Biphasic response to retinoic acid dose in differentiation of F9 cells into primitive endoderm-like cells

Experimental Cell Research 179 (1988) 344-351

Biphasic

Response to Retinoic Acid Dose in Differentiation Cells into Primitive Endoderm-like Cells KIYOSHI

Institute for Biochemical

MIKI

of F9

and YASUO KITAGAWA’

Regulation, Nagoya

School of Agriculture, 464-01, Japan

Nagoya

University,

The differentiation of F9 cells, as monitored by the secretion of basement membrane proteins, was biphasic depending on the dose of retinoic acid (RA) used. Secretion of laminin A reached a plateau at about 10 nM and increased again at more than 100 n&f RA. A similar biphasic response was observed in the secretion of tissue-type plasminogen activator as well. In the presence of RA alone, this biphasic pattern of differentiation was stable for a long period of time, but the addition of 1 mA4 dibutyryl CAMP changed it to monophasic after 12 days of exposure. @ 1988 Academic Press, Inc.

Embryonal carcinoma (EC) cells resemble stem cells of the early embryo and in vitro can differentiate into cells similar to definitive embryonic cells; thus, they can be used as a model for the analysis of early mammalian development 111.Of the many EC cell lines, F9 [2] has been classified as mullipotent, because it does not differentiate spontaneously. However, Strickland and Mahdavi 131 have shown that the differentiation of F9 cells into primitive endoderm-like cells can be triggered by a treatment with retinoic acid (RA). RA-primed F9 cells differentiate into parietal endoderm-like cells when exposed to dibutyryl CAMP [4], and into visceral endoderm-like cells when allowed to aggregate [5]. Retinoids are potent agents controlling cellular differentiation (for review, see Ref. [6]). RA can disturb the pattern formation of regeneration blastema of the limbs of amphibians [7-91. The local application of RA to chick limb bud mimics the function of polarizing region [lo, 111. This suggests that RA can replace a putative morphogen concentration gradient formed in the polarizing region, which commits precursor cells to differentiate into various cell types. Cultured EC cell lines are a good in vitro system for the study of this function of RA. The differentiation of a pluripotent EC cell line, P19, into various cell types was governed in part by the concentration of RA in the culture medium [12, 131. In these experiments, however, the spontaneous differentiation of the cells interferes with the interpretation of the effect of RA. In this regard, quasinullipotency of F9 cells may have an advantage, because their differentiation can be triggered only by RA. This makes the interpretation of the function of RA in differentiation unequivocal.

’ To whom reprint requests should be addressed at Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya-shi, Aichi-ken 464-01, Japan. Copyright @ 1988 by Academic Press, Inc. AU rights of reproduction in any form reserved 0014-4827/88 $03.00

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In this communication, the effects of RA on the differentiation of F9 cells into primitive endoderm cells were studied in detail. The differentiation was biphasic depending on the RA dose. MATERIALS

