Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.     

Synthesis and secretion of lecithin-cholesterol acyltransferase by the human hepatoma cell line HepG2

The human liver cell line HepG2 was investigated for its synthesis and secretion of lecithin-cholesterol acyltransferase. The cells were grown to confluency in Eagle's minimal essential medium plus 10% fetal bovine serum. At the onset of the study, fetal bovine serum was removed and cells were grown in minimal essential medium only. At 6, 12, 24, and 48 h the cells were harvested, and the culture medium collected at each time point was assayed for lecithin-cholesterol acyltransferase mass and activity, cholesterol esterification rate, and apolipoprotein A-I mass. The rate of the enzyme secretion measured by both mass and activity was linear over 24 h of culture. The enzyme mass by radioimmunoassay was 1.7, 4.1, 7.9 and 13.7 ng/ml culture medium (or 8.3, 19.9, 38.5 and 66.7 ng/mg cell protein), respectively, and enzyme activity using an exogenous source of phosphatidylcholine/cholesterol liposomes containing apolipoprotein A-I as substrate was 85, 170, 315, and 402 pmol cholesterol esterified/h per ml culture medium (or 414, 828, 1534 and 1957 pmol cholesterol esterified/h per mg cell protein) for 6, 12, 24, and 48 h of culture, respectively. The endogenous cholesterol esterification rate of the culture medium was 47, 104, 224 and 330 pmol/h per ml and apolipoprotein A-I mass was 305, 720, 2400 and 3940 ng/ml culture medium over the same time frame. In contrast to culture medium, low levels of enzyme activity (approximately 10% of that in culture medium at 24 and 48 h) were observed in the extracts of HepG2 cells. The enzyme secreted by HepG2 was found to be similarly activated by apolipoprotein A-I, apolipoprotein E, or apolipoprotein A-IV, and was similarly inhibited by phenylmethylsulfonyl fluoride, dithiobisnitrobenzoate, p-hydroxymercuribenzoate, or iodoacetate as compared to human plasma enzyme. High-performance gel filtration of the culture medium revealed that the HepG2-secreted enzyme was associated with a fraction having a mean apparent molecular weight of approximately 200,000. We concluded that human hepatoma HepG2 cells synthesize and secrete lecithin-cholesterol acyltransferase, which is functionally homologous to the human plasma enzyme.     

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.     

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

Production of proteoglycans by human lung fibroblasts (IMR-90) maintained in a low concentration of serum.

Maintenance of fibroblasts in 0.5% serum results in viable but non-proliferative cells that may be analogous to fibroblasts in vivo. The synthesis of proteoglycans by human embryo lung fibroblasts in Eagle's minimal essential medium with 0.5% newborn-bovine serum or with 10% serum has been compared. A similar amount of [35S]sulphate-labelled glycosaminoglycan per cell was secreted by fibroblasts in 10% or 0.5% serum. 35SO42-incorporation into sulphated glycosaminoglycans was enhanced in 0.5% serum when expressed per mg of cell protein, but [3H]glucosamine incorporation was decreased. The charge density of these glycosaminoglycans was not changed as determined by ion-exchange chromatography. It was concluded that decreased protein/ cell resulted in an apparent increase in 35S-labelled glycosaminoglycan synthesis/mg of cell protein, whereas decreased uptake of [3H]glucosamine resulted in a decrease in their glucosamine labelling. The proteoglycans secreted by fibroblasts in 0.5% serum were similar in glycosaminoglycan composition, chain length and buoyant density to the dermatan sulphate proteoglycan, which is the major secreted component of cells in 10% serum. Larger heparan sulphate and chondroitin sulphate proteoglycans, which comprise about 40% of the total secreted proteoglycans of cultures in 10% serum, were greatly diminished in the medium of cultures in 0.5% serum. The proteoglycan profile of medium from density-inhibited cultures in 10% serum resembles that of proliferating cultures, indicating that lack of proliferation was not responsible for the alteration. The dermatan sulphate proteoglycan, participating in extracellular matrix structure, may be the primary tissue product of lung fibroblasts in vivo.     

Developmental regulation of a secreted gelatin-binding protein during myogenesis in vitro

Collagen has a stimulatory effect on the differentiation of skeletal muscle cells in culture. Putative collagen-binding proteins were isolated from detergent-solubilized cultures of the L6 rat muscle cell line and primary clonal cultures of human skeletal muscle satellite cells, using gelatin-Sepharose affinity chromatography. In addition to fibronectin, which has been reported by others to be synthesized by cultured muscle cells, we found that muscle cultures synthesized gelatin-binding proteins of lower apparent molecular weight. Only one of these proteins was secreted into the growth medium and bound to type I collagen. Binding of this protein to gelatin and collagen-Sepharose was resistant to repeated washing with 1 M NaCl and nonionic detergent. The secreted gelatin-binding protein had an apparent molecular weight of 63,000-72,000, depending upon the conditions of electrophoresis. The lack of reactivity of the secreted protein with polyclonal antisera against fibronectin, the lack of effect of protease inhibitors on its appearance in the medium, and the rapid de novo production of the protein during pulse labeling with radioactive methionine indicated that it was not a fibronectin fragment. The rate of synthesis of the secreted gelatin-binding protein increased markedly during the myogenesis of rat and human cultures.     

