Negative regulatory role of overexpression of ... - The FASEB Journal

Negative regulatory role of overexpression of PLC␥1 in the expression of early growth response 1 gene in rat 3Y1 fibroblasts SOON YOUNG SHIN,*,† JESANG KO,‡ JONG-SOO CHANG,§ DO SIK MIN,储 CHAN CHOI,¶ SUN SIK BAE,†† MYUNG JONG KIM,†† DAE SUNG HYUN,* JUNG-HYE KIM,* MI YOUNG HAN,‡‡ YOUNG-HO KIM,† YONG SIK KIM,§§ DOE SUN NA,** PANN-GHILL SUH,†† AND YOUNG HAN LEE*,1 *Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, Korea 705–717; †Department of Microbiology, Kyungpook National University, Daegu, Korea 702–701; ‡Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul, Korea 138 –736; §Department of Biology, Daejin University, Pochon-gun, Kyeonggido, Korea 487– 800; 储Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul, Korea 137–701; ¶Chonnam National University Research Institute of Medical Sciences, Kwangju, Korea 501–746; ††Department of Life Science, Pohang University of Science and Technology, Pohang, Korea 790 –784; ‡‡Green Cross Institute of Medical Genetics, Seoul, Korea 135–260; §§Department of Psychiatry, Clinical Research Institute, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea 151-742; and **Department of Biochemistry, University of Ulsan College of Medicine, Seoul, Korea 138 –736, The early growth response 1 (Egr-1) gene product is a transcription factor that functions as an oikis factor. Loss of Egr-1 expression is closely associated with tumor formation. Phospholipase C␥1 (PLC␥1) is overexpressed in some tumors, and its overexpression causes anchorage-independent growth. Here we report that overexpression of PLC␥1 and SH2-SH3 domain of PLC␥1 decreased induction of Egr-1 and the Egr-1-regulated genes TSP-1 and PAI-1. Results from the nuclear run-on assay and transfection experiment with the proximal 455 base pair region of the Egr-1 promoter (ⴚ454 to ⴙ1) showed that Egr-1 transcriptional activity was suppressed in PLC␥1–3Y1 cells whereas decay of Egr-1 mRNA was similar in both cell lines. Serum response element- and ternary complex factor Elk-1-mediated transcriptional activation of the reporter gene in response to EGF were also inhibited in PLC␥1–3Y1 cells. Pretreatment with the protein synthesis inhibitor cycloheximide (CHX) partially abrogated the serum-induced suppression of Egr-1 transcription in PLC␥1–3Y1 cells, suggesting that a CHXsensitive factor(s) is involved in the suppression of Egr-1 transcription in PLC␥1–3Y1 cells. Our results demonstrated that overexpression of PLC␥1 functions as a negative modulator of the tumor suppressor Egr-1 gene expression, possibly through inhibition of Elk-1-dependent transcriptional activity.—Shin, S. Y., Ko, J., Chang, J.-S., Min, D. S., Choi, C., Bae, S. S., Kim, M. J., Hyun, D. S., Kim, J.-H., Han, M. Y., Kim, Y.-H., Kim, Y. S., Na, D. S., Suh, P.-G., Lee, Y. H. Negative regulatory role of overexpression of PLC␥1 in the expression of early growth response 1 gene in rat 3Y1 fibroblasts. FASEB J. 16, 1504 –1514 (2002) ABSTRACT

Key Words: Egr-1 䡠 tumor suppressor 䡠 PLC␥1 overexpression 1504

The early growth response 1 (Egr-1) gene, also known as NGFI-A, zif268, krox 24, and Tis8, is a transcription factor that has three Cys2-His2 type zinc finger-containing DNA binding domains in the carboxyl-terminal portion of the molecule (1–5). Egr-1 protein preferentially binds to GC-rich regulatory elements with the consensus sequence of GCGGGGGCG or TCCTCCTCCTCC (5), leading to induction or repression of its target genes. Several groups have reported that Egr-1 is rapidly and transiently induced by many growth factors, cytokines, cellular stress, and differentiation signals, suggesting a key regulatory role of Egr-1 in cell growth, differentiation, and development (6, 7). The variety of functions of Egr-1 suggests that regulation of expression level of this gene plays an important role in regulation of important cellular processes. It has been hypothesized that down-regulation of the Egr-1 gene is closely associated with tumor formation. 1) Egr-1 is poorly or not expressed in some tumor cells (8 –10). 2) Stable exogenous expression of Egr-1 inhibits cell proliferation and soft agar growth in v-sis-transformed NIH3T3 as well as tumor cells including fibrosarcoma HT1080 cells, breast carcinoma ZR-75–1, glioblastoma U251, osteosarcoma Saos-2 (8, 11). 3) Expression of the Egr-1 gene into HT1080 cells inhibits cell growth and tumorigenicity by induction of TGF-␤1, fibronectin, and plasminogen activator inhibitor 1, which are important in the growth control and stabilization of extracellular matrix proteins (12, 13). 4) 1 Correspondence: Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, 317–1, Daemyung-Dong, Nam-Gu, Daegu 705–717, South Korea. E-mail: [email protected]

0892-6638/02/0016-1504 © FASEB

Conversely, expression of antisense Egr-1 mRNA in v-sis-transformed NIH3T3 cells leads to an enhanced transformed phenotype (11). These results support the notion that functional loss of Egr-1 may contribute to tumorigenic potential. Despite progress in identifying and characterizing the signaling that positively regulate the Egr-1 gene expression, much less is known about negative regulatory mechanism responsible for loss of Egr-1 expression. It has been reported that PLC␥1 is overexpressed in some human hyperproliferative tissues, including breast carcinoma (14), human skin under hyperproliferative conditions (15), colorectal carcinoma (16), familial adenomatous polyposis (17), and highly metastatic colorectal tumor cell lines (18). Overexpression of PLC␥1 causes anchorage-independent growth in soft agar and induces tumors after injection into nude mice (19, 20). These results suggest that abnormal expression of PLC␥1 may be associated with tumor development in some tumors, yet a detailed role of PLC␥1 overexpression has not been fully elucidated. To assess the signaling mechanism for altered expression of Egr-1, we investigated the effect of PLC␥1 overexpression on Egr-1 expression in 3Y1 fibroblasts. Our results show that both Egr-1 mRNA and protein levels are suppressed in PLC␥1 overexpressed 3Y1 fibroblasts, suggesting that overexpression of PLC␥1 functions as a negative regulator of tumor suppressor Egr-1 gene in 3Y1 fibroblasts.

