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Endocrinology Vol. 138, No. 7 2689-2696
Copyright © 1997 by The Endocrine Society

ARTICLES

Alternative Signaling Mechanism of Leukemia Inhibitory Factor Responsiveness in a Differentiating Embryonal Carcinoma Cell

Takashi Takeda, Hirohisa Kurachi, Toshiya Yamamoto, Hiroaki Homma, Kazushige Adachi, Kenichirou Morishige, Akira Miyake and Yuji Murata

Department of Obstetrics and Gynecology, Osaka University Medical School, Osaka 565, Japan

Address all correspondence and requests for reprints to: Dr. Takashi Takeda, Department of Obstetrics and Gynecology, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leukemia inhibitory factor (LIF) is a cytokine that plays an important role during mouse embryogenesis. We showed that adenovirus E1A represses the interleukin-6 signal transduction pathway that uses the same JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor as LIF. Here, we report that the LIF-JAK-STAT signal transduction pathway is blocked in cellular E1A-expressing undifferentiated F9 cells, and that the block is overcome by retinoic acid-induced differentiation. LIF failed to stimulate the expression of the acute phase response element (APRE)-driven luciferase gene in undifferentiated F9 cells, whereas the luciferase activity was remarkably increased by LIF treatment in differentiated F9 (dF9) cells. We analyzed the mechanism of the APRE regulation and found that the LIF-induced APRE-binding activity was regulated in a differentiation-dependent manner. The protein levels and the tyrosine phosphorylation of JAK1, JAK2, and STAT3 in F9 cells were not different from those in dF9 cells. The exogenous expression of activated c-Ha-ras partially recovered the LIF responsiveness of the APRE-luciferase gene in F9 cells, but the dominant negative ras N-17 did not repress the LIF-induced activation of APRE-luciferase in dF9 cells. These results suggested that an unknown coactivation process that is partially compensated by Ras is required for STAT3-APRE binding in F9 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEUKEMIA inhibitory factor (LIF) is a multifunctional cytokine that plays important roles in a wide range of biological activities, such as the synthesis of acute phase proteins in hepatocytes, osteoblast formation, hematopoiesis, and neuronal differentiation (1). In addition, LIF maintains the developmental potential of embryonic stem (ES) cells (2), and it has been reported that the transient expression of LIF is essential for implantation in mice (3). These facts suggest a critical role for this molecule in early embryogenesis.

The embryonic carcinoma cell line, F9 is a useful model system with which to analyze early differentiation events that are similar to those of the early mammalian embryogenesis. Retinoic acid (RA)-treated F9 cells differentiate into parietal extraembryonic endoderm-like cells (4). The activated c-Ha-ras oncogene (Ha-rasVal-12) also induces the differentiation of F9 cells (5). Undifferentiated F9 cells contain an E1A-like activity that is lost upon RA-induced differentiation (6, 7, 8). The exogenous expression of the adenovirus E1A gene reverses the terminal differentiation and the expression of differentiation specific genes in F9 cells (7). A cellular E1A-like activity and an adenoviral E1A are thought to use similar mechanisms to repress the interferon-{alpha} (IFN{alpha}) signal transduction pathway (9).

We showed that adenovirus E1A represses interleukin-6 (IL-6) induced gene activation in the hepatoma cell line HepG2 (10). LIF shares striking similarities with IL-6 in terms of biological activities (reviewed in Ref. 11). LIF and IL-6 bind to respective receptor complexes, both of which contain the same signal transducer gp130 (12). Both cytokines use the recently characterized JAK protein tyrosine kinases (JAK1, JAK2, and Tyk2), as well as the STAT (signal transducer and activator of transcription) transcription factor signal transduction pathway and activate the same acute phase response element (APRE) in the rat {alpha}2-macroglobulin promoter by binding the acute phase response factor (APRF) (13, 14, 15, 16). It is reported that although F9 cells have high affinity LIF receptors, as do ES cells (2), LIF has no biological effect on undifferentiated F9 cells (17). This suggests that the E1A-like activity might repress the LIF-JAK-STAT pathway in undifferentiated F9 cells.