Multiple mechanisms of dissociated epidermal cell spreading

To test the possibility that epidermal cells use a common basement membrane protein whenever they spread, in vitro experiments were conducted using trypsin-dissociated guinea pig epidermal cells and the following proteins: human serum, bovine serum albumin, serum fibronectin, Type IV collagen, laminin, and epibolin (a recently described serum glycoprotein which supports epidermal cell spreading; Stenn, K.S., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6907.). When the cells were added to media containing the specific proteins, all the tested proteins, except for serum albumin, supported cell spreading. Added to protein-coated substrates in defined media, the cells spread on fibronectin, epibolin, and laminin-Type IV collagen, but not on albumin or whole serum. In none of these experiments were the results qualitatively affected by the presence of cycloheximide. Antibodies to a specific protein blocked cell spreading on that protein but not on the other active proteins, e.g. whereas antibodies to epibolin blocked cell spreading on epibolin, they did not affect spreading on fibronectin, collagen, or laminin. In a second assay in which the cells were allowed to adhere to tissue culture plastic before the protein-containing medium was added, the cells spread only if the medium contained epibolin. Moreover, under these conditions the spreading activity of whole serum and plasma was neutralized by antiepibolin antibodies. These results support the conclusion that dissociated epidermal cells possess multiple spreading modes which depend, in part, on the proteins of the substrate, proteins of the medium, and the sequence of cell adhesion and protein exposure.     

Fibronectin and retinyl acetate effects on attachment and spreading of normal and rheumatoid human synovial cells

The action of two effectors - fibronectin (FN) and retinyl acetate (RA) - on cell attachment and spreading of human synoviocytes was investigated by adding these two drugs to the cell culture medium. No relationship was observed between the level of the effectors (FN = 20-80 micrograms/well, RA = 0.50-2 micrograms/well) and the biological effects studied. For normal human synoviocytes, fibronectin was less effective on the adhesion than fetal calf serum (FCS) present in the control culture medium; retinyl acetate, a drug acting on glycoprotein synthesis, led to similar effects to those observed for FCS-treated cells. In the case of rheumatoid synovial cells, the degree of adhesion was similar for drug- and FCS-treated cultures. Moreover, FN and RA had little effect on the spreading compared to FCS. Given these results, it would appear that synoviocytes differ in their behaviour from usual fibroblastic models.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase

We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.     

PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma

Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma.     

Biosynthesis of the carbohydrate portion of immunoglobins. Kinetics of synthesis and secretion of [3H]leucine-, [3H]galactose- and [3H]mannose-labelled myeloma protein by two plasma-cell tumours

The kinetics of incorporation of leucine, galactose and mannose into intracellular and secreted myeloma protein, MOPC 21 IgG(1) and MOPC 46 kappa-type light chain, by cell suspensions of two myeloma plasma-cell tumours, MOPC 21 and MOPC 46, were similar. Radioactive galactose was incorporated to over 90% into galactose residues of intracellular and secreted protein, mannose to over 90% into glucosamine and mannose residues of intracellular protein and to over 90% into glucosamine, mannose and fucose residues of secreted protein, but not into galactose residues. The results show that specific residues in the carbohydrate portion of myeloma proteins can be labelled by specific radioactive monosaccharides, and suggest that fucose residues are added, while myeloma protein is in its final stage of secretion from the plasma cell. The kinetics of incorporation indicate at least three sequential precursor-product relationships between different intracellular forms and the secreted form of myeloma protein.     

Substratum influence on collagen and fibronectin biosynthesis by arterial smooth muscle cells in vitro

