Up-regulation of embryonic NCAM in an EC cell line by retinoic acid

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Up-Regulation of Embryonic NCAM in an EC Cell Line by Retinoic Acid MATTHIAS

HUSMANN,*

INGRID G~)RGEN,* CHRISTOPH WEISGERBER,~

AND DIETER BITTER-SUERMANN~

*Institute of Medical Microbiob, Johannes Gutenberg-Universitiit, Mainz, Federal Republic of Germany; and tInstitute of Medical Microbiology, Me&&i&e Hochschule, Hannover, Federal Republic of Gemnang Accepted June 19, 1989 The impact of retinoic acid (RA) on the expression of the neural cell adhesion molecules (NCAMs) and their developmentally regulated polysialic acid (PSA) moiety was studied in embryonal carcinoma (EC) cell lines. These cell lines are known to be capable of RA-induced differentiation into neurons (murine PlS cells) or parietal endoderm (murine FS cells), respectively. Monoclonal antibodies were employed to monitor expression of NCAM and PSA. FS and PlS cells were both found to express NCAM but only PlS cells carried the highly polysialylated “embryonic form” of NCAM (E-NCAM). The amount of NCAM in aggregated PlS cells but not in FS cells was dramatically increased upon treatment with RA. Since NCAMs play an important role in cell interactions during embryogenesis it is tempting to speculate that the regulative impact of RA on NCAMs is related to its morphogenic property. o 1989 Academic PESS, IN. INTRODUCTION

Two major concepts concerning pattern formation in animal tissues have been supported by experimental evidence during the last years: One of them is the concept of cell adhesion molecules. It assumes that differential adhesion between cells is of crucial importance for tissue pattern formation. Adhesion is most likely mediated by a rather limited number of different cell adhesion molecules (CAMS) (for review see Edelman, 1986). The neural cell adhesion molecules (NCAMs) have been shown to be important for a variety of cellcell interactions, e.g., myoblast fusion and neuromuscular synaptogenesis (Covault and Sanes, 1986; Rutishauser et al, 1983). But how NCAM expression is regulated remains largely unknown. Another important concept in morphogenetic theory is the concept of morphogens. Morphogens are compounds, supposed to give positional information to cells by a prepattern formation constituted by local concentration gradients in developing tissue (Turing, 1952; Crick, 1970; Summerbell et aZ.,1973). Recently, retinoic acid (RA) has been suggested to be a good candidate for being a morphogen (Thaller and Eichele, 1987; Gigu&re et aL, 1989; see also Robertson, 1987). Yet, the mechanisms by which RA would exert its morphogenic effect are poorly understood. The close association of both RA and NCAM with differentiation and morphogenic events led us to hypothesize that RA might play a role in regulation of NCAM expression. Several studies dealing with NCAM expression on various cell lines and attempts to alter it in vivo and in vitro have been described (Daniloff et ab, 1986; Friedlander et al, 1986; Prentice et al., 1987; Gennarini et aL, 1986). To our knowledge so far only one chemically defined compound 001%1606/89 $3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

has been reported to induce NCAM expression: Prentice et al. (1987) reported increased levels of NCAM protein in PC12 cells in response to neural growth factor (NGF), while Friedlander et al. (1986) found only the neuronglia cell adhesion molecule (NGCAM) but not NCAM increased upon treatment with NGF in PC12 cells. In our experiments we have made use of embryonal carcinoma (EC) cell lines which resemble normal embryonal cells with respect to their potential for differentiation. Differentiation of these cells can be chemically induced in vitro. The results of our study indicate that the candidate morphogen and differentiation inducer RA is a potent inducer of embryonic NCAM (ENCAM) in murine P19 cells and therefore might constitute an example of a functional link between morphogenie signal and cell adhesion molecule. MATERIALS

AND METHODS

Cell Culture

Cells were grown in tissue culture petri dishes (coated with gelatine in the case of F9 cells) at an initial cell density of lo5 cells/ml in DMEM, 2 mMglutamine, 10% fetal calf serum. For induction of differentiation of P19 cells, cells were cultured for 4 days in the presence of all-trans-retinoic acid (RA) (Sigma) in baeteriologicalgrade petri dishes where they aggregate spontaneously. Medium containing RA was renewed at Day 2. At Day 5, aggregates were collected, washed twice in medium without RA, plated onto coverslips (previously coated with pOly-D-1ySine) or on tissue culture surfaces and cultured for another 3 days if not otherwise stated. For differentiation of F9 cells into endoderm, RA (0.1 pLM) was added to adherently grown cultures.

