Biochitnlca el Blqohysica dcta, 1134 ( 19921 137-142 1992 Elsevier Science P,,bli+hers B.V+ All rights reserved 0167-48b'9/92/$05.00

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Protein kinase C-independent activation of c-jun and c-los transcription by epidermal growth factor C h r i s t o p h e r C . F r a n k l i n a n d A n d r e w S. K r a f t Division of Hematology / Ontology, Department of Medicine, Uni~,ersityof ,4(utntma in ~irminghwn, 8irm61glmra, AL (USA)

(Received 20 Hovrmbcr 1991)

Key words; Epidermal growth factor; Transcription; Protein kinase C; Pwolo-omaTgcn¢induglio~ ,los: )tin Phorbo[ esters, epidermal growth factor (EGF) and serum indnce the transiem expression of Ihe c-jan and c-fog p r o t o - o n ~ o c s

in quiusccnt fibrublasts. While phorbol esters such as phorbo] 12-myristate 13-acetate (PMA) ate thought to induce the tran.suription of these genes by activating protein kinase C (PKC), the signal transduclion pethwa)qs) mediating the effects of EGF and serum are still unclear. We have investigated whether PKC and/or calcium play a role in mediating EGF-sfimulated c.jun and corot RNA and protein eapression in quiescent N]H 3"i"3fibroblasts. PMA, EGF or serum stimulated a rapid, transknt increase in ¢-jun and c-los expression and c.Jun protein synthesis in quiescent NIH 3T3 cells, i~plction of wbole cell PKC activity by pretrcatment with PMA abolished any subsequent resp~mse to PMA, but had no effect on the ability of EGF or serum to induce e+jnn and c-fos RNA and cJun protein expression. Nuclear run-on analysis indicated that EGF-in~med geoe expression was due to an increase in the rate of transcription of c-jun and c-fog in both naive and PKC-depleled cells, The role of calcium in the EGF-induced expression of c.jnn and c-fog was also investigated using an NIH 3"13 cell line (HER-14) overeapressing the wild type human EGF receptor. Removal of e~tracellular cakium by chelation with excess EGTA or use of the ram-specific calcium chennei blocker lanthanid¢, both of which abolish the EGF-induced calcium transient in HER-14 cells, had no effect on 1he PMA or EGF induced e-jun or c-fog response. These findings suggest that EGF induces e-jun and c-fog transcription and c.lun protein synthesis in a manner independent of an increase in intraceilular calcium or activation of PKC in qrieseent NIH 3"1"3cells,

Intreduetion T h e binding of epidermal growth factor (EGF) to its receptor stimulates the tyrosine phcephoUlation of a number of protein substrates implicated in signal transduction, including phospholigmse C-y, Raf-I kinase, M A P kiaase and the gas-binding protein, G A P [1-7]. However, the mechanism by which EGF stimulates gone transcription and controls cell growth remains unclear. Activation of phospholipas¢ C results in the hydrolysis of phoephatidylinositol-4,5-bisphosphate, generating the second messengers diacyiglycerol and irmsitol trisphosphate, which causes the release of intracellular calcium [8]. Both calcium and diacyl-

glycerol are known to activate protein kinase C [9]. EGF-stimulated protein kinase C activity has been documented in several cell lines [10-12], and the EOF receptor itself is a substrate for protein kinase C-mediated serine/threonine pho~horylation in rive after binding EGF [13-16]. This protein kinase C-mediated phospho~lation of the EGF-recepto¢ results in an attenuation of ligand binding affinity [17] and receptor-associated kinase activity [15,16], suggesting a negative feedback regnlatop/pathway. This cross-talk between the EGF=receptor ~rosine protein kinase and protein kinase C suggests an important role for protein kinase C as a mediator in EGF-receptor signaling. A variety of eatracellular stimuli, including EGF, phorbol esters, cakium ionophore~ platclet-derived

growth factor, and serum, stimulate th¢ transoription Abbreviadoam EGF, epidermal growth factor; PMA, pho~bol t2m~ristate D-acetate; AP. !, activator pt~tein-l: DMEM, Delbeoeo's modified Eagle's medium; PIL~, pho~hate-buffgred safine. Correspondence: A.S. Kraft, Division of Hematolo~/Oneolo~, Department of Medicine, Universityof Alabama in Birmingham,Birm-

ingham,AL 35294, USA.

