THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 2, Issue of January 15, pp. 774-780,199O Printed in U.S. A.

Activation of the c-fos Serum-response Element by the Activated c-Ha-rus Protein in a Manner Independent of Protein Kinase C and CAMP-dependent Protein Kinase*

(Received for publication, July 24, 1989)

Yasuo Fukumoto, Kozo Kaibuchi, Naohisa Oku, Yuichi Hori, and Yoshimi Takai$ From the Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan

12-0-Tetradecanoylphorbol-13-acetate (TPA) acti- vated the c-fos gene enhancer linked to the chloram- phenicol acetyltransferase or luciferase reporter gene in the wild type PC-12 cells but not in the variant PC-12 cells that originated from the wild type cells. Transfection of the c-Ha-rusVa112 complementary DNA (cDNA) or addition of dibutyryl CAMP to the wild type PC-12 cells as well as to the variant PC-12 cells acti- vated the c-fos gene enhancer. Prolonged treatment of the wild type PC-12 cells with phorbol-12,13-dibutyr- ate caused down-regulation of protein kinase C. In these cells, TPA did not stimulate the c-fos gene en- hancer any more, but transfection of the c-Ha-raSV8’12 cDNA still stimulated the c-fos gene enhancer to the same extent as induced in the control cells. Transfec- tion of the c-Ha-rusVa112 cDNA or addition of TPA to the wild type PC-12 cells stimulated the serum-re- sponse element but not the CAMP-response element. Dibutyryl CAMP stimulated both the serum-response element and the CAMP-response element in the wild type PC- 12 cells. These results indicate that the c-Ha-

va’12 protein activates the serum-response element iz not the CAMP-response element in the c-fos genl enhancer, and that the signal pathway from the c-Ha- rag”e1l2 protein to the c-fos serum-response element is independent of protein kinase C and CAMP-dependent protein kinase.

ras ~21s’ have been assumed from their structural and biochemical features to serve as a GTP-binding transducer

* This investigation was supported by Grants-in-Aid for Scientific Research and Cancer Resea&h from- the Ministry of Education, Science. and Culture. Janan (1988 and 1989): Grants-in-Aid for Abnormalities in Hoimone Receptor Mechanisms, Cardiovascular Diseases, and for Cancer Research from the Ministry of Health and Welfare, Japan (1988 and 1989); and by grants from the Yamanouchi Foundation for Research on Metabolic Disease (1988), the Research Program on Cell Calcium Signal in the Cardiovascular System (1988 and 1989), and the Princess Takamatsu Cancer Research Fund (1988). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. 1 The abbreviations used are: ras ~21, the rus protein; TPA, 12-

0-tetradecanoylphorbol-13-acetate; NGF, nerve growth factor; Bt,cAMP, dibutyryl cyclic AMP; protein kinase A, CAMP-dependent protein kinase; SRE, serum-response element; CRE, CAMP-response element; CAT, chloramphenicol acetyltransferase; IL3, interleukin 3; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; HEPES, 4-(Z-hydroxyethyl)-l-p$erazineethanesulionic acid; PDBu; phorbol 12,13-dibutyrate; PBS, phosphate-buffered saline; EGF, epidermal growth factor; SRF, serum-response factor; bp, base pair.

for signal transduction across plasma membranes (for a re- view, see Ref. 1). Saccharomyces cereuisiae contains two closely related GTP-binding proteins encoded by the RASl and RAS2 genes that are homologous to mammalian ras ~21s (2). Genetic and biochemical analyses indicate that RASl and RAS2 proteins regulate CAMP levels through activation of adenylate cyclase (3). In mammalian cells, relevance of ras ~21s in the adenylate cyclase system is controversial. There is also evidence that the RAS and ras proteins may regulate the phospholipase C-mediated hydrolysis of phosphoinosi- tides and consequently modulate protein kinase C activity in yeast and mammalian cells such as NIH/3T3 cells and PC- 12 cells (4-8), although the evidence indicating the direct linkage between the phospholipase C and ras ~21s has not been obtained yet.

The c-fos protein is assumed to play crucial roles in cell differentiation and proliferation as a transacting factor for various genes (9). Schiinthal et al. (10) have recently proposed that ras ~21s stimulate c-fos gene expression at the transrcip- tional level and that the c-fos protein is absolutely required for ras p21- and TPA-induced expression of the genes con- taining the TPA response element in NIH/3T3 cells. The c- fos gene is expressed in response to a wide variety of stimuli such as platelet-derived growth factor, NGF, TPA, and BtzcAMP in addition to rus p21 in many cell types (11-14). Therefore, several intracellular signaling pathways are as- sumed to be involved in the c-fos gene transcription (11-14). The c-fos gene enhancer contains the SRE and CRE which are activated by TPA and Bt2cAMP, respectively (for a review, see Ref. 15). We have recently shown that the c-fos gene enhancer linked to the CAT reporter gene (c-fosCAT) is activated by protein kinase C and protein kinase A using the cDNAs of the constitutively active protein kinase C and the catalytic subunit of protein kinase A (16, 17).

