls. In contrast, the expression of this message in cultured hypertrophic scar fibroblasts was not significantly different from that in normal fibroblasts. The results of this study suggest that hypertrophic scarring developing after thermal injury is associated with an overexpression of fibronectin messenger RNA, and interferon alfa-2b may be of therapeutic value to down-regulate the expression of this transcript.

Journal Article↗
PubMed1993

Chicken B cells adhere to the CS-1 site of fibronectin throughout their bursal and postbursal development.

In chickens, the pre-B cell development takes place in a specialized organ, the bursa of Fabricius. In this study, we show that fibronectin can be detected in the bursa at least from the day 15 of embryonic incubation up to 10 weeks of age after which the bursa starts to involute. During the embryonic incubation, fibronectin localizes mainly in the areas between the follicles and later on also in the border zone between the cortical and medullary parts of the lymphoid follicles. By adhesion studies with synthetic peptides we show that immature bursal B cells bind in a dose-dependent manner to the CS-1 site of the type III connecting segment at the carboxy-terminal end of fibronectin throughout their bursal development. Postbursal B cells from chicken spleen also display a similar preference of binding to the CS-1 site of fibronectin, whereas peripheral blood B lymphocytes bind to the 40-kDa fragment of fibronectin, but do not adhere as strongly to the CS-1 site. Instead, they recognize to some extent also the heparin-binding YEKPGSPPREVVPRPRPGV peptide. The adhesion of the bursal B cells to the CS-1 site can partially be blocked by preincubation of cells with a synthetic CS-1 peptide and also by preincubation with a monoclonal anti-fibronectin receptor antibody. In contrast, the RGD motif present in several adhesion-associated stromal molecules did not promote any adherence of bursal lymphocytes. The bursal B cells which adhere to the fibronectin molecule in vitro form only a small fraction of the total number of B cells present in the bursa as only less than 4% of the cells remained bound to the surface coated with fibronectin in the adhesion studies. In conclusion, these results suggest that fibronectin may be involved in the differentiation of pre-B cells in the bursa of Fabricius and also in the adherence of postbursal B cells in the spleen.

Amino Acid Sequence↗
PubMed2006

Promotion by fibronectin of collagen gel contraction mediated by human corneal fibroblasts.

Collagen contraction mediated by corneal fibroblasts (CFs) is implicated in the maintenance of corneal shape. Given that fibronectin is expressed at sites of corneal stromal wounding, we investigated the effect of fibronectin on CF-mediated collagen gel contraction. Human CFs were cultured in a three-dimensional gel of type I collagen in the absence or presence of various extracellular matrix (ECM) components. The contraction of collagen gels mediated by CFs was evaluated by measurement of changes in gel diameter. The formation of stress fibers and focal adhesions in CFs was examined by fluorescence microscopy. The abundance of paxillin, phosphorylated paxillin, integrins alpha5, beta1, and alpha2, and alpha-smooth muscle actin in CFs was examined by immunoblot analysis. Fibronectin promoted CF-mediated collagen gel contraction in a concentration- and time-dependent manner. Other ECM proteins or glycosaminoglycans did not exhibit such an effect. Fibronectin also induced cell spreading, the formation of stress fibers, and the establishment of focal adhesions containing paxillin in CFs cultured in three-dimensional collagen gels. In addition, it increased the amounts of paxillin, phosphorylated paxillin, and integrins alpha5 and beta1 in these cells. The expression of integrin alpha2 and alpha-smooth muscle actin was not affected by fibronectin, however. Furthermore, the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the stimulatory effect of fibronectin on CF-mediated collagen gel contraction. These results suggest that fibronectin promoted CF-mediated collagen gel contraction in a manner dependent on the formation of stress fibers and focal adhesions, the activation of paxillin, and the up-regulation of integrin alpha5beta1. Fibronectin may therefore contribute to the maintenance of corneal shape by CFs during the healing of stromal wounds.

Actins↗
PubMed2000

alpha5beta1 integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury.

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding alpha5beta1 integrin. Whereas alpha5beta1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the alpha5beta1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the alpha5beta1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-beta1 integrin antibody, and an anti-alpha5beta1 integrin antibody, but not by an anti-beta3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the alpha5beta1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the alpha5beta1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-beta1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.

Actins↗
PubMed1995

Beta 1- and beta 3-class integrins mediate fibronectin binding activity at the surface of developing mouse peri-implantation blastocysts. Regulation by ligand-induced mobilization of stored receptor.