In this study, we examined whether the LIF-JAK-STAT signal transduction pathway is regulated in a differentiation-dependent manner in F9 cells. We further analyzed the mechanism of this regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell and cell culture
The embryonal carcinoma cell line F9 (18) was cultured in {alpha}MEM (Life Technologies, Grand Island, NY) containing 10% FCS (CSL, Parkville, Australia). Terminally differentiated F9 cells (dF9) were obtained by growing F9 cells in medium containing RA (0.1 µM; Sigma Chemical Co., St. Louis, MO) and (Bu)2cAMP (0.1 mM; Sigma).

Plasmids
The 4xAPRE-luciferase gene containing four repeats of APRE (IL-6 response element of the rat {alpha}2-macroglobulin promoter), was constructed by subcloning two repeats of the oligonucleotides (5'-TCG-ACATCCTTCTGGGAATTCTGATCCTTCTGGGAATTCTGGGTAC-3')(19) in front of the minimal junB promoter-luciferase gene as previously described (10). The 4xAPRE-luciferase gene containing four repeats of mutated APRE (4xmAPRE) was constructed by subcloning two repeats of the oligonucleotides (5'-TCGACATCCTTCTCTAGATTCTGATCCTTCTCTAGATTCTGGGTAC-3') (13) in front of the minimal junB promoter-Luciferase gene. The activated c-Ha-ras (Ha-rasVal-12) expression vector was pSVEJ6.6 (20). The dominant negative ras N-17 mutant (Asn-17 ras) expression vector was pZIPRasN17 (21). The mouse c-jun expression vector was pRSV-c-jun (22).

DNA transfection and luciferase assays
DNA transfection and luciferase assays were performed as previously described (10, 23). In each experiment, cells were seeded at 5 x 105 cells/6-cm dish, and 24 h later, 2.9 µg of the luciferase reporter plasmid, 1.0 µg pEFlacZ (23) (internal control for transfection efficiency), and 0.7–2.1 µg expression vector were used as required. The total amount of transfected DNA was adjusted to 6.1 µg with pTZ19R (Pharmacia, Milwaukee, WI). Cells were extracted, and luciferase activities were assayed in a MicroLumat luminometer (EG&G Berthold, Postfach, Germany) as previously described (10). ß-Galactosidase activity was determined to normalize the transfection efficiency. All experiments were performed in triplicate and repeated at least three times with essentially similar results. Results are expressed as relative luciferase activity. Data are shown as averages of three independent experiments, with SDs indicated by bars.

Cytokines and antibodies
The cytokines used in this study were human recombinant LIF (Pepro Tech, Rocky Hill, NJ) and human recombinant IL-6 (a gift from Dr. T. Hirano, Osaka University Medical School, Osaka, Japan). Anti-ISGF3 (STAT1-{alpha}/ß) monoclonal antibody (mAb; Transduction Laboratories, Lexington, KY) and the antiserum against STAT3 (a gift from Dr. K. Nakajima, Osaka University Medical School) have been previously described (10, 24). The antibodies used for Western blotting and immunoprecipitation were anti-JAK1 polyclonal antibody (HR-785, Santa Cruz Biotechnology, Santa Cruz, CA), anti-JAK2 polyclonal antibody (Upstate Biotechnology, Lake Placid, NY), antiphosphotyrosine mAb (4G10, Upstate Biotechnology), and anti-STAT3 polyclonal antibody (C-20, Santa Cruz Biotechnology).