MATERIALS AND METHODS Cell lines

␤-galactosidase activity were purchased from Promega (Madison, WI). Western blot analysis Cells were lysed in 20 mM HEPES, pH 7.2, 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 ␮g/mL leupeptin, and 1 mM PMSF. Protein samples (20 ␮g of each) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. Blots were incubated with anti-Egr-1 antibody (Santa Cruz, Santa Cruz, CA, 1:1,000) and developed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech., Piscataway, NJ). The same blot was stripped by incubating in 62.5 mM Tris, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol for 30 min at 50°C and reprobed with anti-Erk1/2 (Santa Cruz, 1:2,000) antibody for an internal control. Northern blot analysis Ten micrograms of total RNA was separated on 1.2% agarose gel containing 6% formaldehyde in 0.02 M MOPS, pH 7.0, 8 mM sodium acetate, and 1 mM EDTA, then transferred to Hybond N⫹ nylon membrane (Amersham Pharmacia Biotech.) by the standard capillary method. Cross-linking was performed by UV irradiation. Egr-1 (1.4 kb long EcoRI fragment purified from the pGEM/TIS8), c-jun (2.5 kb long EcoRI-HindIII fragment from the pBS/c-jun), and GAPDH (0.5 kb XbaI-HindIII fragment from the pUC/GAPDH) probes were labeled with [␣-32P]dCTP (DuPont NEN, 6000 Ci/mmol) by random primer method (Roche Molecular Biochemicals). After prehybridization, blots were hybridized overnight at 42°C in Northern-Max hybridization solution (Ambion, Austin, TX). Blots were washed with 2⫻ SSC/0.1% SDS for 20 min at room temperature, 2⫻ SSC/0.1% SDS at 42°C for 30 min, and 0.5⫻ SSC/0.1% SDS for 30 min at 52°C. For rehybridization, the probes were stripped from the membrane by boiling in 0.1⫻ SSC/0.5% SDS.

3Y1 rat fibroblasts transfected with vector alone (Vector-3Y1), PLC␥1 (PLC␥1–3Y1), SH2-SH2-SH3 domain of PLC␥1 (SH223–3Y1), and v-src (Src-3Y1) were prepared as described previously (19) and maintained in low-glucose Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 100 ␮g/mL hygromycin B (Life Technologies, Gaithersburg, MD). For stimulation, cells were serum-starved in low-glucose DMEM containing 0.5% FBS for 24 h before addition of 20% FBS, phorbol 12-myristate 13-acetate (PMA) (Calbiochem, San Diego, CA), or epidermal growth factor (EGF) (Calbiochem, San Diego, CA) in the absence or presence of various inhibitors.

mRNA stability assay

Plasmids and reagents

Cells from two 150 mm culture dishes were washed twice with ice-cold PBS and lysed with gentle vortexing in buffer A (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40). Nuclei were pelleted at 500 ⫻ g for 5 min; the nuclear pellets were resuspended in 175 ␮L of buffer B (40% glycerol, 10 Mm Tris, pH 7.5, 5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA) and stored in liquid nitrogen. To start nuclear transcription, thawed nuclei were mixed with 45 ␮L of reaction buffer (5 mM dithiothreitol, 1 mM each of ATP, GTP, CTP, 100 ␮Ci of [␣-32P]UTP (NEN, 3000 Ci/mmol) and incubated at 30°C for 30 min with shaking. The reaction was stopped by the addition of 50 units of DNase I (RNase-free) and 10 ␮L of 20 mM CaCl2, followed by incubation at 37°C for 10 min. The samples were subsequently treated with 1 ␮L of 20 mg/mL

The Egr-1 promoter reporter construction p-454egrLuc (by inserting 455 base pair region (from ⫺454 to ⫹1) of the human Egr-1 gene into a pGL2-basic luciferase plasmid) has been described (21). pSRE-Luc cis-acting reporter vector, which contains five repeats of serum response element (SRE); trans -activator plasmid pFA2-Elk1, which encodes the fusion protein and consists of the activation domain of Elk-1 (residues 307– 427) with the yeast GAL4 DNA binding domain (residues 1–147); the reporter plasmid pFR-Luc, which contains five repeats of GAL4 binding elements; and the luciferase gene were obtained from Stratagene (La Jolla, CA). The pCMV/␤-gal plasmid and assay kits for luciferase and SUPPRESSION OF Egr-1 BY PLC␥1

Serum-starved Vector- and PLC␥1–3Y1 cells (with 0.5% serum for 24 h) were treated with 20% serum for 30 min, then actinomycin D (10 ␮g/mL) was added to block RNA synthesis. Immediately after addition of actinomycin D, total RNA was prepared and Northern blot analysis was performed as described. The relative band intensities of Egr-1 and GAPDH mRNA at each time point were quantitated by scanning densitometer and image analysis software (Bio-1D version 97.04). Nuclear run-on assay