of the e-jan and c-fox p r o ~ e n e s in quiescent tibroblasts [18-23]. The protein products of these celia* lar pmto--oncogenes, Jen and Fee, combine to form a heterodimeric complex which is capable of binding to the D N A enhancer sequence AP-1 ( 5 ' - T G A C / G T C A Y ) [24-26]. T h e interaction of this heterodimeric complex with the AP-1 binding site en-

138 hances the transcription of specific genes present down-stream of this sequence, including the e-ion and c-los genes themsclves [27-30]. EGF and phorbol esters have been shown to activate c-los transcription through both an AP-l-like site and the serum response element [29,30|, suggesting that EGF may share a similar mechanism of transcriptional activation with photbol esters, namely activation of protein kinase C. This notion is supported by the recent finding that EGF-stimulated induction of the stromelysin gent is dependent on protein kinase C activation [12]. However, EGF has been shown to stimulate the induction of e-fro [31], as well as the proliferation-associated gone VL30 [32], in the absence of cellular protein kinas¢ C. These observations suggest the presence of both protein kinase C dependent and independent pathways of EGF-stimulated gene transcription. Using parental NIH 3T3 cells and cells overexprossing the human EGF receptor (HER-14), we have investigated the role of protein kinase C and calcium in mediating the transcriptional activation of the c.jun and c-los proto-oncogenes. We found that depletion of cellular protein kinas¢ C activity with phorbol esters had no effect on EGF-induced c-ion or e-los expression or d u n protein ~nthesis. Removal of extraccllular calcium or blocking transmembrane calcium fluxes also had no effect on EGF-stimulated induction of e.jun or e-los. These data suggest that protein kinase C and calcium influx do not play a significant role in the induction of c-ion and c-fos transcription and cJun protein synthesis stimulated by EGF. Materials and Methods

Matedals. EGF (receptor grade) was obtained from AMGen (Thousand Oaks, CA). PMA was purchased from Calbiochem (La Julia, CA). All pmteinase inhibitors were from Sigma (St. Louis, MO). All radiolabeled compounds were purchased from Amel~ham (Arlington, IL), except Tran[a~S]-Iabel which was obtained from ICN Radioehemicals (Irvine, CA). Cell culture. Mouse NIH 3T3 cells and HER-14 cells were maintained in Dulbeceo's modified Eagle's medium (DMEM) supplemented with 10% bovine calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 gg/ml streptomycin in a humidified atmosphere of 5% CO~/05% air at 3"ffC. Confluent cultures were made quiescent by washing tb.¢ cells twice with phosphatebuffered saline (PBS) and incubating in DMEM containing 0.5% bovine calf serum for 24-48 h. HER-14 cells were kindly provided by Dr. J. Schlessinger, New York University Medical Center. RNA anal),sis. Confluent, quiescent cultures were treated as indicated, washed three times with ice-cold PBS and lysed in 4 M guanidinium thiocvanate. Total cytoplasmic gNA was isolated by the method of