In the present studies, we attempted to explore the mode of action of rus p21s for c-fos gene activation in PC-12 cells using the cDNAs of the normal and activated rus ~21s to understand the intracellular signals transmitted from ras p21 to the nuclei. This paper shows that the SRE rather than the CRE is responsible for the c-Ha-rasVs”’ p21-induced activa- tion of the c-fos gene enhancer and that the signal pathway from rus ~21s to the c-fos SRE is independent of protein kinase C and protein kinase A.

EXPERIMENTAL PROCEDURES

Materials and Chemicals-The wild type PC-12 cells were kindly donated by Y. Kaziro (University of Tokyo). The variant PC-12 cells (clone 6) were obtained from the wild type PC-12 cells by the limited dilution method.’ Other control PC-12 cells (PC-12h and PC-12m)

’ The properties of this variant PC-12 cell will be described else- where.

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were from H. Hatanaka (Osaka University, Osaka, Japan) and Y. Teranishi and Y. Matsui (Mitsubishi Kasei Corn., Yokohama, Japan), - _ respectively. The c-Ha-ras and c-Ha-rasVa”Z cDNA clones (ppS3 and ppS22, respectively) were generous gifts from F. Tamanoi (the Uni- versity of Chicago) (18). The pcDSRa expression plasmid was do- nated from K. Arai and Y. Takebe (DNAX Research Institute, Palo Alto, CA) (19). c-fosCAT (FC4) was a gift from I. M. Verma (Salk Institute, San Diego, CA) (20). c-fosCAT (FC4) was a fos-CAT fusion in which the 5’-flanking region of the human c-fos gene between SstII (position -404) and NaeI (position +41) was cloned upstream from the CAT sequence (Fig. 1B) (20). c-fos-luciferase was a gift from K. Arai and M. Muramatsu (DNAX Research Institute, Palo Alto, CA). The 445-bp S&II-NaeI fragment of the c-fos gene was cut by Hind111 from c-fosCAT and cloned into pSVO-luciferase to construct c-fos- luciferaie (Fig. 1C) (21). TKCAT was donated from K. Arai and S. Mivatake (DNAX Research Institute) (22). The PuuII-PstI fragment ,. (from positions -197 to +18) of the promoter region of the thy&dine kinase gene of herpes simplex virus was cloned upstream from the CAT seauence to construct TKCAT (22). TKCAT was used as a control plasmid to standardize the transfection efficiency. The anti- ras ~21 monoclonal antibody (RASK-4) was provided by H. Shiku (Nagasaki University, Nagasaki, Japan) (23). The anti-protein kinase C rnonoclonal antibody was purchased from Amersham Corp. The SRE and CRE olieonucleotides (5’-CAGGATGTCCATATTAGGA- CATCTG-3’ and ?-GAGCCCGTGACGTTTACAC-3’, respectively) were synthesized by a DNA synthesizer (Applied Biosystem, Model 381A). Other materials and chemicals were from commercial sources.

Construction of P2mmids-Two plasmids, pcDSRac-Ha-ra.s and pcDSRac-Ha-ras’““*, were constructed for expression of normal (Gly- 12) and activated (Val-12) human c-Ha-ra.s ~21. respectively. The 0.8.kilobase SmaI fragments of ppS3 and ppS22 containing the entire coding sequences of the c-Ha-ras cDNAs were cloned into the pcDSRol expression plasmid using an EcoRI linker (see Fig. IA). An IL3 promoter (from positions -50 to +lO in the mouse IL3 gene) was synthesized with the XpnI and XbaI cohesive ends and subcloned into the KnnI and XbaI sites of pUC18 to form pUCIL3 (24). SIL3CAT was synthesized by inserting the CAT gene-into the XbaI cut DUCIL~ usine the XbnI linker. SRECAT and CRECAT were synthesized by intioducing a synthetic SRE and three synthetic CREs upstream fro& the IL3 promoter of AILSCAT (Fig. 1D). SREACAT and CREACAT were constructed bv deletion of the IL3 promoter from SRECAT and CRECAT, respectively.

Cell Cultures-PC-12 cells were maintained in DMEM supple- mented with 10% FCS and 5% horse serum. COS7 cells were grown in DMEM supplemented with 10% FCS. PC-12 and COS7 cells were cultured at 37 “C in a humidified atmosphere of 10% CO*.