AND METHODS

Cell culture. F9 cells, provided by Dr. Y. Iwakura (The Institute of Medical Science, The University of Tokyo, Japan), were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nissui, Tokyo, Japan) containing 15% fetal calf serum (FCS; M. A. Bioproducts, Wakersville, MD) under humidified 5% CO* and 95% air at 37°C. For most cultures, dishes from Nunc (Roskilde, Denmark) were preliminarily incubated with 0.1% gelatin for 12 h at 4°C. For inducing the development of visceral endoderm-like F9 cells, untreated polystyrene microtest plates (LinbroiTitertek, No. 76-203-05; Flow Laboratories, Inc., McLean, VA) were used [14]. The cells were transplanted onto 96- or 24-well plates at a density of 2x lo4 cells/cm* and the medium was replaced every 3 days. A 2 mM stock solution of all-tram RA (Type XX from Sigma) in ethanol was prepared and diluted by culture medium to the necessary concentrations. The final concentration of dibutyryl CAMP (Seikagaku Kogyo Co., Tokyo, Japan), when added, was 1 n-&4. Other details of the cell culture were as described before [15-181. F9 cultures were photographed with an Olympus microscopic system IMT2. Labeling of secreted proteins with [35S]methionine. After the experimental culture period, cultures on 96-well plates were washed with phosphate-buffered saline (PBS) and incubated with 30 ul of Eagle’s minimum essential medium (methionine-free; Nissui) containing [35S]methionine (1200 Cii mmol; Amersham, Buckinghamshire, UK) to the amounts indicated. For the suspension culture of embryoid bodies (visceral endoderm-like F9 cells), care was taken to not aspirate the cells during the medium change. After incubation of the cells for 4 h under humidified 5 % CO* and 95 % air at 37”C, the labeled medium was withdrawn. For the analysis of a-fetoprotein secreted, aliquots of labeled medium corresponding to 5 x lo6 cpm of [‘?S]methionine incorporated into trichloroacetic acidinsoluble cellular proteins were immunoprecipitated with anti-mouse a-fetoprotein antiserum (ICN; Lisle, IL) as described [5]. For the analysis of laminin and type IV collagen secreted, whole labeled medium from each well was applied directly to sodium dodecyl sulfate (SDS) gel electrophoresis. SDS gel electrophoresis. The electrophoresis was done by the method of Laemmli [19], with slab gels of 3 % (w/v) acrylamide for the stacking gel, and 4 % (Figs. 1 and 4) or 8 % (Fig. 6) acrylamide for the separation gel. Gels were treated with En’Hance (NEN; Boston, MA), dried, and exposed to Xray films (XAR-5, Kodak, Rochester, NY) at -80°C. The identification of bands corresponding to the laminin subunits A and B and subchains a, and az of type IV collagen was based on our earlier reports [15-181. For the semi-quantitative estimation of the laminin A subunit secreted, densitometry of a fluorogram was done with a Shimadzu dual-wavelength scanner, and the relative integrated density was calculated with the highest density in each experiment taken to be 100. Assay of tissue-type plasminogen actiuutor. F9 cultures grown in DMEM-FCS containing various doses of RA were prepared on 24-well plates. Medium conditioned for 24 h with these cultures was obtained by replacing the medium with 300 ul of fresh DMEM-FCS containing the same dose of RA. Tissue-type plasminogen activator in the conditioned medium was assayed as described by Karlan et al. 1201with a kit from American Diagnostica, Inc. (Spectrolyse; New York, NY), and melanoma twochain tissue-type plasminogen activator was used as activity standard. The activity in fresh DMEMFCS was negligible (0.01 unit/ml) and the results are presented without subtracting this activity. Cell counts. Cultures were treated with 0.1% (w/v) trypsin (DIFCO, Detroit, MI) solution in PBS, and the cells were detached and suspended into Ca*‘- and Mg*’ -free PBS containing 1.3 mM EDTA. Cells were counted on a hemocytometer under a microscope.

RESULTS By monitoring the secretion of tissue-type plasminogen activator, Strickland and Mahdavi [3] indicated that the differentiation of F9 cells was gradually stimulated when the dose of RA was increased from 10 nM to 1 PM. Jetten and DeLuca [21] suggested that 1 nM RA can induce the differentiation of F9 stem

346 Miki and Kitagawa

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Fig. 1. Secretion of basement membrane proteins from F9 cells treated for 5 days with different RA doses. F9 cells plated on a 96-well microtest plate were cultured for 5 days in medium containing the indicated dose of RA without (n) or with (b) 1 m&f dibutyryl CAMP. Cultures were labeled with 2 uCi (a) or 1 uCi (b) of [35S]methionine and the secreted proteins were separated by SDS gel electrophoresis. The dried gel was exposed for 2 days (a) or 1 day (b). A and B indicate the migration positions of laminin subunits A and B; FN indicates that of tibronectin.

cells. In the following experiments, the differentiation of F9 cells was monitored mainly by the secretion of basement membrane proteins [4]. For this, F9 cells were metabolically labeled with [35S]methionine for 4 h and radioactive medium was analyzed by SDS gel electrophoresis and fluorography. As described previously [15, 181,the synthesis of laminin subunits is completely dependent on the differentiation of F9 cells. Low level secretion of type IV collagen can be observed even from F9 stem cells and it increases depending on the differentiation [15]. The quenching of Iibronectin secretion from F9 cells after differentiation [I51 served as an additional index. As shown in Fig. 1 a, after 5 days of RA treatment a detectable effect on the secretion of basement membrane proteins was observed at 320 pM, but it stayed at a low plateau level even when the dose was increased to 32 ti. F9 cells

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Fig. 2. Effect of RA dose on F9 cell proliferation. F9 cells were plated on a 96-well microtest plate at a density of 2~ lo4 cells/cm* and cultured in medium containing indicated concentration of RA without (a) or with (b) 1 mM dibutyryl CAMP. The cell number was counted on the days indicated.

secondarily responded to RA at more than 100 I-LW,and the maximum response was at 1 FM or greater. The response of F9 cells to the RA dose was also biphasic in the presence of 1 mM dibutyryl CAMP, when F9 cells differentiated into parietal endoderm-like cells (Fig. lb). This biphasic effect of the RA dose was not due to a biphasic effect on cell proliferation, because the cell number was essentially the same over the 15day period in cultures containing RA at concen-

Fig. 3. Homogeneous population of differentiated F9 cells in the presence of low and high doses of RA. F9 cells plated on 60-mm dishes were cultured for 8 days in me,dium containing (a) none, (b) 5 nk! RA, or (c) 1 @I RA. The cultures were photographed with an 011‘mpus microscopic system IMT-2. Magnification x 87.