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proteinase K, 25 ␮L SET buffer (5% SDS, 50 mM EDTA, 100 mM Tris, pH, 7.5), and 2.5 ␮L of 20 mg/mL yeast tRNA. After additional incubation at 37°C for 30 min, 1 mL of TRIzol (Life Technologies) was added for RNA isolation according to the manufacturer’s protocol. Purified 32P-labeled RNA was hybridized to Hybond nylon membrane with immobilized spots containing 1 ␮g of Egr-1, 0.5 ␮g of GAPDH, or 1 ␮g of c-jun cDNA inserts. The hybridization was carried out at 42°C for 2 days and the membranes were washed in 2⫻ SSC/0.1% SDS at 65°C for 1 h and in 0.5⫻ SSC/0.1% SDS at 37°C for 30 min before exposure to X-ray film. Luciferase reporter assay For Egr-1 promoter analysis in Vector- and PLC␥1–3Y1 cells, cells cultured on 35 mm dishes were cotransfected 0.5 ␮g of ⫺454 to ⫹1 region of the human Egr-1 gene (p-454egrLuc) with 0.2 ␮g of pCMV/␤-gal plasmid using LipofectAMINE 2000 reagents according to the manufacturer’s instructions. For analysis of SRE-mediated cis-acting transcriptional activity, 0.5 ␮g of pSRE-Luc were cotransfected with 0.2 ␮g of pCMV/␤-gal plasmid. For Elk-1-mediated trans-acting transcriptional activity, 50 ng of pFA2-Elk1 plus 0.5 ␮g of pFR-Luc was cotransfected with 0.2 ␮g of pCMV/␤-gal plasmid (0.5 ␮g). The total amount of DNA was maintained at 1 ␮g with an empty vector pCDNA3.0. Cells were treated with 100 ng/mL of EGF 24 h after transfection. Cells were harvested after 6 to 8 h of EGF treatment and protein extracts were prepared by three cycles of freezing and thawing. One to 5 ␮g of protein was assayed for luciferase and ␤-galactosidase activities. Luminescence was measured using a luminometer model TD 2020 (Berthold, Tubingen, Germany). Transfection efficiencies were normalized by a ratio of luciferase activity to ␤-galactosidase activity obtained from the same sample.

RESULTS Egr-1 is down-regulated in human mammary carcinoma cell lines Egr-1 is down-regulated in rat and human mammary tumor cells, and loss of Egr-1 expression correlates well with transformed growth and tumorigenicity (9). Since PLC␥1 is overexpressed in some human cancer tissues, including colon and breast cancers (14, 16), the role of PLC␥1 in regulating expression of the Egr-1 gene was examined. We confirmed the relative expression of Egr-1 and PLC␥1 in human breast cancer cell lines. The protein level of Egr-1 after stimulation with either serum or PMA in serum-starved cells was examined to demonstrate induction of Egr-1 in response to extracellular stimuli in breast cancer cell lines (Fig. 1A). After exposure to X-ray film, the relative intensities of Egr-1 and Erk-1/2 bands were determined using a quantitative scanning densitometer. Egr-1 was strongly expressed due to stimulation by PMA (Fig. 1A, lane 2) and serum (Fig. 1A, lane 3) in the normal immortalized mammary cell line MCF-10A, as reported by Huang et al. (9). In contrast, the two human mammary carcinoma cell lines, MCF-MDA365 and MCF-MDA231, showed poor responses to serum compared with the normal mammary cell line (Fig. 1A, lane 3 vs. lanes 6 and 9). The serum-induced Egr-1 level decreased by 1506

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Figure 1. Down-regulation of Egr-1 in human mammary carcinoma cells and PLC␥1–3Y1 cells. A) Cells were serumstarved with 0.5% serum for 24 h, then treated with 20% serum or 50 nM PMA for 2 h. Proteins were extracted, separated on 10% SDS-polyacrylamide gels (20 ␮g/lane), and transferred to nitrocellulose membranes. PLC␥1 protein levels were detected by Western blotting using monoclonal PLC␥1 antibody, F7–2 (top panel). Same blot was stripped and reprobed with polyclonal anti-Egr-1 (middle panel) or anti-Erk1/2 antibody as an internal control (bottom panel). Relative expression of Egr-1 was determined by densitometry. Values are expressed as relative intensities against serum in treated MCF10A cells (lane 3). B) Serum-starved 3Y1 fibroblasts transfected with pREFA vector only (Vector) or with rat PLC␥1 cDNA-inserted pREFA (clones 1, 4, and 7) were treated with 20% serum for 2 h. Proteins were extracted and separated on 10% SDS-polyacrylamide gels (20 ␮g/lane). Immunoblotting was performed as described in panel A.

35% in MDA365 cells and by 66% in MDA231 cells compared with that in nontransformed MCF10A cells. However, normal and carcinoma cell lines both showed similar response to PMA (Fig. 1A, lanes 2, 5, and 8). PLC␥1 was highly expressed in breast cancer cells compared with normal mammary cells (Fig. 1A, lanes 1–3 vs. lanes 4 –9). Reprobing the blots with anti-Erk1/2 antibody revealed similar amounts of proteins in all lanes. Our results demonstrate that PLC␥1 levels are up-regulated whereas serum-induced Egr-1 protein is down-regulated in tumor cells compared to nontransformed cells. Egr-1 expression in response to serum stimulation is decreased by overexpression of PLC␥1 The Egr-1 protein level in 3Y1 fibroblast transfectants that stably overexpress PLC␥1 (PLC␥1–3Y1) (19) was