Transfection of DNA to PC-12 Cells-High efficient transfection to PC-12 cells was carried out as described-by Chen and Okayama (25) with a slight modification. Exponentiallv crowing PC-12 cells (7 x lo5 cells per loo-mm plate) were seeded-in lo-ml of DMEM supplemented with 10% FCS and 5% horse serum 16 h before trans- fection. PC-12 cells were transfected with the plasmid DNA (20 ug) containing pcDSRac-Ha-ras or pcDSRac-Ha-ras’““* (5 bg), the CAT or luciferase construct (5 rg), and pUC18 used as a carrier as indi- cated. One ml of the DNA/Ca-PO4 solution was added to the cells, and the incubation was carried out for an additional 16 h at 35 “C in 3% CO,. The cells were then rinsed with 5 ml of PBS twice and fed with 10 ml of DMEM containing 0.5% FCS and incubated for 48 h. For the last 8-h incubation, various agents were added as indicated in each experiment.

Down-regulation of Protein Kinase C by Treatment with PDBu in PC-12 Cells-PC-12 cells (1 X lo6 cells per 60-mm plate) were seeded and incubated with various doses of PDBu in 5 ml of DMEM containing 0.5% FCS for 40 h. [3H]PDBu binding to the cells was then assayed. In another experiment, PC-12 cells (1 X lo6 cells per 60-mm plate) were seeded and transfected with the CAT constructs and/or the r-Ha-ras constructs by addition of DNA/CaPOI solution. After a 16-h incubation, the cells were rinsed with 5 ml of PBS and incubated in 5 ml of DMEM containing 200 nM PDBu and 0.5% FCS for 40 h at 37°C. The cells were washed with 5 ml of PBS twice and incubated in 5 ml of DMEM containing 0.5% FCS for 8 h with various agents as indicated. The CAT activity was then assayed.

Zmmunoblot Analysis-Exponentially growing COS7 cells (1.5 x lo6 cells per loo-mm plate) were seeded 16 h before transfection. COS7 cells were transfected with the plasmids (10 pg) by the DEAE- dextran method and cultured at 37 “C for 60 h in 10 ml of DMEM supplemented with 10% FCS as described (16, 17). Immunoblot analysis was carried out with the anti-rus ~21 or anti-protein kinase

C monoclonal antibody as described previously (17, 26). CAT and Luciferase Assays-PC-12 cells were cotransfected with

the CAT or luciferase reporter gene and the c-Ha-ras or c-Ha-r~Y*‘12 cDNA. After transfection, the cells were harvested, washed, and resuspended in 0.2 ml of 250 mM Tris/HCl at pH 7.8. The cell lysates were prepared as described by Gorman et al. (27). For the CAT assay, the lvsates (200 aa of protein) were incubated in 0.2 ml of the reaction mix&ire containing 5b nM (i4C]chloramphenicol (0.5 &i) and 2 mM acetvl-CoA in 250 mM Tris/HCl at DH 7.8 for 4 h at 37 “C. CAT activity was assayed by thin’layer chromatography as described (27). For the luciferase assay, the lysates were incubated in 0.4 ml of the reaction mixture containing 1 mM ATP, 3 mM MgSO,, and 0.2 mM luciferin in 10 mM HEPES at pH 7.8 at 37 “C. Luciferase activity was measured by a lumiphotometer (Labo Science, Model TD-4000) (21). Under these conditions, both assays were within the linear range.

rH]PDBu Binding Assay--[3H]PDBu binding to intact cells was assaved as described (17). PC-12 cells were washed three times with 5 mlof PBS and incubated in 2 ml of the binding solution containing 1 mg/ml bovine serum albumin and 10 mM HEPES at pH 7.4 in DMEM with 40 nM [3H]PDBu (0.75 &i) for 30 min at 37 “C. The cells were rinsed three times with 5 ml of ice-cold PBS and scraped into 1 ml of PBS. The radioactivity and the cell number were measured. Nonspecific binding was measured in the presence of 200- fold excess of unlabeled PDBu and was between 5 and 10% of total binding. Nonspecific binding was subtracted to determine specific binding.

“‘I-EGF Binding Assay-The lz51-EGF binding assay was per- formed as described (28) with a slight modification. PC-12 cells (1 X lo6 cells per 60-mm plate) were washed three times with 5 ml of PBS and incubated in 2 ml of the binding solution containing 500 pg/ml iZ51-EGF (0.05 r&i) for 1 h at 37 “C with or without 100 nM TPA. The cells were’ rinsed three times with 5 ml of ice-cold PBS and scraped into 1 ml of PBS. The radioactivity and the cell number were measured. Nonspecific binding was measured in the presence of 2000- fold excess of unlabeled EGF.

Protein Determination-Protein was determined by the method of Bradford (29) with bovine serum albumin as a standard protein.