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Fig. 4. Biphasic response to RA dose of laminin A secretion from F9 cells. F9 cells were cultured for 5, 6, 9, or 12 days and the secreted proteins were analyzed as described in Fig. 1. The relative integrated density of the laminin A subunit was calculated with the highest density in each experiment taken to be 100. Results from cultures without (a) and with (b) 1 mM dibutyryl CAMP are presented. Closed and open symbols indicate independent experiments.

trations of 5 nJ4 or 1 u&Z (Fig. 2), where two distinct plateau levels were seen in the secretion of basement membrane proteins. Also the biphasic response was not due to low frequency of differentiation at a low dose of RA, because a homogeneous population of F9 cells with a morphology (Fig. 36) distinct from that of F9 stem cells (Fig. 3 a) was observed by treatment with 5 ti RA for 8 days. Figure 4a summarizes the semi-quantitative assay of laminin A secretion as a function of RA dose and the period of treatment. By prolonging the period of treatment up to 12 days, the RA dose required for a response dropped, but the biphasic pattern itself was a stable characteristic of F9 cells. Although poor resolution on SDS electrophoresis made the quantitation less accurate, the enhanced secretion of laminin B and two subchains of type IV collagen as well suggested that the biphasic pattern is stable for a long period (results not shown). Despite previous results by Strickland and Mahdavi [3], we reexamined the RA

Biphasic differentiation of F9 by retinoic acid

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RA (Ml Fig. 5. Biphasic response to RA dose of the secretion of tissue-type plasminogen activator from F9 cells. F9 cells were cultured on 24-well plates in the presence of the indicated dose of RA. Fresh DMEM-FCS (300 ~1)containing the same dose of RA was added to each culture 24 h before the assay. On the day indicated, tissue-type plasminogen activator (t-PA) activity in the medium conditioned for 24 h was assayed. Averaged activities from two independent cultures are presented.

dose-dependent secretion of tissue-type plasminogen activator from F9 cells (Fig. 5). Detailed titration of RA revealed that the secretion is also biphasic. In this case, however, the dose required for response did not shift in prolonged periods. Instead, the intermediate plateau level of the secretion increased. These results together suggested that there are different cell types depending on the RA dose, and that the dose governs the state at which F9 cells remain. When F9 cells are allowed to form small aggregates in the presence of low doses of RA, the outer surface cells differentiate into visceral endoderm-like cells 151.In our experiments, F9 cells were cultured on dishes preliminarily coated with gelatin (see Materials and Methods), and RA-treated cells always grew in a monolayer (Figs. 3 b and 3 c). However, it is still possible that the intermediate plateau level in the secretion of basement membrane proteins and tissue-type plasminogen activator reflects the development of visceral endoderm-like cells at this dose of RA. In order to exclude this possibility, we exposed F9 stem cells to various doses of RA for 8 days on gelatin-coated or untreated polystyrene plates. The cultures were labeled with [35S]methionine, and the medium was analyzed by SDS electrophoresis after immunoprecipitation with anti-mouse a-fetoprotein antiserum (Fig. 6). When the cells were allowed to aggregate on polystyrene plates, secretion of a-fetoprotein was detected at 5 nM RA, which reached the maximum level at 500 ti. In a contrast, secretion of this protein from the cultures on gelatin-coated plates was not observed at any dose of RA. The addition of 1 mA4 dibutyryl CAMP to the culture medium brought about somewhat different results (Fig. 4b). The transient plateau level of laminin A secretion was stable until Day 6 of the incubation, but this level gradually

350 Miki and Kitagawa

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RA (nM) Fig. 6. Absence of a-fetoprotein in the medium of RA-treated F9 cultures on gelatin-coated plates. F9 cells were cultured on untreated polystyrene (aggregate) or gelatin-coated (monolayer) 96-well plates for 8 days in medium containing the indicated dose of RA. The culture was labeled with 20 uCi of [%l]methionine and labeled medium was collected. Immunoprecipitates with anti-mouse a-fetoprotein were applied to SDS electrophoresis. As size markers, phosphorylase b (93 kDa), bovine serum albumin (66 kDa), and IgG heavy chain (51 kDa) were used. The asterisk indicates the migration position of a-fetaprotein.

