EXPERIMENTAL

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RESEARCH

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(1992)

Expression of c-Kit Protooncogene Is Stimulated by CAMP in Differentiated F9 Mouse Teratocarcinoma Cells YUKIO *Research

NISHINA,*~ YUHKI KOBARAI,* TETSURO SUMI,? MITSUKO SHIN-ICHI NISHIKAWA,~ AND YOSHITAKE NISHIMUNE*

Institute for Microbial Diseases, Yam&a Okn Suita, Osaka Kumnmoto

Osaka University, tDepartment 565, Japan; and *Department University Medical School,

INTRODUCTION

Cell-cell interaction appears to play a major role both in determination of cell fate within the early embryo and in the differentiation of specific cell types during embryogenesis and adult life. The biochemical bases of these cellular interactions, including the nature of the signals, the mechanisms by which cells transmit and receive them, and the subsequent intracellular events that translate signals into developmental decisions, are just beginning to be understood. In vertebrates, functional evidence exists for the role of peptide growth factors in mesoderm induction [l]. In mammals a number of recent in situ hybridization studies report the developmental expression of genes encoding peptide growth factors [2-41. These growth factors produce their effects via specific receptors which translate the signals into reprint

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should

be addressed.

0014~4827/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

of Oral and Maxillofacial Surgery, Osaka University of Pathology, The Institute for Medical Immunology, 2-2-l Honjo, Kumamoto 860, Japan

Dental

School,

the intracellular information for cell growth, movement, or differentiation in mammalian development. Receptors for a variety of growth factors and hormones are transmembrane tyrosine kinases, each with a ligand-binding extracellular domain [5], a transmembrane domain, and an intracellular tyrosine kinase domain. The identification of these types of growth factor receptors further poses the question of their normal physiological role. The c-kit protooncogene encodes a transmembrane tyrosine kinase receptor for a unique ligand and is a member of the colony stimulating factor, platelet-derived growth factor receptor superfamily [68]. Substantial circumstantial evidence suggests that these tyrosine kinase receptors are important in controlling cellular differentiation and cell-cell interactions, particularly during embryogenesis [9, lo]. Less data are, however, available regarding the nature of receptors especially in early mouse embryos. Recently c-kit was shown to be allelic to the W locus of the mouse [ll, 121. Mutations at the W locus affect proliferation, migration, and differentiation of germ cells, pigment cells, and distinct cell populations of the hematopoietic system during development and adult life [131. Orr-Urtreger kt al. [141 reported that the embryonic expression of c-kit in hematopoietic cells of the yolk sac and embryonic liver, in presumptive subepiderma1 melanocytes, and in primordial germ cells is affected in W mutants. Furthermore, accumulation of ckit transcripts is observed in oocytes of both primary and mature ovarian follicles, suggesting that c-kit may play a role in oogenesis and as a maternal message in early embryogenesis. However, it is difficult to study the regulation of the c-kit gene using early embryos. Embryonal carcinoma (EC) cells mimic the early embryo and are a good model to study preimplantation development since these stem cells can be induced to differentiate in vitro into cell types that resemble those found at various stages of embryonic development [1518]. The mouse teratocaricinoma F9 cells undergo only limited spontaneous differentiation under normal culture conditions but convert sequentially into primitive

Protooncogene c-kit, a transmembrane tyrosine kinase receptor, was recently shown to map to the dominant white spotting locus (W) of the mouse. W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. In order to determine the regulation of the c-kit gene in cell differentiation, we investigated its expression during the differentiation of F9 cells. Undifferentiated F9 cells and FS cells treated with retinoic acid (RA) alone or dbcAMP alone showed little expression of c-kit mRNA if any. The subsequent addition of dbcAMP to FS cells treated with RA markedly increased the expression of c-kit mRNA. Furthermore, the effect of dbcAMP on ckit expression is reversible. In differentiated cells treated with RA, c-kit gene expression is induced by agents such as forskolin or theophylline, which are known to elevate cellular CAMP level. These results indicate that the expression of the c-kit gene is regulated by the level of intracellular CAMP in differentiated F9 cells induced by RA. o 1992 Academic PWS, IIXC.