Collagen, fibronectin, and nonfibrous protein biosynthesis were examined in cultures of rabbit arterial smooth muscle cells grown on tissue culture plastic precoated either with rabbit plasma fibronectin or bovine serum albumin. Cells seeded into fibronectin-coated wells appeared to reach confluence more quickly than counterparts grown on albumin-coated surfaces. Measurement 3H-thymidine incorporation into DNA by these cultures suggested that this was probably a consequence of more rapid and efficient cell attachment rather than an increased rate of proliferation of smooth muscle cells grown on fibronectin. In preconfluent cultures, the rates of collagen and fibronectin biosynthesis were reduced to 34 and 57%, respectively, on a per-cell basis in cultures grown on fibronectin-coated surfaces compared with cells grown on albumin-coated plasticware. In preconfluent cultures grown on fibronectin-coated surfaces, a greater percentage of the total fibronectin synthesized was incorporated into the cell layer. The distribution of newly synthesized collagen between culture medium and cell layer, however, was not affected by alteration of substratum composition. There was no difference in the rate of synthesis of noncollagen proteins between the two groups of preconfluent cells. In postconfluent cultures the rates of collagen and fibronectin biosynthesis were equivalent in both albumin- and fibronectin-treated cultureware. In preconfluent cultures, analyses of procollagens showed that the overall amounts of both types I and III procollagens were reduced in fibronectin-treated wells, indicating the reduction in collagen synthesis to be general and not type-specific. Although type V procollagen biosynthesis was not detected in either preconfluent group, it was found in postconfluent cultures. The reduction of fibronectin synthesis in cells grown in fibronectin-coated wells was significant as early as 4 hours after plating. Together, these findings suggest that cultured arterial smooth muscle cells are capable of deriving information from their substratum and regulating the biosynthetic rates of extracellular matrix components in response to the immediate needs of the cell.     

Spreading of human fibroblasts in serum-free medium: Inhibition by dithiothreitol and the effect of cold insoluble globulin (plasma fibronectin)

We have tested the effect of dithiothreitol (DTT) treatment on the initial spreading of human fibroblasts in serum-free medium in tissue culture dishes. Cell spreading was inhibited following treatment of these cells with 10 mM DTT. Inhibition occurred when the cells were treated at 37°C but not at 4° and was reversible metabolically but not by the addition of sulfhydryl oxidizing reagents. The inhibition was overcome when DTT-treated human fibroblasts were plated on cold insoluble globulin (plasma fibronectin)—coated dishes. Under these conditions spreading appeared to be completely normal, including the formation of focal adhesions. Analysis of the fibronectin concentrations in the human fibroblasts following DTT treatment indicated that there was little decrease in the absolute level of activity as determined in a biological assay for BHK cell spreading on culture dishes. Analysis of the fibronectin distribution on the DTT-treated human fibroblasts by indirect immunofluorescence using a specific anti-CIG antiserum revealed that fibronectin was no longer deposited onto the culture dish surfaces. Even when the DTT-treated human fibroblasts spread in the presence of fetal calf serum, the cell fibronectin remained for the most part in a perinuclear location. These results indicate that DTT treatment of human fibroblasts prevents the normal translocation of fibronectin from a perinuclear location to the surface of the culture dish. This study further supports our hypothesis that the initial spreading in serum-free medium of fibroblasts from cell strains depends upon secretion of fibronectin onto the culture dish surface.     

Studies of fibronectin synthesized by cultured chick hepatocytes

We have adapted a chick embryo liver cell system for studying the synthesis of proteins secreted by hepatocytes. In primary liver cell cultures maintained for several days in arginine-deficient medium containing ornithine (0.7 mM) and carbamyl phosphate (1 mM), only hepatocytes demonstrated normal morphological and biosynthetic characteristics, indicating that they possessed a functional ornithine cycle as a source of arginine production. Non-parenchymal liver cells, such as fibroblasts, which lack the ornithine cycle were excluded. Hepatocytes in arginine-deficient or arginine-containing medium synthesized fibronectin (Fn) over several days at a constant rate of 3 micrograms +/- 1 microgram/mg cell protein per day, with fibronectin representing approximately 3% of the total secreted hepatocyte proteins during any culture period after the first 24 h. Pulse-chase experiments indicated that Fn synthesis and secretion was relatively rapid (t1/2 = 45 min) and represented approximately 95% of the intracellularly labelled Fn. This Fn is secreted predominantly as a 450 kD dimer with a subunit size that is indistinguishable from the plasma form as assessed by one-dimensional electrophoretic analysis. Continuous exposure of hepatocytes to insulin caused a moderate decrease (26%) in Fn synthesis, whereas there was no effect of short-term exposure. In contrast, dexamethasone stimulated Fn production 2-3-fold, consistent with its known ability to stimulate hepatocyte production of acute phase proteins. Under these conditions, electrophoretic analyses showed that an increased quantity of intact hepatocyte Fn was produced having the same molecular size of plasma Fn.     

Fibroblast cellular and plasma fibronectins are similar but not identical

Fibronectins are multimeric, adhesive glycoproteins present on cell surfaces and circulating in blood. Cellular fibronectin produced by fibroblasts in vitro and fibronectin isolated from plasma are known to be very similar immunologically and biochemically. We investigated whether or not they are identifical. Purified chicken and human cell-surface fibronectins are 150-fold more active in hemagglutination of fixed erythrocytes than plasma fibronectins. Cell-surface fibronectin is also 50-fold more active in restoring a more normal morphology to transformed cells originally missing the protein. However, in two other assays that measure cell attachment to collagen and cell spreading, cell-surface and plasma fibronectins have identical specific activities. In sodium dodecyl sulfate polyacrylamide gels, the subunits of human and chicken plasma fibronectins have significantly smaller apparent subunit molecular weights than cellular fibronectins present on cell surfaces or secreted into culture media. These differences are also present in a characteristic large subfragment of both forms of fibronectin after limited proteolysis by trypsin. We conclude that by both biological and biochemical criteria, cellular and plasma fibronectins are similar but not identical.     

