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Dexamethasone increases the synthesis and secretion of a partially active fibronectin in rat hepatoma cells.

After treatment with dexamethasone, rat hepatoma-tissue culture cells show a markedly enhanced adhesion to the substratum and increased cell- to-cell interaction. In addition, there is a profound change in the production of secretory glycoproteins. Although the relative synthesis and secretion of a gelatin-binding, fibronectinlike glycoprotein is increased threefold, we do not think this protein is responsible for the improved adhesion properties of the cells because the hepatoma cells do not bind normal fibronectin and because the HTC-produced fibronectin is neither bound by fibroblasts nor has it any affinity for ganglioside-containing phospholipid vesicles. Therefore, these hepatoma cells represent a unique system for studying the regulation of fibronectin synthesis by glucocorticoids. Furthermore, analyses of primary fetal rat hepatocytes have shown that these cells, unlike normal adult hepatocytes, synthesize and secrete fibronectin, which is structurally related to the HTC-cell protein. The comparison of this protein with fibronectin from normal cells will allow a structural characterization of the functional defect in the fibronectin synthesized by transformed cells.

The nature of the support matrix for primary cultures of adult hepatocytes has been found to be an important factor in maintaining the differentiated state of the cells (1,2). Several components are considered to be essential for providing suitable support conditions such as collagen (2,3), fibronectin, and laminin (4,5). Although all these components are integral parts of most basal membranes, only the two former are detectable in significant amounts in the biomatrix of normal-adult liver (6). Due to the surface specialization of hepatocytes (7), only certain portions of the plasma membranes have contact with the extracellular matrix. Excluded are the sinusoidal and bile canicular membranes, which appear to be free of adhesionpromoting proteins. Hepatoma cells have often been used for the biochemical analysis of adhesion and cell-to-cell interaction. These, in contrast to primary hepatocytes, offer the advantage of being a stable and easily manipulable tissue-culture system. The tumor cells, however, poorly approximate the cellsurface domains characteristic of liver cells (8). Rat-hepatoma tissue culture (HTC) ceils, as well as other transformed livercell lines (9), have proven to be very useful in characterizing several liver-specific membrane phenomena (10). These particular cells, however, have an additional unique feature, which is a glucocorticoid-mediated increase in cell adhesion (11,12). We provide evidence in this communication that this hormoneeffected change in adhesion is independent of fibronectin, THE JOURNAl. OF CEEL BIOLOGY. VOLUME 95 OCIOBER 1982 29-40 © The Rockefeller University Press -0021-9525/82/10/0029/12 $1.00 despite the concomitant increase in synthesis and secretion of that protein by the HTC ceils. Preliminary characterization of the hepatoma fibronectin reveals the lack of some of the binding activities considered specific for fibroblast and plasma fibronectin.

Cells
A newly cloned cell line of rat hepatoma-tissue culture cells (SR-HTC cells) and WI-38 human fibroblasts were grown in monolayer culture in Dulbecco's modified Eagle's medium (DME) containing 10% fetal calf serum under an atmosphere of 5% CO2-95% air. Hormonal treatment of HTC cells consisted of the addition of 1/~M dexamethasone to the culture medium. Primary fetal hepatocytes were obtained by collagenase digestion of livers from 19-d fetuses of Buffalo rats following the procedure of Guguen-Guillouzo et al. (13). The cells, showing 95% viability as measured by trypan-blue dye exclusion, were seeded on collagen-coated 3.5-cm plastic petri dishes (2 x l0 s cells in 2 ml) in DME lacking arginine but containing 2 mM L-ornithine, 10% dialyzed fetal calf serum, and 0.1 ttM dexamethasone. The selective medium was changed after 2 h and then daily. The cells were used after 96 h in culture. Fetal rat fibroblasts were obtained by digestion of the eviscerated body trunks of the rat fetuses with 0.05% trypsin in PBS containing 0.02% EDTA. The washed ceils were plated (3 × 105 cells/10 cm 2) and those adhering to the substratum after 2 h, were maintained in DME containing 10% fetal calf serum. After 4 d, secondary cultures were prepared and used for immunofluorescence studies. For fibronectin-binding studies and for collection of metabolically labeled fibronectin, the cells were cultured in medium containing serum that was first passed through a gelatin-Sepharose column in order to remove endogenous fibronectin.