Electrophoretic mobility shift assays (EMSAs)
Nuclear and cytoplasmic extracts were prepared from F9 and dF9 cells according to the method of Sadowski et al. (25). The double stranded oligonucleotides used as probes or competitor in the EMSAs were 5'-GCGCCTTCTGGGAATTCCTA-3' and 5'-GCGCTAGGAATTCCCAGAAG-3' (10). Binding reactions and electrophoresis proceeded as previously described (23). In the competition analysis, cell extracts were incubated with a 10-fold molar excess of unlabeled oligonucleotides for 5 min before adding the labeled oligonucleotides. In the supershift assays, anti-STAT1 mAb, anti-STAT3 antiserum, or a control rabbit serum was added to the binding reaction mixture in the indicated amount.

LIF binding assay
LIF was iodinated, and binding was carried out as previously described (2). Five million F9 or dF9 cells were incubated at 37 C for 40 min in 100 µl RPMI (Life Technologies) containing 10% FCS, 20 mM HEPES (pH 7.4), and [125I]LIF (2 x 103 to 8 x 105 cpm; SA, ~40,000 cpm/ng) with or without excess amounts (500-fold that of [125I]LIF) of unlabeled LIF. After incubation, cell-associated and free radioactivity were separated by centrifugation, and the radioactivity was counted by a {gamma}-counter.

Immunoprecipitation, SDS-PAGE, and Western blotting
Cells (2 x 107) were harvested and lysed for 30 min in 1 ml lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM NaF, 0.75 mM phenylmethylsulfonylfluoride, 15% glycerol, and 10 µg/ml each of aprotinin, pepstatin, and leupeptin] as previously described (15). Proteins in the lysates or the nuclear extracts from 2 x 107 cells were immunoprecipitated with the appropriate antibodies. Immune complexes were separated by SDS-PAGE (7.5%) and transferred to an Immobilon-P nylon membrane (Nihon Millipore, Tokyo, Japan), which was immunoblotted with the relevant primary antibody as previously described (10). Proteins were detected by enhanced chemiluminescence (ECL, Amersham International, Aylesbury, UK). Rehybridization was performed as previously described (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LIF-mediated induction of the APRE-luciferase gene is impaired in F9 cells
We tested the LIF-induced activation of the APRE-luciferase gene in undifferentiated and RA-differentiated F9 (dF9) cells. The 4xAPRE-luciferase or 4xmAPRE-luciferase plasmid was transiently transfected into F9 and dF9 cells, and luciferase activity was assayed after 5 h of LIF stimulation (Fig. 1AGo). Transfection efficiency was normalized by ß-galactosidase activity as described in Materials and Methods. Both cells showed almost the same transfection efficiency. The data are presented by calculating the luciferase activity of unstimulated F9 cells as 1. The basal APRE activity was slightly (~2.5-fold) increased by the differentiation process. Although F9 cells have high affinity LIF receptors (2, 17), the APRE-luciferase gene was not activated by LIF in F9 cells. In dF9 cells, however, LIF largely (~18-fold) activated APRE activity. As LIF did not activate the mAPRE-luciferase (Fig. 1AGo) and minimal junB promoter-luciferase (data not shown) genes in dF9 cells, LIF-induced activation of the APRE-luciferase gene is through activation of the APRE site.



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  		Figure 1. LIF-mediated induction of the APRE-luciferase gene in undifferentiated and differentiated F9 cells. A, Undifferentiated (F9) and differentiated F9 (dF9) cells were transfected with the 4xAPRE-luciferase gene (APRE) or the 4xmAPRE-luciferase gene (mAPRE). The transfected cells were not stimulated (-) or were stimulated (+) with LIF (10 ng/ml) for 5 h, and luciferase activity was determined. B, Dose-dependent activation of the APRE-luciferase gene by LIF in dF9 cells. The dF9 cells were transfected with the 4xAPRE-luciferase gene, then stimulated with LIF or IL-6 (at the concentrations indicated) for 5 h. Thereafter, luciferase activities were determined.