C~omaezynski and Sacchi [33]. RNA samples (20 ~g/lane) were eleetrnphoresed on a 1.2% agarose/ 5.6% formaldehyde gel, transferred to Duraiose-UV membranes (St ratagene) by diffusion blotting, and fixed by ultraviolet crosslinking. Filters were sequentially probed as previously described [34] with e-ion, c-los, and a-tubulin, labeled to a specific activity of approx. 1 - 10~ cpm//~g by nick translation. Nuclear run.on assay. Nuclear run-on assays were carried out as previously described [22]. Nuclei from two 150 mm dishes were isolated for each treatment [22]. Newly transcribed RNA was ~2P-labeled with [a32P]UTF at 30°C for 30 rain. 32P-labeled nuclei were incubated for 5 rain at 30~C with 600 /zl of DNase I (1,0 mg/ml), and then treated with 200/zl of 5% SDS, 0.5 M Tris-HCI (pH 7.4), 0.125 M EDTA and 10 tzl proteinase K (20 mg/ml). Nuclei were then incubated for 30 rain at 42"C and extracted with 1 mi phenol/ chloroform. RNA was TCA precipitated on ice for 30 rain and filtered through a Millipore type HA filter. Filters were incubated with DNase I for 30 rain at 37~C and RNA eluted with 1% SDS, 10 raM Tris-HO. The RNA was extracted once with phenol/chloroform and once with isoamyl alcohol and preciptated with sodium acetate and ethanol overnight, 10 t~g of the plasmid eDNA indicated was iinearized, denatured and spotted on Duralosc-UV membranes using a slot-blot apparatus and subsequently cross-linked by ultraviolet irradiation. Filters containing eDNA probes were prebybridized for 2 h at 65°C and were hybridized at 65*(: for 40-60 h in 2 ml hybridization buffer containing (2-5)-106 cpm of ~2P-labeled RNA. Filters were washed with 2 × SSC, treated with 10 p.g/ml RNAse A, dried and exposed to X-ray film at -70°C with intensifying screens. Cell labeling and cJun immunopreeipitation. Serumstarved, quiescent HER-14 cells were washed once with Tris-buffered saline and preincabated for 30 rain in MEM lacking L.methionine and L-cysteine. Fresh media containing 100 #Ci/ml Tran[aSS]-label was added and cultures treated as indicated for 2 h. Cells were washed twice with ice-cold PBS and crude nuclei isolated in hypotonie cell lysis buffer as previously described [22]. Crude nuclei were lysed for 30 rain at 4°C in 0.5 ml RIPA buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCI, 1% deoxyeholate, 1% NP-40, 0.1% SDS) containing phosphatase and proteinase inhibitors. Lysates were clarified by centrifugation at 13000 × g for 30 rain and the supernatant preeleared twice with 10 #1 of a 50% slurry of Protein A-sepharese beads. ¢Jun protein was immunoprecipitated with ¢J~m-specific poly¢lonal antisera raised against a pGEX-cJun fusion protein (a gift of Dr. J. Woodgett, Ludwig Cancer Research Institute, London). Immune complexes were collected with Protein A-sepharose beads. The beads were spun through a 1 M sucrose

139 cushion and subsequently washed three times with RIPA, once with 0,5 M LiCI/D. I M Tris-HCl (pH 7.9), once with 10 mM Tris-HCI (pH 7.9) and o:~ce with RIPA. The beads were then boiled for 5 m i , in SDSsample buffer and resolved on 10% SDS gels. Gels were exposed Io EN3HANCE (New England Nuclear, Boston, MA), dried and labele, d proteins visualized by autoradiography at - 7 0 ° C with intensifying screens.

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To examine the induction of c-jun and c-los transcription by pborbol esters, EGF and serum, NIH 31"3 cells were made quiescent by serum-starving confluent cultures for 24-48 h in media comaining 0.5% bovine calf serum. Quiescent cells received either 100 aM PMA or 10 n M EGF by direct addition to the spent media and at the indicated times R N A was isolated and the relative abundance of c-jun, c-fos and a-tubufin R N A assessed by Northern blotting. Untreated quiescent cells contained low but detectable levels of e-/un and andetectable levels of c-fos (Fig. 1). PMA and EGF elicited a rapid transient induction of both c-jun and c-fos. The time course of induction of c-jun was similar fer P M A and E G F with maximal induction w,curing at 2 h and I h, respectively (Fig. 1). PMA and EGF also induced c-los with similar time courses with maximal induction occurring at 30 rain. In both cases c.jun and c.fos R N A levels returned to near control levels within 6 h and 2 h, respectively. The doses used were found to be optimal in stimulating both c.jun and c-fos expression (data not shown). Gel loading for all samples was found to be equal as judged by a-tubul/n R N A signal density.

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quiescent Nil] 3"1"3cells. Confluentcultures were made quiescentby incubationfor 24-48 h in medgacontaining0.5~ bovinecalf serum. Cells were then treated for the time indicated (15, 30 rain, t, 2. 6 h) with either I00 aM PMA ot 10 nM EGF. Cells were ha~¢sted and tolal g'ytopl&.~icRNA iso]ntedand analyzed by Nonhero bloning. The mine filter was sequentially probed with c.j~m~c-fos and atu'bulin cDNA pmhe~as indicated.

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w~-m~t: NT I'MA ~ CS Fig. Z The effect of protein kinase C depletion on PMA, EGF and scram induced e-inn and c-fins~ OM,dluem¢NIH ~13 celh were washed and inoubated foe 24 h in media comtaining0.5% b~:in~ call serum in the pt~a:nce of 1 /~M PMA (+) or 0.1% ethanol ,(-) as a solvem coAtml. Cells then received either no treatment (NT) or were treated for 30 rain with 100 nM PMA, I0 nM EGF or 20% calf serum (C~L added freshly to the conditioned media. Total RNA was isolatedand alta[~ for c-junaml c-losby Nc4rtbemblotting as indicated.