RESULTS

Expression of the c-Ha-ras Products in COS7 Cells-The c- Ha-rasand c-Ha-ra.sV""2 cDNAs were cloned into the pcDSR& expression plasmid under the control of the SRa promoter which was composed of the SV40 early promoter and the R- U5 segment of human T cell leukemia virus -1 long terminal repeat (Fig. L4) (19). To identify the products of the cloned cDNAs, the c-Ha-ros or c-Ha-ro.sV8112 construct was first trans- fected to COS7 cells since a high level of expression of the c- Ha-ma products was expected in this cell line (19). The extracts from COS7 cells transfected with these constructs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis using the anti-ros p21 monoclonal antibody. Endogenous ras p21 was identified by this antibody in the extracts from the cells transfected with the control plasmid (Fig. 2). Transfection of the c-Ha-rus or c-Ha-r-o.sVa’12 construct resulted in an increase in the product having the molecular mass value of 21 kDa that was similar to the value calculated from the primary structure (18). The molecular mass values of these products were the same as those of endogenous ras ~21. Under the same conditions, the extracts from COS7 celb were analyzed with the anti-protein kinase C monoclonal antibody. The level of protein kinase C was not affected by transfection of the c-Ha-ras or c-Ha-ra.sY8’12 construct (data not shown). These results indicate that the increase in the c-Ha-ras prod- ucts is due to expression of the transfected plasmid and not due to a general increase in cellular ras gene products.

Differentiation of PC-12 Cells into Neuron-like Cells by Transfection with the c-Ha-rasua’12 cDNA-It has been shown that PC-12 cells are differentiated into sympathetic neuron- like cells to produce neurite-like processes in response to NGF or Bt2cAMP or by transfection with the c-Ha-rasV*1’2 cDNA

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FIG. 1. Construction of the plasmids. A, pcDSRac-Ha-ros and pcDSRnc-Ha-ras’““*. The c-Ha-ras and c-Ha-ra9”* cDNAs were cloned into the pcDSRa expression plasmid using an EcoRI linker to yield pcDSRac-Ha-ras and pcDSRac-Ha-ros’““*. B, c-fosCAT. The SstII-NaeI fragment (from positions -404 to +41) of the 5’-flanking sequence of the human c-fos gene was inserted upstream from the CAT gene to yield c-fosCAT. C, c-fos-luciferase. The 445hp SstII- NaeI fragment of the 5’-flanking sequence of the c-{OS gene was cut by Hind111 from c-fosCAT and inserted into pSVO-luciferase to yield c-fos-luciferase. D, SRECAT and CRECAT. A 25bp oligonucleotide of the SRE and three 19-bp oligonucleotides of the CRE were inserted upstream from the mouse IL3 promoter linked to the CAT gene, producing SRECAT and CRECAT.

(30). Consistent with these earlier observations, the wild type PC-12 cells were differentiated into sympathetic neuron-like cells in response to NGF or Bt2cAMP but not to TPA, as judged by their morphology (data not shown). To determine the effect of our c-Ha-ra.s constructs, the c-Ha-rus or c-Ha- rmV8112 construct was then transfected to the wild type PC-12 cells. Three to five percent of the wild type PC-12 cells out of those transfected with the c-Ha-ra.sYB’12 cDNA but not with the c-Ha-ras cDNA sent out neurite-like processes (data not shown). Similar results were obtained when the variant PC- 12 cells described below were used instead of the wild type PC-12 cells (data not shown). These results indicate that our c-Ha-r~“*“2 construct functions as described previously (30) and that transfection efficiency is at least 3-5s in the wild type PC-12 cells.

c-fosCAT and c-fos-luciferase Expression by Transfection of the c-Ha-ra.s”0”2 cDNA-A fusion gene (c-fosCAT) containing 445 bp of the c-fos gene 5’-flanking sequence ligated to the coding sequence of the bacterial enzyme CAT was used as a reporter of transcriptional activity of the c-fos gene enhancer (Fig. 1B). This construct was expressed in the wild type PC- 12 cells, and expression was stimulated by TPA, Bt*cAMP, or NGF (Fig. 3). This finding is consistent with transcrip- tional regulation of the endogenous c-fos gene by TPA, BtzcAMP, and NGF in PC-12 cells (31). Under the same conditions, cotransfection of c-fosCAT with the c-Ha-rcz.9’a’12 cDNA to the wild type PC-12 cells increased CAT activity to the extent similar to that observed when the cells transfected with c-fosCAT were stimulated with TPA, Bt2cAMP, or NGF (Fig. 3). Transfection of the c-Ha-ras cDNA conferred a

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1 2 3 FIG. 2. Expression of the c-Ha-r- and c-Ha-rasYBu2 cDNAs

in COS’7 cells. The extract from COS7 cells transfected with the c- Ha-ros constructs was subjected to sodium dodecyl sulfate-polyacryl- amide gel electrophoresis followed by immunoblot analysis using the anti-ras p21 antibody as described under “Experimental Procedures.” The arrow indicates the position of ros ~21. The protein markers used were carbonic anhydrase (M, = 39,000), soybean trypsin inhib- itor (Af, = 27,000), and lysozyme (M, = 17,000). 1, with the control plasmid; 2, with the c-Ha-ras cDNA; 3, with the c-Ha-rasvs”* cDNA.