A specificity for cellular fibronectin in its effect on cultured chondroblasts

Abstract. Chick cellular fibronectin has previously been shown to alter the phenotypic properties of cultured chick-embryo vertebral chondroblasts. Over the course of several days, adhesion and spreading on plastic substrata in the presence of serum was stimulated, the morphology of the cells was changed, the synthesis of cartilage-specific type-IV proteoglycan was inhibited, and the synthesis of type-I collagen and fibronectin was induced or stimulated. In the present study, chick plasma fibronectin was isolated and observed to mimic the effect of cellular fibronectin on cell adhesion and spreading. Both kinetic and dose-response relationships were similar between the two isoproteins. In contrast, chick plasma fibronectin, at up to tenfold higher concentrations by weight, did not alter cell morphology or synthesis of type-IV proteoglycan. Control experiments showed that plasma fibronectin could not neutralize cellular fibronectin and that plasma fibronectin did not simply conceal an effect on type-IV proteoglycan production by shifting the balance released into the culture medium. The results suggest that the effect of cellular fibronectin on the differentiated properties of chondroblasts relies on some unique feature not possessed by plasma fibronectin, and thus is not solely dependent on its own ability to stimulate adhesion and spreading.     

Heparin-binding properties of human serum spreading factor

Human serum spreading factor (SF) is a blood glycoprotein that promotes attachment and spreading and influences growth, migration, and differentiation of a variety of animal cells in culture. SF purified from human plasma or serum by chromatographic methods reported previously (Barnes, D. W., and Silnutzer, J. (1983) J. Biol. Chem. 258, 12548-12552) does not bind to heparin-Sepharose under conditions of physiological ionic strength and pH. In a further examination of the heparin-binding properties of human serum SF, we found that exposure of purified SF to 8 M urea altered several properties of the protein, including heparin affinity, and these alterations remained after removal of the urea from SF solutions. Urea-treated SF bound to heparin under physiological conditions, and salt concentrations of 0.4 M or higher were required for elution of urea-treated SF from heparin-Sepharose at pH 7.0. The alteration of heparin-binding properties of SF also was observed upon exposure of the protein to heat or acid. Treatment of SF with urea, heat, or acid resulted additionally in greatly decreased cell spreading-promoting activity of the molecule. The decreased biological activity was associated with a reduced ability of the treated SF to bind to the cell culture substratum, a prerequisite for the attachment-promoting activity of the molecule. Experiments examining the heparin-binding properties of native SF in unfractionated human plasma indicated that the major portion of SF in blood did not bind to heparin under conditions of physiological ionic strength and pH.     

Isolation and characterization of a Mr = 38,000 protein from differentiating smooth muscle cells

In culture, vascular smooth muscle cells grow and form a confluent monolayer of cells. Under appropriate conditions, regions of the monolayer can be induced to draw away from the substrate and form multicellular nodules. The ultrastructure of the cells in the nodules appears to be similar to that of differentiated smooth muscle cells. The process of nodulation is associated with the synthesis of a unique protein whose molecular weight is estimated from gradient gel electrophoresis to be 38,000 (38-kDa Protein). The protein is secreted into the culture medium and can be detected either by metabolic labeling or by staining with Coomassie Blue. Partial purification of 38-kDa Protein was achieved using affinity chromatography. The protein is adsorbed to heparin-agarose, but not to gelatin-agarose. The concentration of 38-kDa Protein in nodular conditioned medium is estimated at 1.9 micrograms/ml and less than 0.01 microgram/ml in conditioned medium made from monolayer cells. The presence of 5% fetal bovine serum in the labeling medium does not affect 38-kDa Protein synthesis. Cross-reactivity with fibronectin was evaluated using polyvalent antibodies to 38-kDa Protein. The 38-kDa Protein is not antigenically related to fibronectin. Furthermore, we establish that the protein is not qualitatively influenced by the presence of ascorbate (50 micrograms/ml), beta-aminoproprionitrile fumarate (50 micrograms/ml) heparin (10 ng/ml), or fibronectin (20 micrograms/ml) in the culture medium. We find that the added components neither suppress 38-kDa Protein synthesis in nodular cultures nor enhance 38-kDa Protein synthesis in monolayer cultures. The 38-kDa Protein is not detected in either monolayer or nodular cell layers and appears to be a secreted protein. Its appearance in nodular conditioned medium during nodulation suggests a relationship with that process.