Phorbol csters such as P M A cause the translucation of protein kinase C from the cytosol to the cell membrane [35]. Long term treatment with PMA, however, leads to the depletion of total cellulm protein kinase C activity [36,37]. To determine whether protein kinase C plays a role in mediating EGF-sfimulatcd c-jun and c-los expression, NIH 3T3 ~ l i s were depleted o f protein kinas¢ C activity by in'olcmged treatnmnt with PMA. Quiescent cells were pretreated with P M A for 24 h and then treated with either 100 nM PMA, l0 nM E G F or 20% serum ~or 30 rain. As in control cells, cells that wore pretreated with PMA for 24 h exin"oited undetectahle levels of c - f ~ message, however, c-j'un was slightly elevated over that seen in untreated cells (Fig. 2). While the P M A respome was abolished in protein kinase C-depleted cells, F_.~F a n d serum were fully capable of stimulating the induction of e-jun and c-los expression (Fig. 2). c-~m levels were not maximally induced by P M A or E O F within 30 rain (Fig. 1), however, this time point was used to maximize the induction of c-fos and enable us to simultaneously measure both e-jun and ¢-f¢¢" levels. The addition of E G F to naive and protein kinase C down-regulated eeUs could i n , ease the level of c-jun and c.fos R N A by either stabilizing their transcripts or by stimulating an increase in the rate of their transcription. To evaluate these possibilities, nuclear run-on analysis was performed using isolated nuclei from qui-

140 pretreat

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FiS. 3. Nuclear run-on analysis of c-jun and c-f us lrenscription. Nuclei were isolat~l from scrum-starvod NIH 3T3 cells that had received no treatment (NT) or i ~M PMA for 24 h and then either not treated (NT) or treated with 10 aM EGF for 30 rain. s2P-labeled RNA transcripts ((2-5l. 10~ clan) were hybridized to t0/~8 of c-jan, c-los and OAPDH eDNA which had been liocarized and blotted otto Duraiose-UV filters.

ascent cells p r c t r c a t e d for 24 h with e i t h e r l p M P M A or 0.1% ethanol as a solvent comrol. E G F s t i m u l a t e d a m a r k e d increase in the rate of c-jan and c-los transcription in both naive and protein kinase C-depleted cells (Fig. 3). The constitutive t a l e of e-jan transcription was e l e v a t e d approx. 3-fold in P M A - p r e t r e a t e d cells, suggesting t h a t a peat'transcriptional control m e c h a n i s m may account for the reduction in steady state levels of c-jan upon prolonged P M A t r e a t m e n t as m e a s u r e d by Northern blotting. T h e s e transcriptional effects are gene specific as P M A and E G F had no effect on glyceraldehyde-3-phosphate d c h y d r o g e n a s e ( G A P D H ) ~ a n s c r i p t i o n (Fig, 3). To investigate the role o f protein kinase C in the regulation of c,Iun protein levels we utilized a n N I H 3T3 cell line, HER-14, which is stably transfected with the wild type h u m a n E G F receptor (300000 r e c e p t o r s / cell) [1]. Q u i e s c e n t cells were metabolically labeled with ~ S - l a b e l e d amino acids a n d t r e a t e d with e i t h e r P M A , E G F or s e r u m for 2 h. Ceils w e r e then lysed, crude nuclei isolated a n d cJun protein levels analyzed by immtmoprecipitation. Each of these agents stimulated an increase in cJun protein levels relative to u n t r e a t e d cells (Fig. 4). D e p l e t i o n of protein kinase C by P M A p r e t r e a t m e n t p r e v e n t e d additional PMA-induced cJun protein synthesis, b u t had no effect on E G F - or s c r a m - i n d u c e d cJun protein synthesis (Fig. 4). E G F stimulates a rapid transient rise in both inositel p h o s p h a t e production and intracellular calcium k v -