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A B C D E F G FIG. 3. Activation of c-fosCAT by cotransfection with the

c-Ha-rasV”12 cDNA in the wild type PC- 12 cells. c-fosCAT was cotransfected with the c-Ha-rus or c-Ha-rosVs”* cDNA to the wild type PC-12 cells as indicated. TPA, BQAMP, and NGF were added to the cells transfected with c-fosCAT alone at the respective final concentrations of 100 nM, 2 mM, and 100 rig/ml 8 h before harvesting the cells as indicated. CAT activity was assayed as described under “Experimental Procedures.” A, mock (without c-fosCAT); B, c- fosCAT alone; C, stimulated with TPA; D, stimulated with Bt2cAMP; E, stimulated with NGF; F, cotransfected with the c-Ha-rasV8”2 cDNA; G, cotransfected with the c-Ha-ras cDNA. Essentially identi- cal results were obtained in three independent experiments. CM and AcCM represent chloramphenicol and its acetylated forms, respec- tively.

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minimal increment of CAT activity. Cotransfection of the c- Ha-ras and c-Ha-raY’s”Z cDNAs showed an additive effect on c-fosCAT expression (data not shown). CAT activity derived from the CAT construct lacking the c-fos 5’-flanking sequence was negligible in the cells cotransfected with the c-Ha-r&a”2 cDNA or supplied with TPA, Bt*cAMP, or NGF (data not shown). Similar observations were obtained in the other con- trol PC-12 cells such as PC-1Zh cells and PC-12m cells (data not shown).

To examine the reporter gene other than the CAT gene, the c-fos gene 5’-flanking sequence ligated to the luciferase gene was employed as another reporter of transcriptional activity of the c-fos gene enhancer (c-fos-luciferase) (Fig. 1C). Furthermore, to standardize the transfection efficiency, TKCAT carrying the thymidine kinase promoter was used as an internal control (22). c-fos-luciferase and TKCAT were cotransfected to the wild type PC-12 cells. Cotransfection of the c-Ha-ra.sVa”2 cDNA or addition of TPA, BttcAMP, or NGF directed c-fos-luciferase expression in the cells trans- fected with c-fos-luciferase and TKCAT (Table I). Cotrans- fection of the c-Ha-ras cDNA conferred a minimal increment of c-fos-luciferase expression. CAT activity derived from TKCAT was almost the same among these cells (Table I). Consistent with the previous observations (lo), these results indicate that c-Ha-ras”B’12 ~21 stimulates the c-fos gene en- hancer linked to the heterologous reporter genes, such as the CAT and luciferase genes, rather than affecting transfection efficiency.

SRECAT Expression by Transfection of the c-Ha-ra.s”a’12 cDNA-The upstream enhancer region of the c-fos gene con- tains two inducible elements, the SRE and CRE (15). To determine which element is responsible for the c-Ha-rosVa’12 p21-induced c-fosCAT expression, the SRE and CRE were synthesized and inserted into the plasmid AIL3CAT that lacked the enhancer elements but contained the intact mouse IL3 promoter region, consisting of the transcriptional initia- tion site and the CAT reporter gene (Fig. 1D). Cotransfection of SRECAT with the c-Ha-ra.sVa112 construct to the wild type PC-12 cells resulted in a marked increase in CAT activity, but cotransfection of CRECAT did not increase CAT activity as shown in Table II. The same results were obtained when the cells were stimulated by TPA or NGF. Bt2cAMP increased CAT activity in the cells transfected with SRECAT and CRECAT. Cotransfection of the c-Ha-rosV8112 construct or addition of TPA, Bt,cAMP, or NGF did not increase CAT activity in the cell transfected with AIL3CAT, SREACAT, or

TABLE I Activation of c-fos-luciferase by cotransfection with the c-Ha-m.s”a’12

cDNA in the wild type PC-12 cells c-fos-luciferase and TKCAT were cotransfected with the c-Ha-ras

or c-Ha-rasVs”2 cDNA to the wild type PC-12 cells as indicated. TPA, BtgcAMP, or NGF was added to the cells trasnfected with c-fos- luciferase and TKCAT alone 8 h before harvesting the cells. Lucif- erase and CAT activities were assayed as described under “Experi- mental Procedures.” Each value for luciferase activity represents the relative light unit measured by a lumiphotometer. Each value for CAT activity represents the percentage of conversion of chloram- phenicol into the acetylated form. The numbers in parentheses indi- cate -fold induction. Essentially identical results were obtained in three independent experiments.

c-fos-iuciferase TKCAT

None 1 0.45 (1.0) + TPA (100 nM) 180 0.50 (1.1) + B&AMP (2 mM) 200 0.50 (1.1) + NGF (100 rig/ml) 200 0.48 (1.1) + c-Ha-ras”“” cDNA 230 0.55 (1.2) + c-Ha-ros cDNA 15 0.45 (1.0)

TABLE II Activation of SRECAT by cotransfection with the c-Ha-ra.s”““2 cDNA

in the wild type PC-12 cells SRECAT or CRECAT was cotransfected with the c-Ha-ras or c-

Ha-ras’““* cDNA to the wild type PC-12 cells as indicated. TPA, BtpcAMP, or NGF was added to the cells transfected with the CAT constructs alone 8 h before harvesting the cells. CAT activity was assayed as described under “Experimental Procedures.” Each value represents the percentage of conversion of chloramphenicol into the acetylated form. The numbers in parentheses indicate -fold induction. Essentially identical results were obtained in three independent ex- periments.