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Immunoblotting NP-40 extracts were separated on a linear 7.5% SDS-PAGE under nonreducing conditions. The lysing buffer contained 50 mM Tris-HCL, pH 7.5, 150 mM NaCl, 0.5% (v/v) NP-40, 200 U/ml aprotinin, 5 mM PMSF. Insoluble material was removed by centrifugation. Prior to SDS-PAGE, samples were heated for 10 min to 60°C. Immunoblots of the gels were incubated with mAb 735 (mouse anti-PSA, 50 &ml) or mAb H28 (rat anti-mouse NCAM, 10 pg/ml), respectively, followed by rinses with PBS and incubation with iZI-labeled rabbit anti-rat IgG. For detection of bound mAb 735, a rat anti-mouse antibody (Dianova, Hamburg, FRG) was used as the second antibody before applying the radiolabeled reagent. Bound radioactivity was detected by autoradiography.

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anti-mouse Ig. Two-color staining was performed using mAb H28 and biotinylated mAb 735; detecting reagents were FITC-conjugated goat anti-rat Ig absorbed with mouse serum (Dianova) and phycoerythrin-conjugated streptavidin (Coulter), respectively. Cells were analyzed on a FACS scan (Becton-Dickinson). RESULTS

Embryonic and Adult Form of NCAM Are LQTerentially Expressed on P19 and F9 Cells

For detection of NCAM we have used H28, a monoclonal antibody, directed to a determinant present on the 120-kDa the 140-kDa, and the 180-kDa isoforms of murine NCAM (Hirn et al, 1981) and mAb 735, a monoclonal antibody, specifically binding to polysialic acid (PSA) (Frosch et al, 1985; Roth et aL, 1987,1988; Miragall et aL, 1988). PSA is a developmentally regulated carbohydrate moiety, characteristic of the embryonic Immunoprecip&ation of Biosynthetically form of NCAMs (E-NCAMs) and has been detected in Labeled E-NCA M mammalians exclusively on NCAM (Finne et al., 1983; Immunoprecipitation of NCAM from NP-40 lysates of Rutishauser et al, 1985). We have screened a number of biosynthetically labeled cells was performed as follows: cell lines, including EC cells, for E-NCAM expression: cells were incubated for 2 hr in medium with 10% FCS, Fluorescence-activated cell sorter (FACS) analyses relacking methionine. Subsequently lo8 cells were biosyn- vealed different patterns of NCAM/PSA expression: thetically labeled by incubation for 4 hr in medium AtT-20 cells have previously been shown to constitulacking cold methionine to which [YS]methionine (100 tively express the heavily polysialylated embryonic &i/ml; sp act, 1150 Ci/mmole) was added. Cells were form of NCAM (E-NCAM) (Rougon et al, 1986) and thus washed in culture medium and then solubilized in ice- served as a positive control in our experiments. We have cold Trig-HCl, pH 7.5, containing 150 mM NaCl, 1 mM found expression of NCAM and PSA on the majority EDTA, 1 mM EGTA, 200 U/ml aprotinin, 5 mM PMSF, (>90%) of these cells. Murine P19 EC cells (McBurney and 0.5% (v/v) NP-40. Unsoluble material was removed et ak, 1982; Jones-Villeneuve et al, 1982) were found to by ultracentrifugation and the supernatant was diluted express much lower levels of NCAM and PSA (about (to 0.05% NP-40). After preabsorption with protein A- 50% weakly stained cells), whereas the majority of F9 Sepharose (Pharmacia) overnight at 4”C, ion exchange cells (another murine EC cell line) appeared to carry chromatography purified antibody mAb 735 was added relatively high levels of NCAM but no PSA. The fibroat a final concentration of 20 pg/ml and the mixture blast-like cell line L929 did not bind either of the two was shaken on ice for 2 hr. Subsequently protein A- monoclonal antibodies. Sepharose was added and incubation at 4°C was continued for a further 2 hr. Unbound material was washed RA Induces Enhanced Expression of PSA and NCAM off and precipitates were dissolved in sample buffer, in P19 but Not in F9 Cells boiled for 10 min, and electrophoresed on a linear 7.5% SDS-PAGE. Subsequently the gel was blotted onto niIn order to determine whether induction of differentrocellulose and bands were visualized by fluorography. tiation leads to alteration of NCAM expression, a differentiation protocol described by McBurney et al. (1982) was applied, where P19 cells are cultured, aggreIndirect ImmunoJuorescence Staining for FACS gated in the presence of nontoxic doses of RA, and subStaining of cells detached from the culture dishes by sequently plated on tissue culture dishes. Previous studbrief incubation with trypsin/EDTA was performed ies with P19 cells have shown that this treatment reaccording to standard procedures, using H28 or mAb sults in the development of neurons, glia cells, and 735 as the first antibody. For staining with H28, second fibroblast-like cells. Staining for FACS analysis of thus antibody was a F(ab’)z fragment of mouse anti-rat Ig treated cells was performed 8 days after the initiation (Dianova), the detecting antibody for H28 and mAb 735 of the experiments. At that time, neurons growing out was a FITC conjugate of a F(ab’), fragment of goat from RA-treated aggregates had become visible in cul-