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Fig. 4. Effect of PMA, EGF and serum on cJun protein synthesis in naive and PKC~ep~eted HER-I4 cells. Serum-starved HER-14 cells were pretreated In 1he absence or presence of ] ,aM PMA for 24 h and then metabolically labeled with Tran[~S]-lab¢l end treated with either 100 nM PMA. 10 nM EGF or 20% calf serum for 2 h. Crude nuclei were isolated and cJun immunoprecipitates prepared as described in Materials and Method.. Labeled proteins were resolved by SDS-polyacrylamidegel electrophoresis, flu0rographed and exposed to X-ray Fdm at -70% with intensifying screens. els in several cell types [38-43]. This increase in intracellular caicium appears to be dependent on the presence o f extracel]ular calcium [38-43]. Removal o f extraceilular calcium by chelation with excess E G T A or p r e t r e a t m e n t with non-specific calcium channel antagonists such as L a 3+ o r M n 2+ blocks the calcium transient induced by E G F in both m o u s e [38,421 and r a t fibroblasts [40,41], a n d A431 e p i d e r m o i d cells [39]. T o d e t e r m i n e w h e t h e r a n increase in intracellular calcium is necessary for E G F - s t i m u l a t e d c-jan and c-los transcription, we have utilized the H E R - 1 4 ceil line which has previously b e e n shown to exhibit a calcium t r a n . slant in response to physiological levels of E G F [43]. T r e a t m e n t of H E R - 1 4 cells w i t h P M A or E G F stimulated c-jan a n d c-los expression with kinetics identical to those observed in p a r e n t a l N I H 3T3 cells ( d a t a not shown). To examine the role of calcium influx in E G F stimulated c-jun and c-los expression, H E R - I 4 ceLls Z~S. : c-j',n 188-

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Fig. 5. "£herole of calcium influx in PMA- and EGF-stimnlated c.jun and c-los expression in cells overexpressins the haman EGF receptor, Confluent quiescent HER-14 ¢¢11swere pretreated f~r tO rain in the absence ( - ) or presence of either 100 uM I.~CIs (La~+ ) oz 3 mM EGTA priox to exposure to t00 nM PMA (P) or l0 nM EGF (El for 30 rain. Total cel|ular RNA was then isolated and analyzed by Nor thex~,blottius as indicated.

14I were preincubatcd with either 100 g M LaCI 3 or 3 mM EGTA, which lowers extracellutar free calcium concentrations to < 1 ~M and abolishes the EGF-induced calcium response [43]. Both treatments appeared to have no effect on the induction of c-jun or c-fos expression in response to either PMA or EGF (Fig. 5). Discussion

EGF increases intracellular ino~[tol phosphate and calcium levels and stimulate.~ protein kina,~ C activity in several cell lines, including Rat-1 fibroblasts [12], PCI2 cells [10], and B A L B / M K keratin0cyles [11]. Since activation of protein kinase C is apparently required for EGF-stimulated stromelysin gene expression [12], and both EGF and PMA stimulate c-los gene expression through similar enhancer sequences present in the c-los promoter ~29,30], protein kinase C may play a role in mediating EGF-induced gene transcription. To determine whether protein kinasc C is involved in the transcriptional effects induced by EGF, cells were depleted of protein kinase C by prolonged treatment with PMA. Sbort-term ( < 2 h) treatment of ceils with PMA drastically reduces EGF receptor hum+ her and affinity and attenuates EGF-stimulated increases in inositol phosphate and calcium levels [17,38,39]. After prolonged treatment with PMA, how* ever, EGF receptor number and affinity return to normal levels [17]. Treatment of quiescent NIH 31"3 cells with PMA for 24 h resulted in a depletion of whole cell protein kinase C activity to 10% of control, but had no effect on EGF receptor number (data not shown). EGF was still able to induce both c-jun and c-los e~ression in protein kinase C down-regulated ceils. Similar results have been observed in Rat.2 ft. broblasts [12]. Nuclear run-on anal/sis was performed to determine whether the effects of EGF on c-jun and e-los RNA accumulation was secondary to prolonged messenger haft.life or to transcriptional activation. Both EGF and PMA were found to stimulate the rate of e-jun and c-los transcription, CeLls pretreated with PMA for 24 h also exhibit an enhanced rate of e-jun transcription. This is in contrast to results obtained from Northern blot analysis in which comparable levels of e-jun mRNA were detected in both untreated cells and ceils treated with PMA for 24 h. These results indicate that PMA-indu~d changes in c:jH RNA Icy. els are regulated both transcriptionally and p0sttranscfiptionally. Even though ~ e constitutive level of c-jun t~ption was elevated in PMA.pret~eated cells, EGF was found to stimulate the rate of transcription of c-jun and c-los in both naive and protein kinase C

Protein kinase C-independent activation of c-jun and c-fos transcription by epidermal growth factor.

Phorbol esters, epidermal growth factor (EGF) and serum induce the transient expression of the c-jun and c-fos proto-oncogenes in quiescent fibroblast...
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