 
We examined the dose dependence of the LIF-induced APRE activation (Fig. 1BGo). LIF activated the APRE in a dose-dependent manner in dF9 cells. LIF-induced activation reached the maximal level at a concentration of 10 ng/ml. In F9 cells, APRE-luciferase was not activated even at a concentration of 50 ng/ml (data not shown). IL-6 also uses the gp130-JAK-STAT (STAT3) signal transduction pathway and activates the APRE (14, 15). We, therefore, examined whether this factor activates the APRE-luciferase gene in F9 and dF9 cells. IL-6 did not activate the APRE in F9 cells as LIF (data not shown). IL-6 activated the APRE to a much lesser extent than LIF in dF9 cells even at a concentration of 100 ng/ml (Fig. 1BGo). IL-6-induced APRE activation reached the maximal level at 100 ng/ml (data not shown). These findings suggest that the region upstream of the JAK kinase is impaired in IL-6 signal transduction pathways in dF9 cells. The {alpha}-subunit function of the IL-6 receptor may be impaired in dF9 cells.

APRF activation is regulated in a differentiation-dependent manner in F9 cells
To examine which part of the LIF-JAK-STAT signal transduction pathway is disturbed in F9 cells, we investigated whether APRF is activated in LIF-stimulated F9 cells using EMSAs (Fig. 2Go, A and B). LIF-induced DNA-binding activity of dF9 cells was detected in the nucleus (Fig. 2AGo, lanes 3 and 4) and in the cytoplasm (Fig. 2BGo, lanes 3 and 4). This binding complex was competed out by an excess of the unlabeled APRE oligonucleotides (Fig. 2Go, A and B, lane 5). In F9 cells, LIF-induced DNA-binding activity was not detected in either the nucleus (Fig. 2AGo, lanes 1 and 2) or the cytoplasm (Fig. 2BGo, lanes 1 and 2). These results suggest the absence of an APRE activation process in F9 cells. The LIF-induced APRE binding complex in dF9 cells was supershifted by anti-STAT3, whereas anti-STAT1 and control serum had no effect (Fig. 2CGo). These results indicated that STAT3 is dominant in LIF-induced DNA-binding activity in dF9 cells.



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  		Figure 2. Differentiation-dependent activation of APRF in F9 cells. Undifferentiated F9 (lanes 1 and 2) and dF9 (lanes 3–5) cells were not stimulated (lanes 1 and 3) or were stimulated (lanes 2, 4, and 5) with LIF (10 ng/ml) for 15 min, then nuclear and cytoplasmic extracts were prepared. For each reaction, 12 µg nuclear (A) or 20 µg cytoplasmic (B) extracts were incubated with a 32P-labeled APRE probe. Competition analysis (lane 5) proceeded as described in Materials and Methods. The reactions were followed by electrophoresis in a 4.5% polyacrylamide gel. The arrows in A and B indicate the position of APRF in dF9 cells. C, Analysis of APRE-binding proteins in dF9 cells. Nuclear extract (12 µg protein) prepared from dF9 (lanes 1–5) cells stimulated with LIF (10 ng/ml) for 15 min was incubated with an APRE probe. Competition analysis (lane 2) proceeded as described in Materials and Methods. Where indicated, control rabbit serum (cont Ab, lane 3), anti-STAT3 antiserum ({alpha}STAT3, lane 4), or anti-STAT1 mAb ({alpha}STAT1, lane 5) was added at final dilutions of 1:50, 1:50, and 1: 5, respectively. Arrow 1 indicates the APRF in dF9 cells, as described in A and B. Arrow 2 indicates the position of the supershifted complex of APRF by anti-STAT3 antiserum.

 
Tyrosine phosphorylation and the protein levels of JAK1, JAK2, and STAT3 are the same in undifferentiated F9 and dF9 cells
We analyzed which part of the LIF-JAK-STAT signal transduction pathway was disturbed in undifferentiated F9 cells. To examine the possible absence of LIF receptor in F9 cells, we conducted [125I]LIF binding studies (Fig. 3Go). Dissociation constants (Kd) were 100 pM in both F9 and dF9 cells. The maximal numbers of binding sites were 380 and 330/cell in F9 and dF9 cells, respectively. Specific [125I]LIF binding was observed at similar levels in both F9 and dF9 cells. These results suggested the disturbance of LIF-JAK-STAT pathway in undifferentiated F9 cells was not due to the absence of LIF receptors.