Labeling and Isolation of Fibronectin
To obtain radiolabeled medium proteins, confluent cell monolayers were washed three times with serum-free or fibronectin-free medium and incubated for 24-48 li in the same medium (1.3 ml/10 cm 2 monolayer) containing either L-[5,6-SH]leucine (1.8 Ci/mmol), L- [6-aH]fucose (20 Ci/mmol), or L-[~Sl-methionine (20 Ci/mmol). The culture media were centrifuged for 5 rain at 1,000 g and then for 60 rain at 200,000 g. The supernatant fractions were used directly for separation on two-dimensional gels (14) or for isolation of fibronectin. To label rat-plasma flbronectin, 1 ml of citrated rat plasma was iodinated using I mCi ~2BI and 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril as catalyst (15). The radioactive plasma-proteins were separated from the unincorporated label by passage through a column of Sephadex G-25 (Pharmacia Fine Chemicals, Piscataway, N J). Fibronectin was purified from tissue-culture media or plasma by affmity chromatography on immobilized gelatin (16) following the procedure described by Sekiguchi et al. (17). The amount of protein was determined according to Lowry et al. (18) using albumin as a standard.

Gel Electrophoresis
The separation of medium proteins or ftbronectin was carried out by twodimensional gel eiectrophoresis (14) using in the second dimension a uniform 7.5 or 10% polyacrylamide gel. To compare proteins separated by two-dimensional gels, the spots were localized by autoradiography, cat out of the dried gel, and subjected to partial proteolytic digestion within a 15% polyacrylamide gel using Staphylococcus aureus V8 protease (0.1 ~tg protease per well, digestion for 30 rain at 37°C) (19). The radio-labeled pattern was visualized by fluorography (20).

Immunological Methods
The total mixture of proteins that are secreted by dexamethasone-treated HTC cells into serum-free culture medium, was used for immunization of a rabbit. Six subcutaneous injections, at 2-wk intervals, were carried out: the first was in complete, and the subsequent ones in incomplete Freund's adjuvant, and each contained 1 mg of protein. During the following 3 me, the animal was bled at 2wk intervals. The pooled sera were used in this study. To obtain fibronectinspecific antibodies for immune fluorescence studies, the immunoglobulins were collected from the above serum pool by twice precipitating with 40% saturated (NH4)2SO4. The immunoglobulins were redissolved in 0.2 M sodium carbonate, pH 9.0 (30 mg/ml), and reacted with fluorescein isothiocyanate (modification: 1.5 mol/mol immunoglobulins) (2 I). The fluorescent antibodies were then passed through a l-ml column of Sepharose 4B-CL to which 2 nag of purified rat plasma fibronectin was covalently coupled (16). The bound immunoglobulins were eluted in 0.2 M glycine-HCl, 4 M guanidine HCI, pH 3.0, and then chromatographed on Sepliadex G-25 in PBS. For immunoftuorescence studies, HTC cells, and fetal rat flbroblasts were grown on glass cover slips placed in 3.5-cm dishes. The cells were washed three times with culture medium and then incubated for 30 min at 4* in 1 ml medium containing 10 #g fluorescent antifibronectin immunoglobulin. The cells were inspected using a Leitz-fluorescence microscope.
To quantify the absolute amount of secreted fibronectin, confluent monolayers (75 cm 2) of HTC cells were incubated for 24 h in 10 ml of flbronectin-free culture medium. The ultracentrifuged media were dialyzed for 24 h against 50 mM NH41-ICOa containing 0.1 mM phenylmethylsuffonyl fluoride (PMSF) and then freeze-dried. The residues were redlssolved in 1 ml of PBS containing 1% NP-40, 0.1 mM pheaylmethylsalfonyl fluoride. Aliquots of 15 #1 of concentrated media or purified fibronectin were analyzed by standard crossed immuaoelectrophoresis on 1% agarose gels (10 × 10 cm) in 45 mM Tris, 100 mM glycine, 40 mM barbital buffer, pH 8.5, containing 1% NP-40 (22). The first dimension was run at 5 V/cm for 4 h and the second at 2 V/cm for 22 h into an agarose gel containing 5% (vol/ vol) of rabbit antiserum against HTC-cell secretory proteins. The pauerns were visualized by staining with Coomassie Brilliant Blue or by fluorography. The fibronectin peak was quantitatively analyzed by measurement of the area under the precipitin line and compared to known quantities of fibronectin. The values are expressed in micrograms of fibronectin secreted per day and per milligram of total cell protein measured at the time of medium collection.