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investigated in order to determine whether a decrease of Egr-1 expression in response to serum in tumor cells is associated with overexpression of PLC␥1. Three different PLC␥1–3Y1 clones were used to eliminate the possibility of random gene aberrations during the transfection procedure. As shown in Fig. 1B, the seruminduced Egr-1 protein level was significantly decreased after stimulation with serum in PLC␥1–3Y1 cells compared with empty vector-transfected cells (Vector-3Y1), indicating that overexpression of PLC␥1 is sufficient to cause a decrease in the level of Egr-1 in response to serum stimulation. Time course of Egr-1 induction after serum or EGF stimulation The time kinetics of Egr-1 expression were examined to exclude the possibility that overexpression of PLC␥1 alters the time of Egr-1 induction (Fig. 2). Egr-1 protein levels gradually increased in Vector-3Y1 cells; they peaked 2 h after serum stimulation (Fig. 2A) and 1 h after EGF stimulation (Fig. 2B). Egr-1 levels declined thereafter. A notable decline in the Egr-1 level was observed in PLC␥1–3Y1 cells whereas Erk1/2 and EGF receptor levels were comparable in the two cell lines. Northern blot analysis was performed to examine whether the decrease in Egr-1 expression occurs at the mRNA level. Egr-1 mRNA was rapidly induced within 15 min, with a maximum level observed 30 min after serum stimulation in Vector-3Y1 cells (Fig. 2C). In contrast, Egr-1 mRNA was barely detected in PLC␥1– 3Y1 cells. After detaching, the same blot was hybridized with c-jun and GAPDH probes as an internal control. The induction of c-jun mRNA was comparable between the two cell lines whereas GAPDH was clearly induced to a greater degree in PLC␥1–3Y1 cells. Results from EMSA revealed that the DNA binding activity of Egr-1 was suppressed in PLC␥1–3Y1 cells compared with Vector-3Y1 cells (data not shown). These results demonstrate that inhibition of Egr-1 expression in response to mitogenic stimuli is a specific event in PLC␥1–3Y1 cells. Taken together, these data indicated that suppressed expression of Egr-1 is an early event that is dependent on overexpression of PLC␥1. Expression of Egr-1-dependent genes is suppressed by overexpression of PLC␥1

Figure 2. Time-dependent changes of Egr-1 expression after serum or EGF stimulation. Vector-3Y1 cells (Vector) and PLC␥1–3Y1 cells (PLC␥1) were serum-starved with 0.5% for 24 h, then treated with 20% serum (A, C) or 100 ng/mL EGF (B) for the times indicated. A, B) Western blot analysis. Whole cell lysates (20 ␮g/lane) were analyzed by Western blotting with rabbit anti-Egr-1 (A, B, top panel), anti-Erk1/2 (A, lower panel; B, middle panel) or anti-EGF receptor antibodies (B, bottom panel). C) Northern blot analysis. Total RNAs were isolated from cells, electrophoresed on 1% agarose gels (5 ␮g/lane), capillary transferred to nylon membranes, and subjected to Northern blotting. The blot was hybridized with the 32P-labeled Egr-1 probe (top panel). The same blot was stripped and reprobed sequentially with 32P-labeled c-jun (middle panel) and GAPDH (bottom panel) probes.

Previous studies have demonstrated that Egr-1 preferentially binds the GC-rich regulatory elements of the thrombospondin 1 (TSP-1) and transforming growth factor ␤1 (TGF-␤1) promoters and causes trans-activation of these genes (22–24). TGF-␤1 upregulates expression of the plasminogen activator inhibitor 1 (PAI-1) in an autocrine loop (10). Since the expression level and DNA binding activity of Egr-1 were suppressed in PLC␥1–3Y1 cells, we investigated whether Egr-1-dependent genes can be influenced in these cells. Cells were serum-starved and treated with 20% serum before the mRNA levels of

TSP-1 and PAI-1 were examined by Northern blotting (Fig. 3). PAI-1 and TSP-1 mRNA levels were both significantly increased by 6 h after stimulation with serum in Vector-3Y1 cells. In contrast, there were no apparent increases in the levels of either PAI-1 or TSP-1 mRNA whereas GAPDH mRNA levels were highly expressed in PLC␥1–3Y1 cells. These findings indicate that overexpression of PLC␥1 decreased mRNA levels of the Egr-1-dependent genes TSP-1 and PAI-1, confirming a role for PLC␥1 in negative regulation of Egr-1 expression.

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essary for suppression of serum-induced Egr-1 expression in 3Y1 fibroblasts. Effects of actinomycin D and cycloheximide on serum-induced Egr-1 expression

Figure 3. Suppression of Egr-1-regulated genes in response to serum stimulation in PLC␥1–3Y1 cells. Vector-3Y1 cells (Vector) and PLC␥1–3Y1 cells (PLC␥1) were serum-starved with 0.5% for 24 h, then treated with 20% serum for the times indicated. Total RNAs were isolated from cells, electrophoresed on 1% agarose gels (10 ␮g/lane), capillary transferred to nylon membranes, and subjected to Northern blotting. The blot was hybridized with the 32P-labeled TSP-1 probe (top panel). The same blot was stripped and reprobed sequentially with 32P-labeled PAI-1 (middle panel) and GAPDH (bottom panel) probes.