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  		Figure 3. Scatchard analysis of [125I]LIF-specific binding in F9 and dF9 cells. Cells were incubated with increasing amounts of [125I]LIF as described in Materials and Methods. The ratio of the amount of [125I]LIF specifically bound vs. free [125I]LIF was plotted. Scatchard plots for F9 cells (A) and dF9 cells (B) are shown.

 
Next we examined whether LIF induces the tyrosine phosphorylation of JAK1 and JAK2 in F9 and dF9 cells (Fig. 4Go, A and B). LIF induced the tyrosine phosphorylation of JAK1 and JAK2 in F9 cells as effectively as in dF9 cells (Fig. 4Go, A and B, upper panel), and the protein levels of these kinases in F9 and dF9 cells were the same (Fig. 4Go, A and B, lower panel). These results suggested that the LIF-induced JAK activation process is not disturbed and that functional LIF receptors are present in F9 cells.



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  		Figure 4. Tyrosine phosphorylation of JAK family protein kinases by LIF. F9 and dF9 cells were incubated for 10 min without (-) or with (+) LIF (50 ng/ml). Cell lysates were immunoprecipitated with anti-JAK1 (A) or anti-JAK2 (B). Immune complexes were separated by SDS-PAGE and analyzed by blotting with antiphosphotyrosine (upper panel). The blots were then stripped and reprobed sequentially with anti-JAK1 (A, lower panel) or anti-JAK2 (B, lower panel).

 
We then examined whether STAT3 is tyrosine phosphorylated in LIF-stimulated F9 cells, and the STAT3 protein levels in undifferentiated and differentiated F9 cells were compared (Fig. 5AGo). LIF induced the phosphorylation of STAT3 to a similar extent in F9 and dF9 cells, and the protein level in F9 cells was not different from that in dF9 cells. Besides STAT3 protein, we detected three tyrosine-phosphorylated proteins (190, 145, and 130 kDa). It has been reported that the LIF receptor ß-subunit (LIFR-ß) and gp130 coprecipitate with STAT3 under nonreducing conditions and that they correspond to the 190- and 145-kDa proteins, respectively (15). The similarity of the molecular mass suggests that the 130-kDa protein is a JAK kinase. These three proteins were also tyrosine phosphorylated by LIF stimulation in both F9 and dF9 cells to a similar extent. These findings indicate that in F9 cells, LIF induces the tyrosine phosphorylation of gp130, LIFR-ß, JAKs, and STAT3. Under unstimulated conditions, a small amount of tyrosine-phosphorylated proteins was found in F9, but not dF9, cells (Fig. 5AGo). They were slightly activated in the unstimulated condition by an unknown mechanism.



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  		Figure 5. Tyrosine phosphorylation of STAT3 by LIF. F9 and dF9 cells were treated for 10 min without (-) or with (+) LIF (50 ng/ml). Cell lysates (A) or nuclear extracts (B) were immunoprecipitated with anti-STAT3. Immune complexes were separated by SDS-PAGE and blotted with antiphosphotyrosine (A, upper panel, and B). The blots were then stripped and reprobed with anti-STAT3 (A, lower panel).

 
In the JAK-STAT signal transduction pathway, STATs are tyrosine phosphorylated in the cytoplasm and translocate to the nucleus (27). As it is possible that the translocation of the phosphorylated STAT3 protein from cytoplasm to nucleus is impaired in F9 cells, we analyzed whether phosphorylated STAT3 is present in the nucleus of undifferentiated cells. As shown in Fig. 5BGo, STAT3 phosphorylated by LIF stimulation was present in the nuclear protein fraction from undifferentiated F9 cells. These findings together with the results of the EMSA (Fig. 2Go) suggested that tyrosine-phosphorylated STAT3 cannot bind to the APRE site in undifferentiated F9 cells. The presence of a binding inhibitor or the absence of a coactivation mechanism in undifferentiated F9 cells is thus indicated.