Cell Attachment Assay
The assays were carried out essentially as described by Sekigucki and Hakemoil (23). In brief: plastic tissue culture plates with six wells (3.5-cm diameter; Costal Data Packaging, Cambridge, MA) were incubated at room temperature with 2 ml of PBS containing 1 mM CaCh, 1 mM MgClz, and 0. I mM PMSF and 40 ~g of flbronectin from either rat plasma or HTC cells. After 4 h, the wells were washed extensively with PBS. Some wells were then incubated for 1 h at room temperature with 2 ml of buffer containing 100 ~g of affinity-purified rabbit anti-rat fibronectin (see above). WI-38 fibroblasts and HTC ceils were released from monolayers by treatment with trypsin-EDTA and washed once with fibronectin-free culture medium followed three times with PBS. Aliquots of 2 ml ofcen suspension (6 × 105 WI-38 fibroblasts and 1.5 x l0 s HTC cells) were added to the test well and incubated for 1 h at 37°C. The adherent cells were photographed using a Nikon inverted phase-contrast microscope.

Binding of Fibronectin to Reconstituted Phospholipid Vesicles
Phospholipids used for reconstitution were purified from rat-liver plasma membrane fractions or [32P]phosphate labeled HTC-cell membranes as described elsewhere (24). Total gangliosides were purified from rat brain as outlined by Salto and Hakomori (25). The gangliosides in the final preparation were separated by thin-layer chromatography and visualized by staining with resorcinol (26). Sealed and unilamellelar phospliolipid vesicles were reconstituted by deoxycholate dialysis using lmg of phospliolipids, alone or combined with 80 t~g gangLiosides/ml reconstitution mixture (24). The formed vesicles were diluted with 50 mM Tris-HCl, 140 mM NaCI, and 1 mM CaCh, pH 7.6, (= TBS) and brought to 40% (wt/vol) with solid sucrose resulting in a concentration of 500 t~g phospholipid vesicles/mL Aliquots of I ml of vesicle suspension were placed into 12-ml nitrocellulose ultracentrifuge tubes for rotor SW41 (Beckman Instruments, Inc., Spinco Die., Pale Alto, CA). The vesicles were sequentially overlaid with 0.5 ml of 35% sucrose in TBS containing 1.5-2 pg isotopically labeled fibronectin, 6 ml of 30% sucrose in TBS, and 4 ml of TBS. After centrifugation for 16 h at 20@000 g at 20°C, the contents of the tubes were removed by an Isco fractionator and collected in 370 #1 fractions. Aliquots of 50 td from each fraction were used for radioactivity measurements by scintillation counting.

RESULTS
HTC cells, which have been used in previous studies (27,28), were found to have a rather weak interaction with the substratum and between one another when grown in monolayer culture. The cells remain isolated and loosely attached to the culture dish and would not form confluent monolayers. When we established new cloned cell lines from the wild-type SR-HTC cells (27), we observed several clones that exhibited a preference to grow in clusters. One of these cell lines was selected and used in this study. The cells responded to dexamethasone with an increase in adhesiveness to the substratum, as has already been reported for the original SR-HTC cells (12), and also with tighter cell-cell interaction. This feature is most apparent in the altered morphology of the monolayer (Fig. 1). This enhanced adhesion and spreading of HTC cells after dexamethasone treatment is reminiscent of the morphological alterations seen in primary hepatocytes when fibronectin is provided as culture substratum (5). Possible explanations for these dexamethasone-induced phenomena include: (a) an increased amount of fibronectin in the HTC cell culture, (b) an improved ability of the cells to utilize the fibronectin present in the culture medium, or (c) an expression of membrane properties that are independent of fibronectin.
To differentiate between these possibilities, we first analyzed the effect of the steroid hormone on the pattern of proteins secreted by the hepatoma cells into the culture medium. As shown in Fig. 2, within 24 h dexamethasone elicited a drastic change in the qualitative and quantitative composition of fucose-labeled medium proteins. Some of these alterations were identical to those detected in other cell lines of SR-HTC cells (27). Besides the increased synthesis and secretion of the two glycoproteins, gp50 and at-acid glycoprotein (= gp35-50) (28), a third glycoprotein species with apparent molecular weight of 230,000 was also present in an elevated amount (marked with FN in Fig. 2). To demonstrate that this spot represents a protein with properties of fibronectin, we collected the culture media from HTC cells metabolicaUy-labeled with [aH]leucine and chromatographed it on a gelatin-Sepharose colunm. A steroiddependent increase in the radioactivity recovered in the gelatin-    Table I). The media were chromatographed on 1-ml columns of gelatin-Sepharose. The gelatin-bound material was eluted and one-half was separated on a 7.5% polyacrylamide gel. The distribution of the radioactivity on the gel was determined in 2-ram thick slices. The inset shows the fluorographic picture of the same fibronectin preparation separated on a 5% polyacrylamide gel either under nonreducing (1) or reducing (2) conditions. MW, mol wt.