1 To whom

KOSAKA,*

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endoderm and parietal endoderm like cells upon treatment with retinoic acid (RA) and dibutyrylcAMP (dbcAMP) [19]. The present study was undertaken to investigate the expression of c-kit at various stages of F9 cell differentiation. MATERIALS

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Cell culture. The stock culture of F9 cells was grown on gelatincoated culture dishes in Eagle’s minimal essential medium (EMEM) containing 5 m&f glutamine, 1 m&f sodium pyruvate, and 10% fetal calf serum (FCS) at 37°C [20]. For experimental cultivation, the stock culture was treated with 0.125% trypsin and 0.5 n&f EDTA in phosphate-buffered saline (PBS) at 37°C for 10 min. The cells were seeded at an appropriate concentration per culture dish. To induce differentiation, cells were incubated in the standard medium containing RA with or without dbcAMP. RA stock solution (1 mh4 in ethanol) was added to the standard medium to a final concentration of 0.1 PM, dbcAMP (20 mM in PBS) or theophylline (20 mM in PBS) was added to a final concentration of 0.5 mM; forskolin (100 mAfin ethanol) was added to a final concentration of 0.1 n-&f. Whenever dbcAMP was added or removed during the experimental culture, cells were fed with a new medium. Even if the culture was maintained in medium containing the same drugs, new medium was administered on Day 3. RNA isolation and gel blot analysis. After treatment with various inducers, the cells were washed three times with PBS, and total cellular RNA was isolated by guanidium isothiocyanate extraction [21]. For RNA gel blot analysis, RNA was fractionated on formaldehyde agarose gels in MOPS buffer and transferred to nitrocellulose filters in 20X SSC as described by Maniatis et al. [22]. The filters were prehybridized in 50% formamide, 4~ SSC, 5X Denharts’ solution, 0.2% SDS, and denatured sonicated salmon sperm DNA (120 pglml) at 42°C for 2 h and hybridized with radiolabeled DNA probe (l-5 X 10’ cpm/ml) under the same conditions for 24 h. After hybridization, the filters were washed at 55°C with 0.1X SSC, containing 0.1% SDS. The probes used here were mouse cDNA of t-PA [23], laminin Bl [24], c-kit [25], and cytoskeletal p-actin [26]. They were labeled with [asp]dCTP using the random primed method. Metabolic labeling of cells and immunoprecipitattin analysis. At the end of experimental cultures, cells were rinsed twice with PBS and incubated for 1 h at 37’C with methionine free EMEM supplemented with 10% dialyzed FCS. [?l]methionine (200 &i/ml) was added, and incubation was continued for 3 h. The cells were then rinsed twice with PBS and lysed in lysis buffer (50 mM Tris [pH 7.41, 150 n&f NaCl, 20 m&f EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mA4 phenylmethylsulfonylfloride, and leupeptin [20 rgl ml]) and the extract was clarified by centrifugation in an Eppendorf centrifuge at 100,OOOg. Equal amounts of protein (TCA precipitable counts) were immunoprecipitated by incubation with anti c-kit monoclonal antibody ACK-2 [27] or normal rat IgG for 30 min at 4°C. Protein A-Sepharose beads (Pharmacia) were added and the mixture was incubated overnight at 4°C. Immunoprecipitants were washed once at 4°C in 50 n&f Tris, 500 mM NaCl, 5 mM EDTA, 0.2% Triton X-100, three times in 50 mM Tris, 150 n&f NaCl, 5 n&f EDTA, 0.1% Triton X-100,0.1% SDS, and once in 10 mMTris, 0.1% Triton X-100, and resuspended in SDS/gel sample buffer (62.5 n&f Tris [pH 6.81, 2% SDS, 2% mercaptoethanol, 10% glycerol), boiled for 2 min, and analyzed by SDS-PAGE [28] and autoradiography. RESULTS

Induction of c-kit mRNA by CAMP in Differentiated Cells Induced with RA

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F9 cells were induced to differentiate and convert sequentially into primitive endoderm- and parietal endo-

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FIG. 1. Expression of c-kit, t-PA, and laminin Bl mRNA in differentiated F9 cells. F9 cells were grown in the absence (lane 1) or presence of various drugs for 2 days (lanes 2-5) or 6 days (lanes 6-10). Total RNA was isolated and examined by Northern blot analysis with ‘*P-labeled cDNA probes, t-PA (a), laminin Bl (b), and c-kit (c) as described under Materials and Methods. Each lane contains 35 pg of total RNA. Ribosomal RNA subunits were used as molecular size standards (arrowheads). Lanes 2 and 6, RA, lanes 3 and 8, RA plus dbcAMP, lanes 4 and 9, dbcAMP; lanes 5 and 10, theophylline. Lane 7 shows the cells treated with RA, dbcAMP, and theophylline for 6 days. Lane 11 shows the cells which were grown in the medium containing RA plus dbcAMP for 6 days, then changed to the medium containing RA minus dbcAMP for 1 day.

derm-like cells upon treatment with RA alone or RA plus dbcAMP [ 191. To know the role and the expression of c-kit receptor in teratocarcinoma cell differentiation, mRNA levels of c-kit in F9 stem cells and their differentiated cells were analyzed by Northern blotting. The mRNA levels of both t-PA (Fig. la) and laminin Bl (Fig. lb), as endodermal cell markers, were markedly in-

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FIG. 2. Effect of dbcAMP on the expression of the F9 cells treated with RA or in PYS-2 cells. (a) F9 cells with RA alone for 0 days (lane l), 2 days (lanes 2,5), 4 6), 6 days (lanes 4,7), and then the same cells (lanes 5-7) treated with dbcAMP for 1 day. (b) PYS-2 cells were absence (lane 1) or presence of dbcAMP for 1 day (lanes by a l-day incubation without a drug (lane 3). Total RNA and Northern blot analysis was performed as described to Fig. 1.