Negative transcriptional regulator was not found in F9 cells
Some human cancer cell lines express a negative transcriptional regulator, termed transcriptional knockout, that causes a lack of IFN-stimulated gene factor-3; thus, these cells show defects in IFN-induced gene expression (28). We examined whether F9 cells contain such a direct inhibitor that prevents APRF binding to APRE. The addition of the nuclear extracts prepared from untreated F9 cells had no effect on the LIF-induced binding complex in dF9 cells (Fig. 6Go, lanes 1 and 2). Moreover, addition of the nuclear extracts from LIF-treated F9 cells did not affect the LIF-induced DNA-binding activity in dF9 cells (data not shown). These results suggested that a negative regulator, such as transcriptional knockout, is not present in F9 cells.



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  		Figure 6. Absence of a negative transcriptional regulator in F9 cells. Nuclear extract from untreated F9 cells (F9 -, 8 µg) was mixed with nuclear extract (8 µg) from untreated (dF9 -) or 10 ng/ml LIF-treated (dF9 +) dF9 cells (lanes 1 and 2). Nuclear extract from untreated dF9 cells (dF9 -, 8 µg) was mixed with nuclear extract (8 µg) from untreated (F9 -) or 10 ng/ml LIF-treated (F9 +) F9 cells (lanes 3 and 4). These nuclear extract mixtures were incubated with a 32P-labeled APRE probe. The arrow indicates the same complexes as those described in Fig. 3Go, A and B.

 
In IFN-{alpha} signaling, E1A-mediated repression is attributed to a defect in the DNA-binding subunit (IFN-stimulated gene factor-3 {gamma} component) that is restored by the complementation of this component (29). F9 cells might have such a defective DNA-binding subunit. Therefore, we tested whether the nuclear extract from the untreated dF9 cells restores the LIF-induced DNA-binding activity in F9 cells (Fig. 6Go, lanes 3 and 4). Addition of the nuclear extract prepared from the dF9 cells did not restore the LIF-induced DNA-binding activity in F9 cells, suggesting that F9 cells do not have a defective DNA-binding subunit.

Ha-rasVal-12 restored the LIF-induced APRE activation in F9 cells, but the Ras itself was not involved in LIF-JAK-STAT pathway in dF9 cells
The above data suggest a possible defect in the coactivation pathway for APRE activation in undifferentiated cells. As the stable expression of Ha-rasVal-12 induces the differentiation of F9 cells to endoderm-like cells as does RA (5), it was interesting to examine whether Ha-rasVal-12 could induce the LIF responsiveness of the APRE-luciferase gene in F9 cells. The 4xAPRE-luciferase or 4xmAPRE-luciferase plasmid was transiently transfected with a Ha-rasVal-12 expression vector (0.7 µg) into F9 cells, and the luciferase activities were assayed after 5 h of LIF stimulation. As shown in Fig. 7AGo, the LIF-induced APRE activity was partially restored by Ras expression; although the LIF-induced APRE activation was less in the Ras-expressed F9 cells than in dF9 cells (~18-fold, see Fig. 1Go), approximately 4-fold induction was obtained by Ha-rasVal-12 transfection in F9 cells. As LIF failed to induce mAPRE activity by Ras expression (Fig. 7AGo), Ras-induced restoration of the APRE-luciferase gene may be through activation of the APRE site. When we transfected higher doses of Ras expression vector (up to 2.2 µg), the LIF responsiveness of the APRE-luciferase in F9 cells did not change (data not shown). The dominant negative Ras (N-17) (22) did not restore the LIF responsiveness of APRE (Fig. 7AGo). These results show that the Ras-dependent pathway might be involved in the coactivation process of the APRE activation in F9 cells. Next we examined the effect of the N-17 on the LIF-induced activation of APRE in dF9 cells (Fig. 7BGo). The 4xAPRE-luciferase was transiently transfected with an N-17 expression vector (2.1 µg) into dF9 cells, and the luciferase activities were assayed after 5 h of LIF stimulation. As shown in Fig. 7BGo, the LIF-induced APRE activity was not repressed by N-17. The effect of N-17 in dF9 cells was confirmed by the fact that N-17 completely repressed the c-jun-induced activation of the activator protein-1 site-luciferase (30) (data not shown). These results suggested that Ras itself was not involved in the LIF-JAK-STAT signal transduction pathway. An unknown coactivation process that could be partially compensated by Ras expression may be involved in this process.