bound fraction was observed. PAGE of the affinity-purified material under reducing conditions revealed that the majority of the radioactivity appeared in a single band migrating with Mr 230,000, and under nonreducing conditions in two bands, a minor one at Mr 230,000 and a major one at Mr 450,000 (Fig.  3). The same molecular weights are found for the fibronectin purified from rat plasma (data not shown). These values are in agreement with those molecular weights reported for the monomeric and dimeric forms of plasma flbronectin from other sources (17,29). Quantitative determination of the radioisotope in the gelatin-bound material (Table I) reveals that dexamethasone treatment elicits a two-to fourfold increase in the relative synthesis and secretion of that protein, depending upon the type of precursor provided.
The use of radioactive metabolites to determine the influence of the glucocorticoid on the fibronectin synthesis is, however, complicated by the effect of the hormone on the uptake of the precursors (see legend to Table I and references 10, 27). Although valid estimates of the hormone effect on fibronectin production can be obtained by expressing the measurements as relative values, we determined the absolute amount of fibronectin secreted by HTC cells using immunological means. To do so, we immunized a rabbit with the secretory proteins of dexamethasone-treated HTC cells. The antiserum obtained was used to analyze the antigenic composition of conditioned HTC-cell culture medium by crossed immunoelectrophoresis (Fig. 4). The antigens in the three major precipitin lines were identified by reelectrophoresis of the material recovered from crossed immunoelectrophoresis plates on two-dimensional polyacrylamide gels (data not shown). The migration of affinity purified rat-plasma fibronectin on crossed immunoelectrophoresis plates is identical to that of HTC ceils (Fig. 4 C). Both fibronectins share all antigenic determinants as judged from tandem crossed-immunoelectrophoresis analysis and they yield the same peak area per p.g of applied protein (data not shown). Furthermore, we could not detect any cross reaction of the antiserum with bovine or human plasma fibronectin. gAUMANN Fig. 3). Values from two identically treated monolayers are shown and represent specific radioactivities (cpm/#g of total cell proteins).  The immunoelectrophoretic analysis and quantification of fibronectin in several HTC-cell medium preparations indicate a threefold increase in the fibronectin concentration after treatment of HTC cells with dexamethasone (Table I). This t'mding agrees closely with the results of the metabolic labeling studies.

34
THE IOURN^t OF CELL BIOLOGY, VOLUME 95,1982 Although HTC cells release more fibronectin into the surrounding milieu after exposure to dexamethasone, monolayers of control or hormone-treated cells appear to bind little or no fibronectin, as judged by the very low level of detectable fluorescence when the cells were probed in situ with fluoresceinlabeled antifibronectin immunoglobulins (Fig. 5 A and B). Nor does the addition of rat-plasma fibronectin to dexamethasonetreated HTC cells lead to an enhanced fluorescence (Fig. 5 C). The absence of externally bound fibronectin was confirmed by cell-surface labeling procedures. The treatment of HTC-cell monolayers with either lactoperoxidase-Na I25I or neuraminidase-galactose oxidase-NaB[3H]4 did not result in labeling of a protein comigrating with fibronectin on two-dimensional polyacrylamide gels (data not shown, see similar results in references 30, 31).