Serum-induced Egr-1 expression was tested for dependence on RNA and protein synthesis. Cells were pretreated with either the transcription inhibitor actinomycin D (ActD) or the protein synthesis inhibitor cycloheximide (CHX) for 2 h before serum stimulation. Treatment with ActD alone or ActD plus serum completely blocked accumulation of Egr-1 mRNA in both cell lines (Fig. 5A, lanes 3– 4 and 9 –10), indicating that serum-induced Egr-1 expression is transcriptionally regulated. In contrast, CHX alone increased the level of Egr-1 mRNA (Fig. 5A, lanes 5 and 11) and CHX plus serum yielded a pronounced superinduction of Egr-1 mRNA in both cell types (Fig. 5A, compare lanes 2 and 8 with 6 and 12). Pretreatment of cells with CHX

Overexpression of the SH2-SH2-SH3 domain of PLC␥1 inhibits serum-induced Egr-1 expression The SH2-SH2-SH3 domain of PLC␥1 resides between the X and Y domain catalytic regions and is involved in various cellular functions including propagation of mitogenic signals independent of PLC␥1 catalytic activity (19, 25–27). Since activation of PKC and Ca2⫹/ CaM/CaMK-II signaling pathway did not contribute to inhibition of Egr-1 expression in PLC␥1–3Y1 cells (data not shown), we next examined whether the SH2-SH3 domain of PLC␥1 is involved in regulation of Egr-1 expression using 3Y1 cells overexpressed the SH2-SH2SH3 domain of PLC␥1 (SH223–3Y1). We used v-srctransformed 3Y1 cells (Src-3Y1) as a positive control cell line because expression of TSP-1 is repressed in v-srctransformed fibroblasts (28). Accumulation of Egr-1 mRNA peaked after 30 min of serum stimulation in Vector-3Y1 cells and gradually decreased thereafter (Fig. 4A), similar to the result shown in Fig. 2C. Egr-1 induction was compared with TSP-1, an immediateearly gene that is directly regulated by Egr-1. The TSP-1 mRNA level gradually increased through the first 120 min of stimulation, then gradually decreased for up to 12 h (Fig. 4A, lanes 9 and 13). Maximum TSP-1 induction was slightly later than Egr-1 induction (2 vs. 0.5 h) and return to the basal level was also delayed. Egr-1 and TSP-1 mRNAs were strongly suppressed in PLC␥1–3Y1, SH223–3Y1, and Src-3Y1 cells vs. Vector3Y1 cells. In accordance with the observed decrease of Egr-1 mRNAs, Egr-1 proteins were barely detectable by Western blotting in PLC␥1–3Y1, Src-3Y1, and SH223– 3Y1 cells (Fig. 4B). These results are consistent with the hypothesis that an aberrant elevation of the PLC␥1 level is associated with inhibition of Egr-1 expression. The SH2-SH2-SH3 domain of PLC␥1 is probably nec1508

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Figure 4. Inhibition of Egr-1 and TSP-1 expression in response to serum in SH223–3Y1 cells. Vector-3Y1 (Vector), PLC␥1– 3Y1 (PLC␥1), SH223–3Y1 (SH223), and Src-3Y1 (Src) cells were serum-starved with 0.5% for 24 h, then treated with 20% serum for the times indicated. A) Northern blot analysis. Total RNAs were isolated from cells, electrophoresed on 1% agarose gels (5 ␮g/lane), capillary transferred to nylon membranes, and subjected to Northern blotting. The blot was hybridized with the 32P-labeled Egr-1 probe. After washing, the same blot was reprobed with 32P-labeled TSP-1 probe without stripping (upper panel), then GAPDH probe (lower panel). B) Western blot analysis. Whole cell lysates (20 ␮g/lane) were analyzed by Western blotting with rabbit anti-Egr-1 antibody (upper panel). The same blot was stripped and reprobed with rabbit anti-Erk1/2 antibody as an internal control (lower panel). Arrow indicates the positions of Egr-1 protein.

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Figure 5. Effects of serum and CHX on Egr-1 mRNA expression and stability. A) Steady-state level of Egr-1 mRNA. Vector-3Y1 (Vector) and PLC␥1–3Y1 (PLC␥1) cells were serum-starved with 0.5% serum for 24 h, preincubated with or without 10 ␮g/mL ActD or 10 ␮g/mL CHX for 2 h, then either untreated or treated with 20% serum. After additional incubation for 30 min, total RNAs were isolated from cells and subjected to Northern blotting as described in Fig. 2C. B) Egr-1 mRNA stability assay. Serum-starved cells were treated with 20% serum for 30 min, followed by addition of 10 ␮g/mL ActD at time 0. C) Effect of CHX on Egr-1 mRNA stability. Serum-starved cells were pretreated with 10 ␮g/mL CHX for 1 h, treated with 20% serum for 30 min, followed by addition of 10 ␮g/mL ActD at time 0. At the indicated time intervals, total RNAs were isolated and Northern blot analysis was performed. Graph shows the remaining Egr-1 mRNA quantitated by scanning densitometry after normalized to corresponding amounts of GAPDH mRNA. Error bars represent the mean ⫾ sd of 3 independent experiments.

partially abrogated inhibition of serum-induced Egr-1 mRNA expression by overexpression of PLC␥1 but did not affect mRNA expression of other immediate-early genes, like c-jun (Fig. 5A, lanes 6 and 12). The half-life of Egr-1 mRNA was measured in ActDtreated cells to determine whether acceleration of mRNA decay is involved in the decrease of the Egr-1 mRNA level in PLC␥1–3Y1 cells. Serum-starved cells were first treated with 20% serum for 30 min, then RNA synthesis was stopped by addition of ActD (10 ␮g/mL). Egr-1 mRNA levels were measured from the time of ActD addition for up to 240 min. Despite different starting levels the half-life value in Vector-3Y1 cells was not significantly different from the value obtained in PLC␥1–3Y1 cells; the half-life was 52 ⫾ 5 min and 45 ⫾ 10 min (n⫽3) in Vector-3Y1 and PLC␥1–3Y1 cells, respectively (Fig. 5B). To test whether short-lived new protein synthesis affects Egr-1 mRNA stability in PLC␥1–3Y1 cells, cells were pretreated with CHX for 1 h and 20% serum was added to culture media for 30 min before addition of ActD. Egr-1 mRNA levels were measured from the time of ActD addition up to 6 h. As shown in Fig. 5C, CHX profoundly stabilized the serum-induced Egr-1 mRNA to 3 ⫾ 0.5 h in Vector-3Y1 cells and to 2.5 ⫾ 0.5 h in PLC␥1–3Y1 cells (Fig. 5C), indicating that a postulated CHX-sensitive factor promotes decay of serum-induced Egr-1 mRNA, but seems unlikely to be involved in the down-regulation of Egr-1 in PLC␥1– 3Y1 cells. SUPPRESSION OF Egr-1 BY PLC␥1