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  		Figure 7. Presence of an unknown coactivation pathway in the LIF-JAK-STAT pathway. A, Ha-Ras expression recovered the LIF responsiveness in F9 cells. F9 cells were transfected with the 4xAPRE-luciferase (APRE) or 4xmAPRE-luciferase (mAPRE) gene and the expression vector, pSVEJ6.6 (Ha-Ras), or its dominant negative mutant pZIPRasN17 (N17). B, Dominant negative Ras (N17) did not repress the LIF-induced activation of APRE-luciferase in dF9 cells. The dF9 cells were transfected with 4xAPRE-luciferase and pZIPRasN17 (N17). The transfected cells were not stimulated (-) or were stimulated (+) with LIF (10 ng/ml) for 5 h. Luciferase activity was then determined.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here, we showed that 1) the LIF-JAK-STAT signal transduction pathway is blocked in undifferentiated F9 cells; 2) LIF-induced APRF activation is regulated in a RA-induced, differentiation-dependent manner; 3) tyrosine-phosphorylated STAT3 is present in the nuclei of undifferentiated F9 cells, but it does not bind to the APRE site; 4) the exogenous expression of Ha-rasVal-12 partially rescues the responsiveness to LIF in undifferentiated F9 cells; but 5) Ras itself is not involved in LIF-JAK-STAT signal transduction pathway in dF9 cells.

F9 cells have high affinity LIF receptors, like ES cells, and both cells bind similar levels of LIF (2). Although LIF is necessary for maintenance of the developmental potential of ES cells (2, 31), LIF has no biological effect on F9 cells (17). Both F9 and dF9 cells have the same number of LIF receptors and level of affinity (17). Moreover, under our conditions, LIF activated JAK1 and JAK2 in F9 cells to the same extent as in dF9 cells, suggesting that the LIF receptor is also functional in F9 cells. Therefore, the LIF-JAK-STAT signal transduction pathway in F9 cells is likely to be blocked downstream of the LIF receptor. Our data showed that the critical step of this repression was the disturbance of tyrosine-phosphorylated STAT3 binding to the APRE site and suggested the presence of an unknown coactivation process that is regulated in a differentiation-dependent manner.

It has been reported that the H7 (serine/threonine kinase inhibitor)-sensitive pathway is involved in the IL-6, LIF signal transduction pathway (23, 32, 33) and that serine phosphorylation at the mitogen-activated protein kinase (MAPK) site of STAT3 is required for STAT3-DNA complex formation (34). Therefore, an H7-sensitive coactivation process may be required for STAT3-DNA complex formation, which is disturbed in undifferentiated F9 cells. As Ras activates the Raf/MAPK signal transduction pathway (35, 36, 37), we studied the exogenous expression of Ha-rasVal-12 in F9 cells. We found that Ras restored LIF-induced APRE activation in undifferentiated F9 cells, suggesting that STAT3 phosphorylation at the MAPK site may be required to bind to APRE. We also found that the LIF-JAK-STAT pathway was Ras independent in dF9 cells. Therefore, Ras itself seemed not to be involved in the coactivation pathway in dF9 cells. A Ras-independent and MAPK-dependent pathway was reported in 3T3-L1 adipocytes in the insulin signal pathway (38), and such alternate routes of signal transduction may be involved in the coactivation pathway in dF9 cells. We cannot rule out the possibility that the Ras effect is indirect, and the restoration is the result of differentiation itself, but in the previous study, the Ras-induced differentiation of F9 cells was observed in the stable transfectants and took at least 4–6 days for the morphological differentiation (5). In our study the expression of Ras was transient, the time from transfection through harvest was 40 h, and morphological differentiation was not observed.