To determine whether SR-HTC cells indeed do not bind fibronectin and/or whether the fibronectin produced by HTC cells lacks actual cell-binding activity, we purified fibroneetin known to have all binding activities (see reference 17) and that could also be metabolically labeled in a manner comparable to that of HTC cells. We chose WI-38 human-diploid fibroblasts as a source of medium fibronectin, because among all fibroblast-cell lines these ceils were found to synthesize the greatest amount of cell-bound and secreted fibronectin (32). Using [3H]-fucose for metabolic labeling and gelatin aft'ralLy chromatography, we obtained fibroblast fibronectin, which appeared on two-dimensional gel as a single spot and did not differ significantly in its electrophoretic mobility from the hepatoma product (data not shown). These two fibronectin preparations were subsequently used in bioassays (33, 34) to test the binding activites of both the cells and the fibronectins (Table II). To exclude the possibility that there are speciesspecific binding properties, we also studied 125I-labeled ratplasma fibronectin (Experiment II in Table II). It appears that HTC cells, with or without dexamethasone treatment, do not bind appreciable amounts of either their own fibronectin or that of fibroblasts. In addition, the HTC-cell-derived fibronectin was bound to WI-38 cells much less efficiently than the fibroblast or rat-plasma fibronectin.
Despite the lack of binding of soluble fibronectin of HTC ceils to cells, the possibility still exists that this protein can promote cell attachment and spreading. Therefore, we coated culture substrata with fibronectin from either rat plasma or HTC cells. Using WI-38 fibroblasts and dexamethasonetreated HTC cells as test cells for the activity of the prepared surfaces, we observed that the adhesion of fibroblasts was promoted only by plasma-derived fibronectin and that this activity could be abolished by pretreatment of the coated plate with antibodies against fibronectin (Fig. 6). The attachment and spreading of HTC cells, however, were not significantly influenced by the nature of the culture substratum. A similar test employing hormone-untreated HTC-cells yielded the same results (data not shown).
To corroborate the partial activity of HTC-cell fibronectin using another system, we took advantage of the observation of Kleinman and co-workers (35,36) that fibronectin has a specific affinity to more highly sialylated gangliosides. This hypothesis is attractive with respect to the hepatoma-cell system, for HTC cells do not contain any ganglioside species with more than one sialic acid residue per molecule (26). The lack of these membrane constituents might explain the absence of fibronectin binding, despite the observation that highly sialylated gangliosides were minimally present in nontransformed fibroblasts,   (Experiment  II), we prepared 1251-labeled fibronectin from rat plasma. The final preparations had the following specific radioactivities: HTC-cell tibronectin, 20700 cpm/#g (Experiment I); and 8,400 cpm/#g (Experiment II); Wl-38 fibronectin: 4,300 cpm/#g; and rat plasma fibronectin: 87,000 cpm/#g. HTC cells, with or without prior dexamethasone treatment, as well as WI-38 fibroblasts were released from monolayers by trypsin. The cells were washed once with fibronectin-free medium and resuspended in the same medium yielding densities of 1 x 10 a HTC cells/ml and 5 X 105 Wl-38 fibroblasts/ml. 2 ml of the cell suspensions were placed into 2S-cm 2 plastic dishes. Before use, these culture dishes were incubated with 5 ml fibronectin-free medium for 2 h at room temperature. To each culture (in duplicates), 15 #g of radiolabeled fibronectin was added. We tested the binding specificity of fibronectin to fibroblasts by adding in Experiment II, to some of the Wl-38 cultures, 1251labeled rat-plasma fibronectin together with an excess (250,ug) of unlabeled fibronectin. After 24 h in culture, the cells attached to the dishes were washed three times with PBS. The acid-precipitable radioactivities bound to the cell monolayers as well as the total amount of cell protein were determined. Based on the known specific activities of the fibronectin preparations, the p,g amounts of fibronectin bound per mg of total cell protein were calculated. To estimate the degradation of radioactive fibronectin, the cells from the culture media after a 24-h incubation were passed through gelatin-Sepharose columns and the bound material analyzed on polyacrylamide gels. In all cases, 92-96% of the radioactivities present in the media after 24 h were still associated with intact fibronectin.
and with the subsequent conclusion by Rauvala et al. (37) that these plasma-membrane constituents do not act as true cellsurface receptors for fibronectin. Nevertheless, we used this peculiar binding property to analyze the activity of hepatoma and fibroblast fibronectin. The assay system consisted of phosphovesicles carrying higher sialylated gangliosides that were centrifuged through a layer of [3H]fucose-labeled fibronectin. If binding of fibronectin to the vesicles takes place, it can be detected by comigration of tritium with the phospholipid vesicles (Fig. 7). To mark precisely the position of the isopycnically concentrated vesicles in the centrifuge tube, we utilized radiolabeled vesicles prepared by addition of trace amounts of 32p_ 36 T,E JournAL OF CELt BIOLOGY. VOLUme 95, 1982 labeled phospholipids to the reconstitution mixture. Although this assay system allows only a qualitative assessment of binding activity, it clearly shows that the fibronectin from HTC cells, unlike that from fibroblasts, lacks any detectable interaction with the artificial membrane. This result, combined with the previous data in Table II, indicates that although it is structurally similar to fibroblast fibronectin, hepatoma fibronectin shares only partial activity with that for fibroblasts.