increased by prolonging the incubation period. The transient plateau level disappeared after 12 days of incubation and the dose curve became monophasic. In the eventual monophasic pattern, the half-effective dose of RA was about 1 nM. DISCUSSION The secretion of basement membrane proteins and tissue-type plasminogen activator from F9 cells was biphasic depending on the RA dose. A plateau level of laminin A secretion at an RA dose of about 10 ti was stable for at least 12 days of culture. For the differentiation of F9 cells induced by RA, primitive endodermlike cells have been the only stage of differentiation recognized [41. Our results strongly suggested that there is an additional stage of differentiation between F9 stem cells and primitive endoderm-like cells. Therefore, the differentiation of this quasinullipotent EC cell line is basically the same as that of the pluripotent EC cell lines, in which various cell types can be induced by a concentration gradient of RA [12, 131.Our suggestion of a novel stage of differentiation of F9 is based on quantitative differences in the secretion of basement membrane proteins and tissue-type plasminogen activator. The morphology of F9 cells at low concentrations of RA was indistinguishable from that of primitive endoderm-like cells (Figs. 3 b and 3 c). To substantiate that this stage of F9 cell differentiation is novel, further characterization with many other phenotypes is needed. The presence of dibutyryl CAMP in addition to RA made this transient stage unstable and increased the laminin A secretion to a level similar to that of parietal

Biphasic differentiation of F9 by retinoic acid

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endoderm-like cells after 12 days of incubation. We showed that the differentiation of RA-primed (primitive endoderm-like) F9 cells into parietal endoderm-like cells is reversible and dependent dibutyryl CAMP [ 15, 181.This supports the idea that RA commits the differentiation of F9 stem cells, whereas dibutyryl CAMP stimulates expression of the phenotypes of parietal endoderm. The present result suggests that parietal endoderm-like cells can be induced not only from primitive endoderm-like cells induced by a high RA dose, but also from cells produced by a low RA dose. This also provides an explanation of the biphasic effect of the RA dose. It is possible that a low RA dose commits F9 stem cells to differentiate, but that a high RA dose stimulates the full expression of the phenotypes of the primitive endoderm. In contrast to the mechanism of committing differentiation, this secondary action of RA could involve multiple mechanisms. The shift of the effective dose (Fig. 4a) and of intensity (Fig. 5) during prolonged RA treatment may be due to a sequential operation of such mechanisms on different phenotypes. Although these questions are open to further analysis, it is worthwhile to notice that this type of interconversion of cells dependent on local signals can be an important element of embryogenesis. This work was supported in part by a Grant-in-Aid for Scientific Research (62560078)from the Ministry of Education, Science, and Culture, Japan, by the Aichi Cancer Research Foundation, and by the Ishida Foundation. K.M. is a recipient of an Okumura Scholarship.

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3. Strickland, S., and Mahdavi, V. (1978) Cell 15, 393-403. 4. Strickland, S., Smith, K. K., and Marotti, K. R. (1980) Ce// 21, 347-355. 5. Hogan, B. L. M., Taylor, A., and Adamson, E. (1981) Nature (London) 291, 235-237. 6. Spom, M. B., and Roberts, A. B. (1983) Cancer Res. 43, 3034-3040. 7. Maden, M. (1982) Nature (London) 295, 672-675. 8. Maden, M. (1983) Dev. Biol. 98, 409416. 9. Thorns, S. D., and Stocum, D. L. (1984) Dev. Biol. 103, 319-328. 10. Tickle, C., Alberts, B., Wolpert, L., and Lee, J. (1982) Nature (London) 296, 564-566. 11. Tickle, C., Lee, J., and Eichele, G. (1985) Dev. Biol. 109, 82-95. 12. McBumey, M. W., Jones-Villeneuve, E. M. W., Edwards, M. K. S., and Anderson, P. J. (1982) Nature (London) 299, 165-167. 13. Edwards, M. K. S., and McBumey, M. W. (1983) Dev. Biol. 98, 187-191. 14. Grover, A., Oshima, R. G., and Adamson, E. D. (1983) J. Cell Biol. 96, 1690-1696. 15. Morita, A., Kitagawa, Y., and Sugimoto, E. (1985) J. Cel[ Physiol. 125, 263-272. 16. .Morita, A., Kitagawa, Y., and Sugimoto, E. (1985) J. Biochem. 98, 561-568. 17. Morita, A., Sugimoto, E., and Kitagawa, Y. (1985) Biochem. J. 229, 259-264. 18. Miki, K., Sugimoto, E., and Kitagawa, Y. (1987) .I. Biochem. 102, 385-392. 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 20. Karlan, B. Y., Clark, A. S., and Littlefield, B. A. (1987) Biochem. Biophys. Res. Commun. 142, 147-154. 21. Jetten, A. M., and DeLuca, L. M. (1983) Biochem. Biophys. Res. Commun. 114, 593-599. Received April 5, 1988

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