Implanting mouse blastocysts adhere through their abembryonic surfaces to the endometrial extracellular matrix. Because blastocysts cultured on fibronectin in vitro dissociate to form trophoblast outgrowths, it is unclear whether this adhesion is initially mediated by fibronectin receptors on the apical or basolateral surface of the trophectoderm. Intact blastocysts were examined in a ligand binding assay utilizing the fibronectin cell binding domain attached to fluorescent microspheres. Fibronectin binding activity on the apical surface of the trophectoderm was confined to the abembryonic pole of the blastocyst, where trophoblast differentiation initiates, and was regulated temporally in accordance with blastocyst outgrowth. Soluble fibronectin (IC50 = 0.2 microM) or Gly-Arg-Gly-Asp-Ser-Pro, but not laminin, competitively inhibited fibronectin binding activity. Addition of antibodies against the alpha v, alpha 5, beta 1, or beta 3 integrin subunits also inhibited binding activity. Blastocysts cultured in the absence of an adhesive substratum exhibited fibronectin binding activity only after exposure to immobilized or soluble ligand. Potentiation of binding activity by ligand was unaffected by cycloheximide but was sensitive to brefeldin A inhibition of protein trafficking. These findings suggest that the interaction of fibronectin with the trophectoderm induces a translocation event that up-regulates fibronectin binding beta 1- and beta 3-class integrins on the apical surface.

Amino Acid Sequence↗
PubMed1985

Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts.

Plasma fibronectin binds saturably and reversibly to substrate-attached fibroblasts and is subsequently incorporated into the extracellular matrix (McKeown-Longo, P.J., and D. F. Mosher, 1983, J. Cell Biol., 97:466-472). We examined whether fragments of fibronectin are processed in a similar way. The amino-terminal 70,000-mol-wt catheptic D fragment of fibronectin bound reversibly to cell surfaces with the same affinity as intact fibronectin but did not become incorporated into extracellular matrix. The 70,000-mol-wt fragment blocked binding of intact fibronectin to cell surfaces and incorporation of intact fibronectin into extracellular matrix. Binding of the 70,000-mol-wt fragment to cells was partially abolished by cleavage into 27,000-mol-wt heparin-binding and 40,000-mol-wt gelatin-binding fragments and more completely abolished by reduction and alkylation of disulfide bonds. Binding of the 70,000-mol-wt fragment to cells was not blocked by gelatin or heparin. When coated onto plastic, the 70,000-mol-wt fragment did not mediate attachment and spreading of suspended fibroblasts. Conversely, fibronectin fragments that had attachment and spreading activity did not block binding of exogenous fibronectin to substrate-attached cells. These results indicate that there is a cell binding site in the 70,000-mol-wt fragment that is distinct from the previously described cell attachment site and is required for assembly of exogenous fibronectin into extracellular matrix.

Binding Sites↗
PubMed1986

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells.

Many hemopoietic cell lines were examined for their ability to adhere to culture dishes coated with extracellular matrix proteins. Adhesion assay was performed with murine and human leukemic cell lines representative of different stages of differentiation along both erythroid and myeloid lineages. All the hemopoietic cell lines tested adhered to fibronectin but not to laminin, types I, III, and IV collagen, serum-spreading factor, and cartilage proteoglycans. In addition to immortalized cell lines, immature erythroid and myeloid mouse bone marrow cells adhered to fibronectin. To define the fibronectin region involved in hemopoietic cell adhesion, proteolytic fragments, monoclonal antibodies, and synthetic peptides were used. Among different fibronectin fragments tested, only a 110-kD polypeptide, corresponding to the fibroblast attachment domain, was active in promoting adhesion. Moreover, a monoclonal antibody to the cell binding site located within this domain prevented hemopoietic cell adhesion. Finally, the tetrapeptide Arg-Gly-Asp-Ser, which corresponds to the fibronectin sequence recognized by fibroblastic cells, specifically and competitively inhibited attachment of hemopoietic cells to this molecule. The cell surface molecule involved in the interaction of mouse hemopoietic cells with fibronectin was identified as a 145,000-D membrane glycoprotein by adhesion-blocking antibodies. This glycoprotein was found to be antigenically and functionally related to the GP135 membrane glycoprotein involved in the adhesion of fibroblasts to fibronectin (Giancotti, F. G., P. M. Comoglio, and G. Tarone, 1986, Exp. Cell Res., 163:47-62). On the basis of these data, we conclude that interaction of hemopoietic cells with fibronectin involves a specific fibronectin sequence and a 145,000-D cell surface glycoprotein. We speculate that this property might be relevant for the interaction of hemopoietic cells with the bone marrow stroma, which represents the natural site of hemopoiesis.