To study further the structural properties of HTC-cell fibronectin, we needed for appropriate comparison a fibronectin molecule that is synthesized by nontransformed cells (34). Because the HTC cells are of hepatic origin, the logical source of such fibronectin would be parenchymal cells of the liver. Previous labeling experiments have shown, however, that primary cultures of fully differentiated adult rat hepatocytes, whether or not they are treated with dexamethasone, do not synthesize or secrete detectable amounts of fibronectin (28). Therefore, we analyzed primary cultures of fetal rat hepatocytes. As shown in Fig. 8A, fibronectin represents a significant portion of the proteins synthesized and secreted by these cells. We have confirmed that this protein binds to gelatin and is recognized by the anti-rat fibronectin immunoglobulins (data not shown). For structural comparison, [3~S]methionine-labeled, monomeric fibronectin from fetal rat hepatocytes and from HTC cells were recovered from two-dimensional polyacrylamide gels and analyzed by partial proteolytic mapping (Fig. 8 B). To demonstrate the validity of proteolytic comparison for proteins that are shared between fetal hepatocytes and the HTC cells, we included a similar digestion of gp50, a glycoprotein that is, like fibronectin, regulated by dexamethasone in hepatoma cells and is exclusively expressed by fetal hepatocytes (for data on adult hepatocytes, see reference 28). The peptide patterns of the proteins from the two former sources show almost perfect homology indicating structural similarity of the polypeptide moieties. Close inspection of the fragmentation pattern of the fibronectin proteins, however, reveals one band differing in mobility (indicated by arrow in Fig. 8 B). Whether this structural difference is connected with the reduced biological activity of the hepatoma fibronectin remains to be analyzed. Furthermore, we do not yet have sufficient data to determine whether or not fibronectin synthesis and secretion in fetal hepatocytes are regulated by glucocorticoids.

DISCUSSION
We have attempted to determine whether fibronectin plays any role in the adhesion and cell-cell interaction of rat hepatoma cells. Two distantly related phenomena have stimulated this line of investigation: first, nontransformed adult rat hepatocytes require for adhesion and spreading either collagen (3) or fibronectin (5), both components of the liver matrix (2), and secondly, hepatoma cells exhibit a marked increase in adhesion to the substratum upon dexamethasone treatment (12). An additional intriguing observation is that dexamethasone elicits in HTC cells an increased synthesis and secretion of a glycoprotein with gelatin-binding activity, similar in size and charge to the soluble fibronectin of fibroblasts and containing the same antigenic determinants as rat plasma fibronectin. Two results strongly suggest that the steroid-induced adhesion and spreading properties of HTC cells are not mediated by fibronectin. First, in HTC cells, no significant amount of fibronectin protein is accessible to immunoglobulin probing (Fig. 4) or to radioactive modification by cell-surface labeling techniques.   (3), glycoprotein with Mr 50,000 (gpS0) and fibronectin (FN). B: Concentrated media containing [3SS]methionine-labeled proteins of fetal rat hepatocytes and dexamethasone-treated HTC cells (each preparation containing 1.2 x 106 cpm) were separated as in A and the spots corresponding to fibronectin and to gp50 were analyzed by partial proteolytical digestion with S. aureus protease on a 15% polyacrylamide gel (19). The lanes represent 1, rat-hepatocyte gp50 without protease; 2, rat-hepatocyte gp50; 3, HTC-cell gp50 with 3 N-g[ycans (see reference 28); 4, rat-hepatocyte fibronectin without protease; 5, rat-hepatocyte fibronectin; 6, HTC-cell fibronectin. To achieve comparable intensities, the various lanes were exposed for periods ranging from 2 to 14 d. Arrow indicates the difference in the peptide pattern of HTC-cell fibronectin.