As Ras could not fully activate LIF-induced APRE in F9 cells, another coactivation process(es) might be considerable. A coactivator, cAMP response element binding protein (CREB)-binding protein (CBP) has been found in the protein kinase A-cAMP response element binding protein signal transduction pathway (39). CBP has a striking homology with p300, and adenoviral E1A binds both proteins (40, 41). Recently, it was reported that RA induces serine and threonine phosphorylation of p300 during the differentiation of F9 cells (42), and that p300/CBP cooperate with STAT2 in the IFN-{alpha} pathway (43) and with STAT1 in the IFN-{gamma} (44) signal transduction pathway. Because the LIF-JAK-STAT3 pathway closely resembles the IFN-{gamma}-JAK-STAT1 pathway, it might be interesting to examine whether a coactivator such as CBP or p300 is involved in the LIF-JAK-STAT3 pathway. It might be possible that in undifferentiated F9 cells, the p300-like coactivator is inactive; thus, the tyrosine-phosphorylated STAT3 cannot bind to the APRE site.

EMSA analysis in this study showed that in F9 cells the LIF-induced APRE-binding activity was not present, and complementation of the extracts from dF9 cells could not restore this defect. These results were consistent with those of our previous study (10); in a rat fibroblast cell line stably expressing adenoviral E1A (45), the IL-6-induced STAT3 binding to APRE was greatly reduced compared with that in the parental 3Y1 cells (46). A reduction of the STAT3 protein level in E1A3Y1 cells is one of causative factors for the reduction in APRE binding. In this study, however, we observed a distinct difference; the STAT3 protein level in F9 cells with cellular E1A was not different from that in dF9 cells without E1A. These inconsistencies might suggest a difference between the cellular E1A activity present in F9 cells and the adenoviral E1A. However, in the E1A3Y1 cells, exogenous expression of STAT3 protein did not restore the IL-6-induced APRE activation (our unpublished data), suggesting that the reduction in the STAT protein levels may only partly cause the repressive effect of the adenoviral E1A. The adenoviral E1A may also block the coactivation process of STAT-APRE binding as does the cellular E1A of undifferentiated F9 cells.

LIF-deficient mice generated by gene targeting have shown that LIF is essential for implantation (3). LIF is expressed in the uterine endometrial glands on the fourth day of pregnancy in mice (47). The true target of the maternal LIF is not yet known, but it may directly affect the blastocyst to be implanted. The RA-induced differentiation process of F9 cells is thought to correspond to the events that occur around the day of implantation (4, 48). This differentiation process is thought to correspond to the event in the inner cell mass of the 4.5-day postcoitum embryo (4, 48). It is possible that the maternal LIF stimulates the JAK-STAT signal transduction pathway in the preimplantation embryo and that this signal is critical for implantation.

An adenovirus E1A-like activity is present in preimplantation stage mouse embryos (49). The LIF-JAK-STAT pathway may be blocked in these cells. The differentiation-dependent activation of the LIF-JAK-STAT pathway may occur in the early embryo, and its regulation may play an important role in implantation. It would be of interest to test whether the regulation found in F9 cells also exists in the early embryo. Further examination of this mechanism will provide new insight into the regulation of early embryogenesis and implantation.


   Acknowledgments
 
We are grateful to Dr. T. Hirano for the gift of the IL-6, and to Dr. K. Nakajima for providing the anti-STAT3 antiserum, ras, and c-jun expression vectors. We thank Ms. I. Iida and Ms. K. Ogami for excellent secretarial assistance.

Received January 2, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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