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tures of P19 cells (Fig. 1) and DNA synthesis as measured by rH]thymidine incorporation had decreased by 75% (not shown). FACS analysis revealed that more than 95% of the cells treated with RA now gave positive signals with both anti-NCAM or anti-PSA antibodies; average staining intensity with either of the two antibodies had markedly increased (Fig. 2). In contrast, RA

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FIG. 2. FACS analysis with P19 (3,4) or F9 cells (‘78) prior to differentiation and after RA-induced differentiation (5,6 and 9,10), respectively. Staining was performed using mAb 735 (anti-PSA; 3,5,7,9) or H28 (anti-NCAM; 4,6,8,10).Individual control traces (FITC-conjugate alone) are indicated for each panel. (1,2) Forward light scatter, revealing standard distribution of apparently equal cell size with untreated or treated P19 cells (Day 8 of RA treatment).

treatment of F9 cells did not result in a significant alteration of the expression of NCAM or PSA and the addition of 1 mM dibutyryl CAMP even led to a decreased NCAM expression (Fig. 3b). Indirect immunofluorescence with mAb 735 or H28 of differentiating P19 cells on poly-D-lysine-coated coverslips revealed staining of cells of neuronal and nonneuronal morphology (not shown). Time and Dose Requirements NCAM/PSA Expression

FIG. 1. Photomicrographs of cell cultures (400-fold magnification) of P19 cells prior to and after differentiation induction with RA. Neurones, grown out from RA-treated aggregates after attachment to tissue culture surface are shown in b (Day 8 after initiation of the experiment); the majority of these cells expresses high levels of ENCAM (see Fig. 2). Untreated P19 cells are shown in a.

for RA-Induced

Time courses for the increased synthesis and surface expression of NCAM/PSA were done in immunoblot and FACS experiments, respectively. By Day 2 of RA treatment of aggregated P19 cells, slightly enhanced synthesis of NCAM and PSA was seen in immunoblot, this was followed by a steep increase between Day 4 and Day 8 (Fig. 3a). FACS analyses revealed similar time courses for surface expression of NCAM and PSA (Fig. 4a). Double-