SRECAT CRECAT

None + TPA (100 nM) + Bt2cAMP (2 mM) + NGF (100 rig/ml) + c-Ha-ra.sY8’12 cDNA + c-Ha-ras cDNA

0.1 (1) 0.1 (1) 2.9 (29) 0.1 (1) 3.7 (37) 9.5 (95) 3.9 (39) 0.2 (2) 6.3 (63) 0.2 (2) 0.4 (4) 0.2 (2)

fk 00 0 200 400 0 200 400

PDBu (nM) PDBu (nM) FIG. 4. Decrease of [‘H]PDBu binding to PC-12 cells by

prolonged treatment with PDBu. PC-12 cells pretreated with various doses of PDBu for 40 h were incubated with 40 nM [3H] PDBu. After the 30-min incubation, the amount of cell-associated [3H]PDBu was measured. Detailed experimental conditions are de- scribed under “Experimental Procedures.” Essentially identical re- sults were obtained in three independent experiments. A, in the wild type PC-12 cells; B, in the variant PC-12 cells.

CREACAT (data not shown). SREACAT and CREACAT are the CAT constructs that have the respective enhancer ele- ments but not the IL3 promoter upstream from the CAT gene. Similar observations were obtained when the PC-12h and PC- 12m cells were used instead of the wild type PC-12 cells (data not shown).

Effect of Down-regulation of Protein Kinase C on c-fosCAT Expression-ras p21 has been proposed to control the phos- pholipase C-mediated hydrolysis of phosphoinositides and subsequent protein kinase C activation (4-8). We next ex- amined whether protein kinase C was involved in the c-Ha- rasva’12 p21-induced c-fosCAT expression. It has been shown that prolonged treatment of various cells with PDBu induces down-regulation of protein kinase C (32-34) and that the down-regulation of protein kinase C abolishes c-myc gene expression induced by TPA in Swiss 3T3 cells (35). Treatment of the wild type PC-12 cells with PDBu caused a decrease in protein kinase C activity, which was estimated by measuring [3H]PDBu binding to the cells, as described for other cell types (Fig. 4A) (32-34). The level of this down-regulation was partial. The reason for this partial down-regulation is not

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known, but similar phenomena have been observed in other cell types such as vascular smooth muscle cells (34). In the PDBu-treated cells, TPA did not activate c-fosCAT expres- sion whereas transfection of the c-Ha-rasV8112 cDNA or addi- tion of BtzcAMP or NGF induced c-fosCAT expression to the same extent in both the control and PDBu-treated cells (Table III). Similar observations were obtained when SRECAT was transfected to the PDBu-treated cells (data not shown).

c-fosCAT Expression in the Variant PC-12 Cells-The mu- tant PC-12 cell line, A126-lB2, which is deficient in CAMP- dependent protein kinase II, has been utilized for dissection of the mechanism for CAMP-dependent c-fos gene expression (36). It has been shown that c-fos gene expression induced by forskolin is reduced greatly in this mutant cell line (36). This observation indicates that CAMP-dependent protein kinase II is responsible for c-fos gene expression by the action of forskolin. To take the similar molecular genetic approach for dissection of the mechanism for the c-Ha-rcsVa’12 p21-induced c-fosCAT expression, we isolated a novel variant PC-12 cell line from the wild type PC-12 cell line by the limited dilution method.* In the variant PC-12 cells, c-fosCAT was not acti- vated by TPA (Table IV). Transfection of the c-Ha-rasVB”2 cDNA or addition of Bt*cAMP or NGF to this cell line directed c-fosCAT expression. The normal c-Ha-ras cDNA was less active in this capacity. Similar observations were

TABLE III

Effect of down-regulation of protein kinase C on c-fosCAT expression in the wild type PC-12 cells

c-fosCAT was cotransfected with the c-Ha-ma or c-Ha-ras’s”2 cDNA to the wild type PC-12 cells as indicated. The cells were incubated with or without 200 nM PDBu for 40 h. After washing with PBS, TPA, Bt,cAMP, or NGF was added to the cells transfected with the c-fosCAT alone 8 h before harvesting the cells. CAT activity was measured as described under “Experimental Procedures.” Each value represents the percentage of conversion of chloramphenicol into the acetylated form. The numbers in parentheses indicate -fold induction. Essentially identical results were obtained in three independent ex- periments.