Suppression of Egr-1 induction in PLC␥1–3Y1 cells is controlled at the transcription level To investigate whether overexpression of PLC␥1 can influence transcription of the Egr-1 gene, a nuclear run-on assay was performed using purified nuclei from Vector-3Y1 and PLC␥1–3Y1 cells treated with serum. After treatment with 20% serum for 30 min, nuclei were isolated from Vector-3Y1 and PLC␥1–3Y1 cells and in vitro transcription was performed. Northern dot-blot analysis was performed using a plasmid containing the full-length cDNA probe of either Egr-1 or GAPDH. Densitometric analysis indicated that transcription rate of the Egr-1 gene increased 9.1 ⫾ 0.7-fold (n⫽3) for Vector-3Y1 and 4.5 ⫾ 0.4-fold (n⫽3) for PLC␥1–3Y1 cells due to serum stimulation (Fig. 6A). In contrast, transcriptional activity of the GAPDH gene in PLC␥1– 3Y1 cells more than doubled. These results indicate that differences in steady-state mRNA levels between Vector-3Y1 and PLC␥1–3Y1 cells are due at least in part to differential transcriptional activity of the Egr-1 gene. Inhibition of protein synthesis induces transcriptional activation of the Egr-1 gene in Rat1 fibroblasts, suggesting that fibroblasts contain a labile protein that actively represses basal transcription of the Egr-1 gene (28). Based on this finding, it is possible that a CHXsensitive factor(s) control important steps for Egr-1 transcription in PLC␥1–3Y1 cells. To explore this possibility, we examined the effect of CHX on the transcription rate of Egr-1 gene using a nuclear run-on assay. Because c-jun mRNA levels induced by serum 1509

PLC␥1–3Y1 cells compared to Vector-3Y1 cells and that CHX partially overcomes the inhibition of serum-induced Egr-1 transcription in PLC␥1–3Y1 cells. This result suggests that ongoing protein synthesis is probably involved in the suppression of serum-induced Egr-1 expression in PLC␥1–3Y1 cells. Overexpression of PLC␥1 regulates Egr-1 promoter activity through inhibition of Elk-1-dependent trans-activation

Figure 6. Effects of serum and CHX on relative transcription rates of Egr-1 gene. A) Vector-3Y1 (Vector) and PLC␥1–3Y1 (PLC␥1) cells were serum-starved with 0.5% for 24 h, and treated with 20% serum for 30 min. B) Vector and PLC␥1 cells were serum-starved with 0.5% serum for 24 h, preincubated with 10 ␮g/mL CHX for 1 h, then treated with 20% serum. Nuclei were prepared and performed transcriptional run-on assay. [32P]UTP-labeled RNAs were isolated and hybridized to plasmid DNA (1 ␮g/dot) containing Egr-1, GAPDH (A) or c-jun (B) cDNA insert. Upper blots show a representative of 3 independent experiments. Autoradiograms were scanned using a densitometer, and relative transcription rates are expressed as an arbitrary unit (lower graph). Error bars represent the mean ⫾ sd of 3 independent experiments.

were similar between Vector-3Y1 and PLC␥1–3Y1 cells (Fig. 2C), densitometry values were normalized to c-jun in Vector-3Y1 cells. The Egr-1 transcription rate in Vector-3Y1 cells consistently exhibited an ⬃twofold greater increase than in PLC␥1–3Y1 cells 30 min after serum stimulation (Fig. 6B). Treatment of cells with serum induced a 4.7 ⫾ 0.6-fold (n⫽3) increase in Egr-1 transcription over control levels in Vector-3Y1 cells and a 2 ⫾ 0.2-fold (n⫽3) increase in PLC␥1–3Y1 cells. CHX alone resulted in an increase of a 3.2 ⫾ 0.6-fold (n⫽3) in Vector-3Y1 cells and a 2.2 ⫾ 0.5-fold (n⫽3) in PLC␥1–3Y1 cells. Serum stimulation in the presence of CHX resulted in an 8.5 ⫾ 0.7-fold (n⫽3) increase in Vector-3Y1 cells and a 5 ⫾ 0.8-fold (n⫽3) increase in PLC␥1–3Y1 cells (Fig. 6B). It was shown that CHX weakly stimulates the Egr-1 transcriptional activation in 1510