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FIG. 3. (a) Time course of E-NCAM synthesis upon RA treatment. Immunoblots of solubilized cells, using mAb 735, were performed as described under Materials and Methods. Two hundred micrograms of total protein was applied per lane. Lane 1, aggregated P19 cells without RA. Lanes 2-5, aggregated P19 cells cultured in the presence of 50 nM RA for 2,4, 6, and 8 days, respectively. Lanes 6 and 7, F9 cells and F9 cell-derived parietal endoderm (treatment with RA for 8 days), respectively. Lane 8, AtT20 cells (positive control). (b) Immunoblot of solubilized cells with antibody H28 (anti-NCAM). Lanes 1 and 2, aggregated P19 cells, cultured in the presence of 50 nMRA for 8 and 6 days, respectively. Lane 3, P19 cells without RA. Lanes 4 and 5, F9 cell-derived parietal endoderm (treatment with RA plus dibutyryl CAMP) and untreated F9 cells, respectively, expressing the 140-kDa isoform of unpolysialylated, so-called “adult” form of NCAM.

staining experiments demonstrated that the increase in NCAM expression directly correlates the increase in PSA expression or vice versa (Fig. 4b). The induction of NCAM was dose dependent in a concentration range from 10 p&f to 50 nM RA (Fig. 5). Concentrations higher than 0.1 &l! led to a decreased viability of P19 cells. It appeared that the requirements for NCAM induction in P19 cells were the same as those reported for the induction of differentiation, i.e., aggregation and simultaneous treatment with RA. Whether the expression of NCAM and/or PSA is in turn required for further differentiation remains to be investigated. E-NCAM

Isofnm

F9 cells expressed the 140-kDa isoform of NCAM (Fig. 3b). Undifferentiated P19 cells expressed only low levels of NCAM and thus the isoform composition remained unclear. In differentiating P19 cells, bands of 120, 140, and 180 kDa were detectable by immunoprecipitation with mAb 735 (Fig. 6), indicating that these proteins are present in the “embryonic” form in these cells. By boiling the samples, PSA is degraded, polydispersity of NCAM is lost, and isoforms become visible as distinct bands. DISCUSSION

NCAM is expressed in derivatives of all three germ layers early in development (Edelman et al., 1983). Thus, it is not surprising that P19 and F9 (both are murine cell lines of embryonal origin) express this pri-

mary CAM. But it is noteworthy that P19 cells in contrast to F9 cells do carry the highly polysialylated embryonic form of NCAM (E-NCAM). Differences between F9 and P19 were also found with regard to their response to RA: while both cell lines did undergo morphological changes, only P19 cells were induced to elaborate significantly increased amounts of PSA and NCAM. What is the molecular base for differential responsiveness of F9 and P19 cells to RA, with respect to NCAM expression? One possible explanation might be a different set of receptors for RA (Evans, 1988). At least three receptors for RA have been identified (Brand et CZL,1988; Benbrook et al, 1988; Petkovich et ah, 1987; Giguere et al., 1987). Specific tissue distribution has been reported (Benbrook et al., 1988). Alternatively, different responsive DNA elements might explain differential effects of RA. Several lines of evidence indicate that E-NCAM but not the adult form is increased upon RA treatment as (i) P19 but not F9 cells gave enhanced E-NCAM expression; (ii) double staining revealed that in P19 cells apparently no significant increase of unpolysialylated NCAM takes place; and (iii) NTZ/Dl (Andrews, 1984), a human EC cell line, like P19 cells expressing E-NCAM, also appears to respond to RA with increased E-NCAM synthesis (unpublished results). In order to test this hypothesis a larger panel of EC cell lines will be required. What are the mechanisms of RA-induced NCAM/ PSA expression? While the activity of several sialyltransferases has been shown to be stimulated by RA

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FIG. 4. Time course of NCAM/PSA surface expression. In a, the effect of RA is plotted against the time of incubation in days. The numbers of RA-treated P19 cells expressing enhanced levels of PSA or NCAM as compared with untreated P19 cells were determined from single fluorescence histograms. Values of samples incubated for 10 days were defined as 100%. A steep increase between Day 4 and Day ‘7during the differentiation process is seen for both NCAM (filled circles) and PSA (triangles). (4b) Original data of two color staining experiments, performed as described under “Materials and Methods” showing NCAM expression (abszissa) and PSA expression (ordinate). Relative fluorescence intensities are given on log scales. Note the increase of intensely double-stained cells with time.