Control cells PDBu-treated cells

None + TPA (100 nM) + BtzcAMP (2 mM) + NGF (100 rig/ml) + c-Ha-rasY8’12 cDNA + c-Ha-ras cDNA

0.1 (1) 0.1 (1) 4.8 (48) 0.5 (5) 6.7 (67) 6.5 (65) 4.5 (45) 4.5 (45) 6.9 (69) 7.0 (70) 0.5 (5) 0.6 (6)

TABLE IV

Effect of down-regulation of protein kinase C on c-fosCAT expression in the variant PC-12 cells

c-fosCAT was cotransfected with the c-Ha-ras or c-Ha-ras’““* cDNA to the variant PC-12 cells as indicated. The cells were incu- bated with or without 200 nM PDBu for 40 h. After washing with PBS, TPA, Bt,cAMP, or NGF was added to the cells transfected with the c-fosCAT alone 8 h before harvesting the cells. CAT activity was measured as described under “Experimental Procedures.” Each value represents the percentage of conversion of chloramphenicol into the acetylated form. The numbers in parentheses indicate -fold induction. Essentially identical results were obtained in three independent ex- periments.

Control cells PDBu-treated cells

None + TPA (100 nM) + B&CAMP (2 mM) + NGF (100 rig/ml) + c-Ha-ros”““* + c-Ha-ras cDNA

0.1 (1) 0.1 (1) 0.15 (1.5) 0.13 (1.3) 7.0 (70) 6.8 (68) 3.5 (35) 3.1 (31) 7.2 (72) 6.7 (67) 0.5 (5) 0.4 (4)

FIG. 5. Effect of TPA on 12’I-EGF binding to PC-12 cells. PC-12 cells were incubated with “‘1-EGF in the presence or absence of 100 nM TPA. After the l-h incubation, the amount of cell- associated “‘1-EGF was determined (hatched rectang.gle). Nonspecific binding was represented as an open rectangle. Detailed experimental conditions are described under “Experimental Procedures.” Essen- tially identical results were obtained in three independent experi- ments. A, in the wild type PC-12 cells; B, in the variant PC-12 cells.

obtained when SRECAT was transfected to the variant PC- 12 cells (data not shown).

The amount of protein kinase C in the variant PC-12 cells, which was estimated by measuring [3H]PDBu-binding to the cells, was almost the same as that in the wild type PC-12 cells (Fig. 4B). Furthermore, prolonged treatment of the variant PC-12 cells with PDBu caused the down-regulation of protein kinase C as observed in the wild type PC-12 cells. Transfec- tion of the c-Ha-rasVB”* cDNA or addition of Bt2cAMP or NGF still stimulated c-fosCAT expression in the PDBu- treated variant PC-12 cells (Table IV). TPA is known to desensitize the EGF receptor to ‘Y-EGF binding in NIH/ 3T3 cells and Swiss 3T3 cells (28, 37). Consistent with these observations, TPA decreased ‘251-EGF binding to both the variant PC-12 cells and the wild type PC-12 cells to a similar extent (Fig. 5). These results indicate that protein kinase C per se functions after stimulation with TPA in the variant PC-12 cells in a manner similar to that in the wild type PC- 12 cells.

DISCUSSION

Activation of the c-fos Gene Enhancer by c-Ha-ras”a’12 p21- We have shown in this paper that TPA, BtzcAMP, and NGF stimulate c-fosCAT and c-fos-luciferase expression in the wild type PC-12 cells. This result is in good agreement with the previous observation that TPA, Bt2cAMP, and NGF activate the endogenous c-fos gene (31). We have also found that transfection of the c-Ha-rasVB’12 cDNA directs c-fosCAT and c-fos-luciferase expression but not TKCAT expression. These results together with the previous observation that the tran- sient induction of c-Ha-rc.sVa”* p21 results in endogenous C-

fos gene expression and c-fosCAT expression in NIH/3T3 cells (10) indicate that c-Ha-rasYa”2 p21 activates the c-fos gene enhancer linked to the heterologous reporter genes rather than stimulates transfection efficiency in the wild type PC-12 cells.

Activation of the SRE by c-Ha-rasua”* p21-The upstream enhancer region of the c-fos gene contains two inducible enhancer elements, the SRE and CRE (15). We have shown

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here that TPA and NGF stimulate the SRE but not the CRE in the wild type PC-12 cells and that BtzcAMP stimulates the SRE as well as the CRE. This notion is consistent with the recent observation that BtzcAMP activates not only the CRE but also the SRE in NIH/3T3 cells (38). Transfection of the c-Ha-rosYB’12 cDNA stimulates the SRE but not the CRE in this cell line. Since this specificity is similar to that of TPA and NGF, it is likely that c-Ha-rusVa’12 ~21, protein kinase C, and NGF activate the SRE in a manner independent of protein kinase A.

