Mar 5, 1990 - Ectopic expression of c-jun leads to differentiation of. P19 embryonal carcinoma cells. Rolf P.de Groot, Frank A.E.Kruyt,. Paul T.van der Saag ...
The EMBO Journal vol.9 no.6 pp.1831 - 1837, 1990
Ectopic expression of c-jun leads P19 embryonal carcinoma cells
Rolf P.de Groot, Frank A.E.Kruyt, Paul T.van der Saag and Wiebe Kruijer Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands Communicated by E.Wagner
The product of the c-jun proto-oncogene, a major component of the transcription factor API, has been implicated in both positive and negative transcriptional control as well as in transformation. The role of c-jun in early development and differentiation processes, however, is still largely unknown. In this paper we show that ectopic expression of exogenous c-jun sequences leads to differentiation and loss of the transformed phenotype of P19 embryonal carcinoma (EC) cells. The mixed populations of endoderm- and mesoderm-like cells that were obtained after the introduction of c-jun morphologically and biochemically resembled P19 cells differentiated in monolayer by retinoic acid (RA). Furthermore, we provide evidence that direct effects of the c-jun gene product on transcription of the retinoic acid receptor (RARI3) gene may be responsible for the differentiationpromoting potential of c-jun. Key words: c-jun/EC cell differentiation/RARf
to differentiation of
pluripotent stem cells from the inner cell mass of early mouse pre-implantation embryos (Graham, 1977; Martin, 1980). Undifferentiated EC cells are tumorigenic cells that exhibit all the characteristics of transformed cells, including anchorage independent growth and proliferation in the absence of growth factors. Differentiation of EC cells in response to chemical agents [e.g. retinoic acid (RA)] is accompanied by loss of tumorigenicity and the transformed phenotype (Strickland and Mahdavi, 1978; Jones-Villeneuve et al., 1983). Previously, we have shown that c-jun is expressed at very low levels in undifferentiated murine P19 EC cells, while its expression is strongly enhanced after differentiation by RA (de Groot et al., 1990). In addition, in P19 EC and F9 EC cells very low levels of API binding activity were found, whereas TPA treatment does not result in trans-activation of the TRE (Kryszke et al., 1987; Chiu etal., 1988). To further determine the role of c-jun in the differentiation of EC cells, we stably transfected a human c-jun gene into P19 EC cells. In this paper we show that ectopic expression of c-jun induces differentiation of P19 EC cells, and that c-jun-induced expression of the RARI3 gene might be the underlying mechanism of this differentiation process.
Results Introduction The nuclear proto-oncogene c-jun encodes one of the polypeptides found in purified transcription factor API (Lee et al., 1987; Angel et al., 1988a; Bohman et al., 1988). The products of the c-jun gene and two related genes, junB and junD, can form homodimeric complexes or heterodimeric complexes with the products of thefos gene family, including c-fos, fosB andfra-1 (Chiu et al., 1988; Halazonetis et al., 1988; Nakabeppu et al., 1988; Rauscher et al., 1988a,b; Sassone-Corsi et al., 1988b; Cohen et al., 1989; Zerial et al., 1989). These complexes bind to the TPA responsive element (TRE/AP1), and can both activate and repress transcription of target genes containing the API site (Distel et al., 1987; Angel et al., 1987a,b,1988b; Kerr et al., 1988; Sassone-Corsi et al., 1988a; Schonthal et al., 1988). jun and fos genes are rapidly and transiently induced by growth factors and phorbol esters in a variety of cell types (Kruijer et al., 1984; Cohen and Curran, 1988; Lamph et al., 1988; Quantin and Breathnach, 1988; Ryder et al., 1988,1989; Ryseck et al., 1988; Zerial et al., 1989), and thus act as nuclear 'third messengers' converting cytoplasmic signals into long-term changes in gene expression. Very little is known about the role of Jun in early mammalian development. To study the role of c-jun in this complex process, we have used P19 embryonal carcinoma (EC) cells as an in vitro model system. EC cells are the malignant stem cells of teratocarcinoma and resemble the Oxford University Press
Isolation of c-jun-expressing P19 clones Previously, we have shown that the proto-oncogene c-jun is expressed at very low levels in P19 EC cells, whereas differentiation by RA causes a strong increase in c-jun expression within 24-48 h (de Groot et al., 1990). To study the significance of the differential c-jun regulation for the differentiation of EC cells, we transfected undifferentiated P19 EC cells with an SV40-driven human c-jun gene together with the selectable neo gene (pSV2neo, Southern and Berg, 1982). The SV40 early promoter works efficiently in undifferentiated F9 EC cells (Gorman et al., 1985) as well as in undifferentiated P19 EC cells (our unpublished data). Of 2 x 106 transfected cells, -40 geneticin-resistant colonies were obtained after selection for 12 days. The overall morphology of the cells in all colonies strongly resembled the mixed population of cell types found after differentiation of P19 EC cells by RA in monolayer (McBurney et al., 1982), while transfection of the expression vector lacking the c-jun gene (pSG5; Green et al., 1988) resulted in the formation of geneticin-resistant colonies with normal EC cell morphology. These results suggest that introduction of an SV40-driven c-jun gene into undifferentiated P19 EC cells leads to differentiation of these cells. In order to further analyse the effects of the exogenous c-jun gene on P19 EC cells, 20 independent colonies were cloned from which 16 neomycin-resistant, stably transfected proliferating cell lines were obtained. DNA from these cell lines was isolated and analysed for the presence of exogenous
1831
R.P.de Groot et al. Table I.Properties of c-jun transfected P19 cells
Cella
Exogenous
c-jun (human)
TRE
expression
binding
TRE transactivationb
SSEA-1
copies
expression
Soft agar growth
Endo A expression
P19 EC
-
-
-
1
++
29.2%
-
pSG5cl 4 pSG5 cl 8 pSG5 cl 9
2 4 4
-
-
1 1 1
++ ++ ++
33.8% 21.2% 24.3%
-
jun cl 2 jun cl 8 juncl 15 juncll8 jun cl 20 jun cl 24 jun cl 25 jun cl 28
10 2 2 5 2 2 15 2
+++ ++ +++ + ++ +++
++ +++ ++ ++
20 8 6 1 5 5 7 5
-