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Expression of the Egr-1 gene in response to several mitogenic stimuli and to the oncogenes v-src and v-raf is mediated primarily through the SRE in the 5⬘-flanking region of the gene (29, 30). The ⫺454 to ⫹ 1 region of the human Egr-1 gene (p-454egrLuc) containing five clusters of the SRE, one cAMP response element (CRE), and the TATA motif (21) was transfected into Vector-3Y1 and PLC␥1–3Y1 cells in order to investigate the Egr-1 transcriptional activity by overexpression of PLC␥1. Increase in luciferase activity normalized for ␤-galactosidase activity were observed. Treatment with EGF resulted in a 2.9 ⫾ 0.3-fold and 1.3 ⫾ 0.2-fold (n⫽3) increase in luciferase activity in Vector-3Y1 and PLC␥1–3Y1 cells, respectively (Fig. 7A), confirming that transcriptional activity of the Egr-1 gene is down-regulated in PLC␥1–3Y1 cells. SRE is composed of two binding sites: a serum response factor (SRF) site for the SRF dimer, and a ternary complex factor (TCF) site that binds Ets family transcription factors SAP1, SAP2, and Elk-1 (32). Once activated, the two transcription factors (SRF and TCF) form ternary complexes on the SRE and mediate transcription activation of Egr-1. We investigated whether protein binding to the SRE is associated with suppression of Egr-1 gene transcriptional activity in PLC␥1–3Y1 cells. The effect of EGF on SRE-dependent transcription was assessed using the pSRE-Luc plasmid containing five tandem repeats of the SRE linked to a minimal promoter. Treatment with EGF resulted in a 2.4 ⫾ 0.3-fold (n⫽3) increase of SRE-dependent transcription in Vector-3Y1 cells. In contrast, there was no augmentation of the EGF response in PLC␥1–3Y1 cells (Fig. 7B). To determine whether Elk-1 transcriptional activity in the SRE can be altered in PLC␥1–3Y1 cells, a fusion construct of the Gal4 DNA binding domain and the activation domain of Elk-1 (pFA2/GAL4dbd-Elk1) was transfected with a luciferase reporter gene containing five repeats of a GAL4 binding site (pFR/GAL4Luc) This assay system allows direct assessment of Elk-1-mediated transcriptional activation in response to different stimuli. When cells were treated with EGF, Elk-1-dependent trans-activation increased ⬃fourfold in luciferase reporter activity in Vector-3Y1 cells (Fig. 7C). Little activating effect was observed in PLC␥1–3Y1 cells. Our results indicate that overexpression of PLC␥1 regulates Egr-1 expression at the transcription level through inhibition of Elk-1-dependent trans-activation.

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Figure 7. Inhibition of Egr-1 promoter activity, SRE-dependent cis-acting and Elk-1-mediated trans-acting transcription activities. A) Egr-1 promoter activity. Vector-3Y1 and PLC␥1– 3Y1 cells were transfected with either 0.5 ␮g p-454egrLuc (A), 0.5 ␮g cis-acting pSRE-Luc (B), or 50 ng trans-activator pFA2-Elk1 with 0.5 ␮g pFR-Luc reporter plasmid (C). The pCMV/␤-gal plasmid (0.2 ␮g) was included as an internal control for normalization of transfection efficiency. After 24 h of transfection, cells were serum-starved with 0.5% serum for 18 –24 h and treated with 100 ng/mL EGF or not. After 6 – 8 h of EGF treatment, luciferase and ␤-galactosidase assays were performed. The activities of luciferase/␤-galactosidase of untreated cells (NT) were set arbitrarily as 1. Error bars represent the mean ⫾ sd of a single experiment performed in triplicate. Similar results were observed in 3 independent experiments.

DISCUSSION In this study, we present evidence demonstrating that the tumor suppressor Egr-1 is a cellular target of the PLC␥1 signaling pathway. Egr-1 expression is profoundly down-regulated at the transcriptional level in PLC␥1 overexpressed 3Y1 cells. SUPPRESSION OF Egr-1 BY PLC␥1

Serum-induced Egr-1 protein is down-regulated in human mammary carcinoma cell lines compared to nontransformed mammary epithelial cells whereas the PLC␥1 level is clearly elevated in carcinoma cell lines (Fig. 1A). Since the PLC␥1 and Egr-1 levels were inversely correlated in the examined samples, we investigated whether ectopic expression of PLC␥1 can regulate Egr-1 expression. Induction of Egr-1 and its target gene in response to mitogenic signals, including serum and EGF, was significantly suppressed in PLC␥1–3Y1 cells (Figs. 2 and 3). Therefore, Egr-1 is probably negatively regulated by ectopic expression of PLC␥1. Since overexpression of PLC␥1 in 3Y1 fibroblasts appears to inhibit Egr-1 expression, abnormal elevation of PLC␥1 expression probably contributes to the observed down-regulation of Egr-1 in breast tumors cells (Fig. 1A). Our results demonstrating a negative regulatory role for overexpression of PLC␥1 in the inhibition of Egr-1 expression initially appear paradoxical. PLC␥1 in PLC␥1⫺/⫺ cells is necessary for maximum expression of many PDGF-induced immediate-early response genes, including Egr-1 and c-fos (33). It is possible that constitutive long-term expression of a high oncogenic level of PLC␥1 in transformed cells leads to inactivation of the tumor suppressor Egr-1 in a negative feedback regulation mechanism. Several lines of evidence support this hypothesis. Chen et al. (34) reported that expression of oncogenic-Ras negatively regulates calcium-dependent c-fos and Egr-1 induction in lymphocytes. Serum-induced Egr-1 induction is suppressed in mutated N-Ras (Gln61 to Lys61)-expressing human fibrosarcoma HT1080 cells whereas transient expression of the dominant active mutant of Ha-Ras (Gln61 to Leu61 stimulates Egr-1 promoter activity (20). We found that the Egr-1 protein is not induced by serum and EGF stimulation in dominant active Ras (Gly12 to Val12)-transformed Rat1 fibroblasts (data not shown). Slack and Bornstein (28) demonstrated that overexpression of v-src results in activation of transcriptional induction of TSP-1. However, the TSP-1 mRNA level was dramatically decreased in Rat1 fibroblasts chronically transformed by v-src. All these results indicate that a much more complex mechanism controls regulation of Egr-1 gene expression between nontransformed and chronically transformed cells. We cannot eliminate the possibility of an indirect adaptive response in the endogenous regulation of the Egr-1 gene during long-term expression of PLC␥1 rather than a direct effect of PLC␥1 overexpression. PLC␥1 propagates mitogenic signals independent of its catalytic activity through the SH2-SH2-SH3 domain (19, 25–27). The SH2-SH2-SH3 domain of PLC␥1 resides between two catalytic regions, the X and Y domain, and this exhibits a striking similarity to the oncogenic adaptor proteins Nck and Crk (35). Microinjection of either a catalytically inactive mutant of PLC␥1 (36) or a noncatalytical module of PLC␥1, SH2-SH2-SH3 domains (26, 27) into quiescent NIH3T3 cells triggers mitogenesis. Transplantation of 3Y1 fibro1511