The signal transduction mechanism from c-Ha-ru.sVa’12 ~21 to the SRE is unknown, but a SRE-binding protein, SRF, is recently found in many cell types (39, 40). SRF is thought to be essential for transcriptional activation of the SRE during the action of TPA and growth factors (39, 41). We assume that c-Ha-ru.sV8’12 ~21, TPA, Bt2cAMP, and NGF stimulate the SRE through the activation of SRF.

Relationship between Protein Kinase C and c-Ha-rasua”* ~21 -induced Stimulation of the c-fos Gene Enhancer-To de- termine the involvement of protein kinase C in the c-Ha- ra.~“~‘~* p21-induced c-fosCAT expression, we have carried out two types of experiments. In one type of experiments, we have used the PC-12 cells whose protein kinase C is down-regulated by PDBu treatment. In the PDBu-treated cells, TPA does not activate c-fosCAT and SRECAT expression anymore, whereas transfection of the c-Ha-ra.sVall* cDNA or addition of Bt*cAMP or NGF induces c-fosCAT and SRECAT expression to the same extent as that induced in the control cells. In the other type of experiments, we have examined the variant PC- 12 cells. In our variant PC-12 cells, TPA is unable to stimulate c-fosCAT and SRECAT expression, although it directs c- fosCAT and SRECAT expression in the wild type PC-12 cells and the other control PC-12 cells. Under the same conditions, transfection of the c-Ha-rasVan2 cDNA or addition of Bt,cAMP or NGF stimulates c-fosCAT and SRECAT expres- sion in both the variant and wild type PC-12 cells. Since PDBu down-regulates protein kinase C and TPA desensitizes the EGF receptor in the variant PC-12 cells as well as in the wild type PC-12 cells, it is likely that protein kinase C functions in response to phorbol ester even in the variant PC- 12 cells. We assume that the signal transduction pathway from protein kinase C to the SRE is impaired in the variant PC-12 cells. All of these results indicate that c-Ha-rusVa”* p21 as well as NGF activates the SRE in a manner independent of protein kinase C and protein kinase A, and that protein kinase A activates both the SRE and CRE in a manner independent of protein kinase C. Thus, c-Ha-ra.sYan2 ~21, protein kinase C, and protein kinase A appear to transmit signals to their respective enhancer elements in independent pathways.

Mode of Activation of c-Ha-ras ~21 during Stimulation of the c-fos Gene Enhancer-k has been considered that c-Ha- ras ~21 serves as a signal transducer for extracellular signals such as growth factors (1). The extracellular signals for the activation of c-Ha-rczs ~21 have not, however, been deter- mined definitely. NGF as well as c-Ha-rasYa112 ~21 induces neuron-like differentiation of PC-12 cells (30). The NGF- induced PC-12 cell differentiation is inhibited by microinjec- tion of the anti-ras p21 antibody (42). We have shown here that NGF stimulates expression of c-fosCAT and SRECAT but not CRECAT. This specificity is similar to that of c-Ha- rusya”2 ~21. Moreover, we have shown here that NGF as well as c-Ha-rasVsn2 p21 stimulate the expression of the CAT constructs in a manner independent of protein kinase C and protein kinase A. All of these observations indicate that a functional ras ~21 is required for NGF action.

It may be noted that transfection of the c-Ha-ras cDNA activates c-fosCAT expression to a small extent compared with that induced by the c-Ha-rosVB1’* cDNA. This difference may be explained by the assumptions that 1) c-Ha-ras ~21 is active as a signal transducer in the GTP-bound form rather than in the GDP-bound form; 2) ras ~21 is present as the GDP-bound form in the absence of the extracellular signals; 3) the extracellular signals induce the conversion from the GDP-bound form of ras p21 to the GTP-bound form of ras ~21; 4) c-Ha-rasVan2 p21 tends to keep the GTP-bound form because of diminished GTPase activity in intact cells even in the absence of the extracellular signals (1). We are currently investigating how c-Ha-ros p21 is regulated by the extracel- lular signals and how c-Ha-ras p21 stimulates the c-fos gene enhancer.

Acknowledgments-We greatly appreciate the supply of the wild type PC-12 cells from Dr. Y. Kaziro, PC-12h cells from Dr. H. Hatanaka, PC-12m cells from Drs. Y. Teranishi and Y. Matsui, the c-Ha-ras cDNA clones from Dr. F. Tamanoi, the pcDSRa expression plasmid from Drs. K. Arai and Y. Takebe, c-fosCAT from Dr. I. M. Verma, c-fos-luciferase from Drs. K. Arai and M. Muramatsu, TKCAT from Drs. K. Arai and S. Miyatake, and the anti-ras ~21 monoclonal antibody from Dr. H. Shiku. We are grateful to J. Yamaguchi and F. Higuchi for their skillful secretarial assistance.

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