However, using the same labeling techniques for fibroblast monolayers, fibronectin appears as the most prominently labeled component (38,39). Second, neither exogenously supplied fibroblast fibronectin nor its own fibronectin were bound by HTC cells (Fig. 5 and Table II). The absence of fibronectin on the cell surface of HTC cells is not unique to this cell type, as two other hepatoma-cell lines, mouse Hepa II cells and rat H-35 cells, do not display surface-bound fibronectin (H. Baumann, unpublished observations). Furthermore, unlike HTC cells, these two hepatoma-cell lines do not synthesize or secrete detectable amounts of fibronectinlike proteins under any known hormonal culture conditions. From these data, the specific identity of the cell-surface component(s) responsible for adhesion either in HTC cells or other hepatoma cells is not apparent. Moreover, we have at the present time no evidence whether these hepatoma cell lines synthesize and secrete a protein that promotes cell attachment, but is structurally different from fibronectin and does not bind to gelatin (40).
Because a role for fibronectin in the adhesion of nontransformed hepatocytes has not been clearly established (3)(4)(5), speculation about a possible loss of fibronectin recognition by hepatoma cells due to transformation is not warranted. However, that transformation can indeed lead to reduced fibronectin-binding has been demonstrated for hamster fibroblasts (34).
Although fibronectin synthesized by HTC cells binds appropriately to gelatin, it appears not to be a completely functional fibronectin, because it binds only partially to fibroblasts and not at all to ganglioside-containing phospholipid vesicles. Whether the binding activities for other molecules such as glycosaminoglycans (41), fibrin (42), DNA (43), S. aureus (44), or actin (45) are still preserved, remains to be determined. The occurrence of functionally defective fibronectin is not unique to HTC cells, as there are reports of the production of similarly inactive fibronectin by senescent human fibroblasts (46) and of the presence of defective plasma fibronectin in a patient with Ehlers-Danlos syndrome (47). This report of a fibronectin protein with partial activity provides the opportunity to study the specific structural or chemical requirements for binding activity.
During the process of transformation and culture selection, HTC cells have retained few properties that are considered to be liver-specific; most noted are the glucocorticoid-regulated FiGURe 7 Binding of fibronectin to ganglioside-containing phospholipid vesicles. Phospholipid vesicles, with or without gangliosides, were prepared as described in Materials and Methods. The vesicles that were used in the separations shown in the top panels, contained in addition 38,000 cpm of [32p] phospholipids. Aliquots of the vesicle preparations were placed into ultracentrifuge tubes overlaid with 0.5 ml of sucrose solution containing either 1.6/~g of HTC-cell fibronectin (=35,000 cpm) or 1.8/~g of fibroblast fibronectin {=7,300 cprn) and centrifuged as outlined in Materials and Methods. After isopycnic centrifugation and fractionation of the gradients, the radioactivities present in 50-/~1 aliquots of each fraction were determined. Inset, left panel shows the thin-layer pattern of the gangliosides incorporated into the phospholipid vesicles (1) next to the GM1 ganglioside standard (2). activity of tyrosine aminotransferase (9) and synthesis of atacid glycoprotein (28). Fibronectin, however, is barely detectable as a secretory product of primary adult hepatocytes from either normal or dexamethasone-treated rats (27,28). The low levels occasionally observed in the culture fluid of primary hepatocytes (48) can be attributed to contaminating fibroblasts in the hepatocyte preparation. For this reason, HTC cells are an interesting system, not only because of the relatively large quantities of soluble fibronectin produced, but also because of the glucocorticoid-regulated synthesis. There is a report that dexamethasone treatment of hepatocytes derived from newborn rats leads to an increased amount of ceil-surface localized fibronectin as judged by fluorescent immunological staining (49). Similarly, a glucocorticoid-dependent enhancement of immunoreactive fibronectin on the surface of transformed human fibroblasts has been observed (50). In neither case, however, has it been determined whether the glucocorticoid treatment affected the synthesis of fibronectin or the affinity of the cell to bind fibronectin. The occurrence of dexamethasoneregulated synthesis of flbronectin in HTC cells, therefore, could be either a property of developmentally deregulated hepatocytes or the consequence of transformation. Future experiments with primary fetal hepatocytes, which are found to synthesize fibronectin, and long-term tissue cultures of primary adult hepatocytes, will give a definite answer. The reason for the partial loss of activity of HTC-cell fibronectin cannot at the present stage be elucidated. One remaining possibility, as yet untested, is offered by the observation that secondary modification of newly synthesized glycoproteins in HTC cells is different from that in adult hepatocytes (28). However, the findings that carbohydrate-free fibronectin (51) or fibronectin with altered oligosaccharide structure (34) exhibit normal activities, make this explanation appear less likely.