Animals↗
PubMed1987

Induction of fibronectin matrix assembly in human fibrosarcoma cells by dexamethasone.

Previous studies have suggested that the assembly of fibronectin into the extracellular matrix of cultured fibroblasts is mediated by specific matrix assembly receptors that recognize a binding site in the amino terminus of the fibronectin molecule (McKeown-Longo, P.J., and D.F. Mosher, 1985, J. Cell Biol., 100:364-374). In the presence of dexamethasone, human fibrosarcoma cells (HT-1080) acquired the ability to specifically bind exogenous plasma fibronectin and incorporate it into a detergent-insoluble extracellular matrix. Dexamethasone-induced fibronectin binding to HT-1080 cells was time dependent, dose dependent, and inhibited by cycloheximide. Saturation binding curves indicated that dexamethasone induced the appearance of 7.7 X 10(4) matrix assembly receptors per cell. The induced receptors exhibited a dissociation constant (KD) for soluble fibronectin of 5.0 X 10(-8) M. In parallel experiments, normal fibroblasts exhibited 4.1 X 10(5) receptors (KD = 5.3 X 10(-8) M) per cell. In the presence of cycloheximide, the induced fibronectin-binding activity on HT-1080 cells returned to uninduced levels within 12 h. In contrast, fibronectin-binding activity on normal fibroblasts was stable in the presence of cycloheximide for up to 54 h. The first-order rate constant (Kt = 2.07 X 10(-4) min-1) for the transfer of receptor-bound fibronectin to extracellular matrix was four- to fivefold less than that for normal fibroblasts (Kt = 1.32 X 10(-3) min-1). Lactoperoxidase-catalyzed iodination of HT-1080 monolayers indicated that a 48,000-mol-wt cell surface protein was enhanced with dexamethasone. The results from these experiments suggest that dexamethasone induces functional matrix assembly receptors on the surface of HT-1080 cells; however, the rate of incorporation of fibronectin into the matrix is much slower than that of normal fibroblasts.

Cell Membrane↗
PubMed1987

Identification of fibronectin receptors on T lymphocytes.

We report the identification of fibronectin receptors on thymocytes and T lymphoma cells. Affinity chromatography of extracts of the T cell lymphoma, WR16.1, on a fibronectin-Sepharose column combined with specific elution using a synthetic peptide containing the cell attachment-promoting sequence, arginine-glycine-aspartic acid, yielded two polypeptide components having apparent molecular masses of approximately 160 kD reduced and 175 and 150 kD nonreduced. Immunoprecipitations from surface-iodinated WR16.1 cells or fibronectin-adherent thymocytes using a rabbit antiserum raised against the fibronectin receptor that is present on human fibroblasts revealed, in each case, the same two radiolabeled components. In contrast, immunoprecipitation from fibronectin-nonadherent T lymphoma cells, designated WR2.3, revealed the presence of only the smaller subunit. Although the lymphocyte receptor and the fibronectin receptor identified on fibroblasts share immunologic determinants, they differ in that the molecular mass of the lymphocyte protein is larger. Moreover, trypsinization of either thymocytes or the WR16.1 T lymphoma cells resulted in a subsequent loss of their ability to adhere to fibronectin-coated substrates and a reduction in the electrophoretic mobility of each of the polypeptide chains of the fibronectin receptor present on their surfaces. These changes, however, were not observed with normal rat kidney fibroblasts or mouse 3T3 fibroblasts in response to trypsinization. The data establish the existence on normal lymphocytes of fibronectin receptors that are quite similar to those found on fibroblasts. The possible function of this molecule on thymocytes is discussed.

Animals↗
PubMed1987

Human osteosarcoma cells resistant to detachment by an Arg-Gly-Asp-containing peptide overproduce the fibronectin receptor.