(Lotan et al, 1984; Deutsch and Lotan, 1983; Shanker and Pieringer, 1983; Moskal et al, 1987) this cannot explain increased NCAM (protein) levels in RA-treated P19 cells. The lag period between RA addition and increased NCAM synthesis makes it unlikely that NCAM expression is a direct consequence of RA treatment (primary response). Instead, secondary response events are likely to be involved; the nature of these is unclear. The 180-kDa isoform, known to be expressed during neural differentiation was detected, and like differentiation of P19 cells, induction of NCAM/PSA by RA required the aggregation of the cells. While recently strong evidence has been presented that RA is the critical compound responsible for for-

mation of the anterior-posterior axis in the chick limb bud (Thaller and Eichele, 1987) the question remains, how positional information, delivered by such a gradient, is manifested. In other words: what are the-direct or indirect-target genes for RA? It is tempting to assume that at least one mechanism involved might be the regulation of cell adhesion. It has been questioned whether a relative shallow gradient of RA-as it has been detected along the anterior-posterior axis in the developing chick limb bud (20 to 50 nM)-can really give positional information (Robertson, 1987). Though our experiments did not indicate a real discontinuous relationship between RA concentration and expression of NCAM it is fair to state that these effects of RA (and also, e.g., the formation of

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neurons in aggregates of P19 cells upon treatment with RA (Jones-Villeneuve et al., 1982)) exhibit strong dose dependence within the concentration range in question. An interesting point regarding morphogenic gradients and NCAM may be added by taking into account the observation of Keane et al. (1988) that the expression of NCAM in cultured neural crest cells correlates with the formation of gap junctions: It is a long-standing theory that direct cell-cell channels are probably required for sufficiently rapid diffusion and for formation of concentration gradients by morphogens (Crick, 1970, Loewenstein, 1987). In the present study we have demonstrated that the candidate morphogen RA may upregulate NCAM; RA has previously been shown to influence adhesion properties of various cell types (Chertow et al, 1983; Kamei, 1983; Jetten and Goldfarb, 1983; Kato and De Luca, 1987; Hyodoh, 1987) and a number of other “factors” including, e.g., interleukin-2 and platelet activating factor have been found to influence cell adhesion (Aronson et al., 1988, Damle et ah, 1987, Kimani et aL, 1988). Via its impact on cell-cell adhesion, RA might influence cell-cell channel formation and thus facilitate diffusion in turn. In any case it will be useful to clarify whether the morphogenic effect of RA relates to its impact on NCAM expression. Finally, thyroxine, like RA acting through a DNA binding protein that belongs to the steroid/thyroid-receptor superfamily (Evans 1988) was shown to downregulate NCAM (Thompson et al., 1987). Moreover, very recently it has been demonstrated that the retinoic acid receptor and the thyroxine receptor bind to a common responsive DNA element (Umesono et ah, 1988). Thus, pathways taking part in up- and down-regulation of

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FIG. 6. Immunoprecipitation of polysialylated NCAM polypeptides from differentiating P19 cells by monoclonal antibody directed to PSA revealing bands at apparent molecular weights of 140,180, and 120 kDa. (lane 1). Untreated P19 cells, shown in lane 2, repeatedly gave signals scarcely above background.

NCAM levels might meet at such common responsive DNA elements. We thank Dr. McBurney and Dr. Andrews for generously providing P19 and NTZ/Dl cells, respectively, and for sharing information on these cells; Dr. Goridis for anti-NCAM antibodies; Mrs. U. Klutentreter for kindly operating the FACS; and Mrs. K. Junker for the typing of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, Grant Bi 154121. REFERENCES ANDREWS, P. W. (1984). Retinoic acid induces neuronal differentia-

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FIG. 5. Dose-response curve for the induction of NCAM by RA in P19 cells. Cells were treated as described under Materials and Methods, harvested at Day 6 after initiation of the experiment, stained for NCAM using mAb H28, and subsequently analyzed by FACS. The number of positive cells as compared with untreated cells is given in percentage of the total cell count.

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