blasts overexpressing the SH2-SH2-SH3 domains of PLC␥1 results in solid tumor formation in nude mice (19). Our data showed that mRNA levels of both Egr-1 and its regulated gene TSP-1 are decreased in SH223– 3Y1 cells (Fig. 4), indicating that the SH2-SH3 domain of PLC␥1 is sufficient for inhibition of Egr-1 expression. Since the SH2-SH3 domain mediates protein–protein association, further work is required for identification of the SH2-SH3 domain interacting protein factors involved in regulation of Egr-1 gene expression. Inhibition of Egr-1 expression by overexpression of PLC␥1 can be regulated at transcriptional inactivation and destabilization of mRNA levels. A nuclear run-on assay and a promoter reporter assay both indicated that the serum-induced transcription activity of the Egr-1 gene is strongly inhibited in PLC␥1–3Y1 cells (Figs. 6A and 7A). However, decay of Egr-1 mRNA in PLC␥1–3Y1 cells is nearly the same as in Vector-3Y1 cells (Fig. 5B). These observations indicate that suppression of Egr-1 mRNA expression in PLC␥1–3Y1 cells is controlled at the transcriptional level, but unlikely at the post-transcriptional level. Slack and Bornstein (28) have reported that transcriptional activation of the Egr-1 gene is induced by inhibition of protein synthesis in Rat1 fibroblasts, suggesting that fibroblasts contain a labile protein that actively represses basal transcription of the Egr-1 gene. We also observed that CHX alone was able to stimulate the Egr-1 transcription in both Vector-3Y1 and PLC␥1– 3Y1 cells; however, the effect of CHX on the activation of Egr-1 transcription was strongly suppressed in PLC␥1–3Y1 cells compared to Vector-3Y1 cells (Fig. 6B). Pretreatment of CHX partially abrogated suppression of serum-induced Egr-1 transcription in PLC␥1– 3Y1 cells (Fig. 6B). A widely accepted interpretation of this result is that a labile transcription repressor exists and this labile repressor would maintain immediateearly response genes in an inactive state. In this model, protein synthesis inhibitors block this short-lived repressor, causing superinduction of immediate-early response genes like c-fos (37, 38). Thus, although it appears that the mechanism of Egr-1 mRNA superinduction by CHX plus serum in PLC␥1–3Y1 cells (Fig. 5A) involves both an improved mRNA stability (Fig. 5B) and an increase in the transcription rate (Fig. 6B), the postulated CHX-sensitive labile factor would play a role in negative regulation of Egr-1 gene transcription by acting as a downstream effector or target gene of PLC␥1. Alternatively, it is possible that CHX leads to transcriptional activation directly or indirectly by stimulation of intracellular signaling pathways similar to those activated by growth factors independent of the ability to inhibit protein synthesis (39), which is inhibited by downstream effector of PLC␥1 at the transcriptional level. The Egr-1 promoter contains several putative regulatory elements, including SP1 binding sites, five clusters of the SRE, and two putative CRE (40). The SRE cluster is implicated in transcriptional activation of Egr-1 in response to various growth factor stimulations (41– 44). 1512

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Egr-1 SRE includes a CArG box that binds the SRF and an Ets motif that binds a TCF family member (44). The TCF family includes Elk-1, Sap-1, and Sap-2/Net/Erp (45). The Ras/MEK/Erk cascade is responsible for phosphorylation of Elk-1, and Elk-1 phosphorylation by Erk correlates with increased transcriptional activation of the Egr-1 gene (46). The results presented here demonstrated that EGF-induced transcriptional activity of a ⫺454 to ⫹1 Egr-1 promoter fragment was inhibited by overexpression of PLC␥1. This region contains five functional SREs and one CRE (21). The CRE located between ⫺57 and ⫺76 is required for induction of Egr-1 in response to GM-CSF (47) but is not functional in response to either EGF or growth hormone (41). Thus, it is possible that overexpression of PLC␥1 suppresses Egr-1 transcription through modulation of Elk-1 activity or binding to Ets motif in the SRE on the Egr-1 promoter, probably via SH2-SH3 domain interacting protein factors or CHX-sensitive factor(s). Further study is necessary to determine whether CHX-sensitive factor(s) is a target of PLC␥1 or binding protein to SH2-SH3 domain of PLC␥1 and whether this factor is associated with loss of Egr-1 expression in other tumor cells. Additional insight into how Elk-1 is regulated by overexpression of PLC␥1 can be achieved. In conclusion, overexpression of PLC␥1 negatively regulates Egr-1 expression possibly through modulation of Elk-1 transcription activity. An understanding of the mechanism of inhibition of Egr-1 expression in PLC␥1-overexpressing cells will help us to understand tumor suppressor Egr-1 gene regulation in transformed cells and the role of PLC␥1 overexpression in tumor development. This study was supported by grants from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (HMP-97-B-3– 0024), and the Ministry of Science and Technology (M10016000021– 01A190002100).

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Received for publication January 4, 2002. Revised for publication May 29, 2002.

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