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c-kit gene in were treated days (lanes 3, were further grown in the 2,3) followed was isolated in the legend

creased by the treatment of F9 cells with RA alone and were further elevated by the addition of dbcAMP as reported by Rickles et al. [23]. In contrast, c-kit mRNA was increased only slightly if at all by the treatment with RA alone (Fig. lc). By the addition of dbcAMP, however, the mRNA level of c-kit was markedly enhanced. These cells contained c-kit mRNA with a majority of 5.8 kb long RNA species in agreement with previous reports [ 7,8]. F9 stem cells and F9 cells treated by dbcAMP or theophylline alone for 6 days showed no expression of c-kit mRNA at all. Since the results of our preliminary experiments indicated that the effect of dbcAMP on c-kit gene expression is rapid, we studied the effect of the duration of RA treatment followed by the dbcAMP addition for 1 day. It was found that the expression of c-kit mRNA after the treatment with dbcAMP was slightly induced in the cells pretreated with RA for 2 days. Then, the level of c-kit mRNA increased, depending upon the duration of RA pretreatment (Fig. 2), and was expressed in association with differentiation markers induced by RA (Figs. la and lb). Furthermore, the expression of c-kit mRNA was increased by dbcAMP in differentiated PYS-2 cells (initially derived from OTT6050 embryonal carcinoma cells) [29] (Fig. 2b). Other modulators such as forskolin or theophylline which elevate intracellular CAMP concentration also induced the expression of c-kit mRNA in differentiated F9 cells, but not in F9 stem cells (Fig. lc; Fig. 3). When dbcAMP was removed from the medium, however, the level of c-kit mRNA decreased rapidly, although the level of t-PA and laminin Bl mRNA remained constant. These results indicate that dbcAMP has a specific inducible effect on the expression of c-kit gene and that this effect is reversible.

FIG. 3. Effect of various compounds elevating intracellular CAMP concentration on the expression of c-kit mRNA in F9 cells. Total RNA was isolated and Northern blot analysis was performed as described in the legend to Fig. 1. Lane 1, F9 stem cells; lane 2, F9 cells treated with RA, lanes 3-7 show RNA from F9 cells treated with RA for 6 days followed by a l-day treatment of RA (lane 3), dbcAMP plus theophylline (lane 4), dbcAMP (lane 5), theophylline (lane 6), or forskoline (lane 7).

Production of c-kit Protein in Differentiated

F9 Cells

To characterize the protein products of the c-kit gene in F9 cells, induced cells were labeled with [“%Imethionine, and immunoprecipitation analysis was carried out using a monoclonal antibody ACK-2 specific to the c-kit protein. SDS-PAGE analysis of immunoprecipitants is shown in Fig. 4. Two bands having molecular weights of 160 and 124 Kd were observed in differentiated F9 cells incubated with RA plus dbcAMP.

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FIG. 4. Analysis of c-kit protein in differentiated F9 cells. F9 cells at different conditions and mast cell line MC/9 were labeled for 3 h with [%]methionine. Protein lysates were precipitated with anti c-kit monoclonal antibody and analyzed by SDS-PAGE and autoradiography. Arrows indicate the relative mobilities of c-kit products. Protein markers are indicated in kilodaltons; Stem, untreated F9 cells; RAGd, treated with RA for 6 days; RACTGd, treated with RA, dbcAMP, and theophylline for 6 days; MC/g, mast cell line.

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These proteins have the same molecular weight as immunoprecipitants detected in the control mast cell line MC/9 [30]. The 160-Kd band very likely corresponds to fully processed neuraminidase-sensitive glycoproteins, and the 124-Kd band may correspond to partially processed glycoproteins containing high mannose structures [31]. These results indicate that the c-kit mRNA of differentiated F9 cells treated with RA plus dbcAMP is translatable and produces the same protein molecules as the mast cell line MC/g. Neither undifferentiated F9 cells nor F9 cells treated with RA alone contained any immunoprecipitable proteins.