MG-63 human osteosarcoma cells were selected for attachment and growth in the presence of increasing concentrations of a synthetic peptide containing the cell attachment-promoting Arg-Gly-Asp sequence derived from the cell-binding region of fibronectin. Cells capable of attachment and growth in 5-mM concentrations of a peptide having the sequence Gly-Arg-Gly-Asp-Ser-Pro overproduce the cell surface receptor for fibronectin. In contrast, these cells show no differences in the numbers of vitronectin receptor they express as compared with the parental MG-63 cells. In agreement with the resistance of the selected cells to detachment by the peptide, 25-fold more Arg-Gly-Asp-containing peptide is required to prevent the attachment of these cells to fibronectin-coated surfaces than is needed to inhibit the attachment of MG-63 cells to the same substrate. However, similar concentrations of this peptide inhibit attachment of both cell lines to vitronectin-coated surfaces. The increase in fibronectin receptor is due to an increase in the levels of mRNA encoding the fibronectin receptor. Because of the nature of the selection process, we reasoned that this increase might be due to amplification of the fibronectin receptor gene, but no increase in gene copy number was detected by Southern blot analysis. The peptide-resistant cells display a very different morphology from that of the MG-63 cells, one that has a greater resemblance to that of osteocytes. The resistant cells also grow much more slowly than the MG-63 cells. The increased fibronectin receptor and altered morphology and growth properties were stable for at least 3 mo in the absence of peptide. The enhanced expression of the fibronectin receptor on the resistant cells indicates that cells are capable of altering the amount of fibronectin receptor on their surface in response to environmental factors and that this may in turn affect the phenotypic properties of the cell.

Amino Acid Sequence↗
PubMed1992

Regulation of fibronectin receptor distribution.

To determine the role of each intracellular domain of the fibronectin receptor in receptor distribution, chimeric receptors were constructed containing the human interleukin-2 receptor (gp55 subunit) as the extracellular and transmembrane domains, in combination with either the alpha 5 or beta 1 intracellular domain of the fibronectin receptor as the cytoplasmic domain. These chimeric receptors were transiently expressed in normal fibroblasts, and their localization on the cell surface was determined by immunofluorescence using antibodies to the human interleukin-2 receptor. The alpha 5 chimera was expressed diffusely on the plasma membrane. The beta 1 chimera, however, colocalized with the endogenous fibronectin receptor at focal contacts of cells spread on fibronectin. On cells spread in the presence of serum, the beta 1 chimera colocalized both with the fibronectin receptor at sites of extracellular fibronectin fibrils and with the vitronectin receptor at focal contacts. The beta 1 intracellular domain alone, therefore, contains sufficient information to target the chimeric receptor to regions of the cell where ligand-occupied integrin receptors are concentrated. The finding that the beta 1 chimeric protein behaves like a ligand-occupied receptor, even though the beta 1 chimera cannot itself bind extracellular ligand, suggests an intracellular difference between occupied and unoccupied receptors, and predicts that the distribution of integrin receptors can be regulated by ligand occupancy. We tested this prediction by providing a soluble cell-binding fragment of fibronectin to cells spread on laminin. Under conditions preventing further ligand adsorption to the substrate, this treatment nevertheless resulted in the relocation of diffuse fibronectin receptors to focal contacts. Similarly, a redistribution of diffuse vitronectin receptors to focal contacts occurred on cells spread on laminin after the addition of the small soluble peptide GRGDS. We conclude that the propensity for receptor redistribution to focal contacts driven by the beta 1 cytoplasmic domain alone is suppressed in heterodimeric unoccupied fibronectin receptors, and that ligand occupancy can release this constraint. This redistribution of integrin receptors after the binding of a soluble substrate molecule may provide a direct means of assembling adhesion sites.

Amino Acid Sequence↗
PubMed1983

Binding of plasma fibronectin to cell layers of human skin fibroblasts.

Human plasma fibronectin bound to confluent cell layers of cultured human-skin fibroblasts in two distinct pools. Initial binding of fibronectin occurred in a deoxycholate-soluble pool (Pool I). Binding in Pool I was reversible and reached a steady state after 3 h. After longer periods of incubation, fibronectin became bound in a deoxycholate-insoluble pool (Pool II). Binding in Pool II was irreversible and proceeded at a linear rate for 30 h. After 30 h of incubation, a significant proportion of fibronectin bound in Pool II was present as disulfide-bonded multimers. HT1080 cells, a human sarcoma cell line, did not bind fibronectin in either pool. Also, isolated cell matrices prepared by deoxycholate extraction did not bind fibronection. Binding of fibronectin in Pool I of normal fibroblasts occurred via specific, saturable receptors. There were 128,000 binding sites per cell, and KDiss was 3.6 X 10(-8) M. Fluorescence microscopic localization of fibronectin bound in Pool I and Pool II was performed using fluorescein-conjugated fibronectin. Fluorescent staining in Pool I was present in a punctate pattern and in short, fine fibrils. Pool II fluorescence was exclusively in coarse, dense fibrils. These data indicate that plasma fibronectin may become incorporated into the tissue extracellular matrix via specific cell-surface receptors.

Binding Sites↗