DISCUSSION

The developmental expression of protooncogene ckit, a tyrosine kinase receptor encoded by the W locus, is observed in fetal liver, bone marrow, mast cell, differentiated melanocyte, ovary, and testis, which are all affected by W mutation [13]. In addition, c-kit is also expressed in several tissues such as brain, placenta, lung, and lymphoid tissue which are not yet known to be affected by W mutations in mice [28]. In order to determine the role of the c-kit gene during embryogenesis, we investigated its expression during the differentiation of F9 cells, because teratocarcinoma cell differentiation in vitro is well known to mimic normal embryogenesis [ 15 181 and is easy to control, whereas it is difficult to study the regulation of the c-kit gene during normal embryogenesis. F9 cell differentiation can be divided into two separate events: the production of primitive endoderma1 cells and p’arietal endodermal cells. When F9 cells are exposed to RA alone, cells become flat and show typical endodermal morphology, which is the functional equivalent of primitive endoderm of normal embryogenesis and increased production of the t-PA and component of the basal lamina such as type IV collagen and laminin Bl (Figs. la and lb) [23,32]. In this type of F9 cell differentiation, c-kit mRNA was expressed at a very low level, if at all. The subsequent addition of dbcAMP to F9 cells treated with RA enhanced the expression of the phenotype induced by retinoic acid, generating cells similar to those of the parietal endoderm in early mouse embryogenesis, as reported by Strickland and Mahdavi [18]. These cells became round and refractile and expressed a high level of t-PA and laminin Bl mRNA. Moreover the level of c-kit mRNA subsequently markedly increased in these cells. Thus, for the expression of c-kit mRNA, the differentiation of F9 cells has to have progressed at least to the primitive endodermal cell stage under the inductive influence of RA for 2-4 days but effective c-kit mRNA expression was only observed after F9 cells had differentiated into parietal endoderma1 cells by treatment with dbcAMP. Furthermore, the expression of c-kit mRNA was dramatically induced

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with dbcAMP alone in the endodermal cell line PYS-2 whose expression of c-kit mRNA is originally low. Since the action of dbcAMP is known to be irreversible in F9 cells induced to differentiate into parietal endodermal cells [ 181, the expression of t-PA and laminin Bl mRNA remained constant even after the removal of dbcAMP from the culture medium. In contrast, c-kit mRNA decreased to the basal level within a day after removal of dbcAMP, implying that the effect of dbcAMP on c-kit gene expression is reversible. Moreover, dbcAMP could reactivate the expression of c-kit mRNA in parietal endodermal cells (data not shown). Thus, dbcAMP seems to have a dual effect on F9 cells: it induces the differentiation of RA-treated cells into parieta1 endodermal cells and modulates the amount of c-kit mRNA in these differentiated cells. Expression of the c-kit gene was induced by agents, such as forskolin or theophylline which are known to elevate the cellular CAMP level, in differentiated cells treated with RA but not in undifferentiated cells. Although the mechanism by which RA affects gene expression remains unknown, treatment of F9 stem cells with RA has been shown to increase the activity of CAMP-dependent protein kinase and adenylate cyclase [33,34]. Since dbcAMP and other agents which increase intracellular CAMP do not affect the c-kit expression in original F9 stem cells, it is speculated that some factors induced in the differentiated F9 cells by RA may play an important role in the responsiveness of these cells to CAMP. Although the CAMP-dependent protein kinase appears to be the sole mediator of CAMP in higher eukaryotes, the specific mechanism of CAMP-PK modulation of gene transcription is still unknown. Recent results strongly implicate the catalytic subunit of the CAMP-dependent protein kinase 2 (R2) as the mediator of CAMP action on gene expression [35]. Whether the R2 subunit is involved in c-kit gene activation or not, it appears certain that some protein factor(s) exists which links kinase activation with transcription of CAMP-responsive genes having specific CAMP-responsible elements [36, 371. Since the c-kit gene is regulated by the level of intracellular CAMP in differentiated F9 cells, the genomic regulatory sequence of c-kit gene may have the capacity to respond to CAMP. Further analysis of the genomic structure of the regulatory sequence of the c-kit gene would give insight into this hypothesis and may help us understand the specific gene regulation in the F9 cell system. It would also help us to understand how the c-kit gene is regulated in embryogenesis. What is a role of the c-kit protooncogene in F9 cell differentiation? The c-kit protooncogene is known to function as the growth factor receptor for the Sl gene product [38]. The c-kit gene product may work in controlling, inducing, or maintaining cell differentiation or in any other functions particular to these differentiated F9 cells, similar to the mast cell system [39]. In mam-

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mals, a number of recent studies report the developmental expression of genes encoding peptide growth factors and their receptors. F9 EC cells will be a useful complement to direct studies on early embryos and would have the potential to yield greater insight into the functional aspect of the c-kit protooncogene in development. We thank Dr. S. Strickland, Dr. K. Tokunaga and Dr. Y. Yamada for providing us the mouse t-PA, p-actin, and laminin Bl cDNA clones, respectively. This work was supported in part by a Grant-inAid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

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