Thrombin Aivation of the 9E3/CEF4 Chemokine Involves TyrosineKinases Including c-src and the Epidermal Growth Factor Receptor*

(Received for publication, March 21, 1997, and in revised form, November 14, 1997)

Sucheta M. Vaingankar and Manuela Martins-Green‡

From the Department of Biology, University of California, Riverside, California 92521

The 9E3/CEF4 gene codes for a chemokine that ishighly homologous to human interleukin-8 and mela-noma growth-stimulating activity/groa. These chemo-kines belong to a family of molecular mediators that areimportantly involved in inflammation, wound healing,tumor development, and viral entry into cells. On thechorioallantoic membrane the 9E3 protein is chemotac-tic for monocyte/macrophages and lymphocytes and isangiogenic. In cultured chicken embryo fibroblasts,which have many of the properties of wound fibroblasts,the gene is stimulated by a variety of agents includingoncogenes, growth factors, phorbol esters, and throm-bin. The strong stimulation of 9E3 by thrombin in cul-ture correlates well with the observation that in youngchicks this gene is stimulated to very high levels infibroblasts upon wounding and remains high through-out wound repair. Activation of 9E3 by thrombin: (i)occurs very rapidly, one minute exposure to thrombin issufficient to initiate the signals necessary for gene acti-vation; (ii) is independent of mitogenesis; (iii) operatesthrough the proteolytically activated receptor forthrombin; (iv) is mediated by tyrosine kinases, includingc-src and the epidermal growth factor (EGF) receptor,rather than Ser/Thr kinases such as protein kinase Cand protein kinase A. Inhibition of either c-src or theEGF receptor tyrosine kinase inhibits the stimulation of9E3 by thrombin. We show here for the first time thatactivation of the EGF receptor through a cell-surfacereceptor that does not have tyrosine kinase activity canlead to expression of an immediate early response genewhich encodes for a secreted protein, a chemokine. Thisrapidly activated tyrosine kinase pathway may be a gen-eral stress response by which in vivo a localized cellpopulation reacts to emergency situations such as viralinfection, wounding, or tumor growth.

The avian gene 9E3/CEF4 encodes a secreted protein that isa member of the chemokine superfamily (1–4). Of all the mem-bers of this superfamily, the 9E3 protein shows the highesthomology to interleukin-8 and also is highly homologous toMGSA1/groa. This gene is constitutively overexpressed inchicken embryo fibroblasts (CEFs) transformed by Rous sar-

coma virus, but its expression in normal quiescent cells istightly regulated (5, 6); serum stimulation of normal CEFsresults in transient expression of 9E3 as a consequence of entryinto the cell cycle (7). In addition to serum, a wide variety ofoncogenes, growth factors, and inflammatory agents also stim-ulate 9E3 expression in normal quiescent CEFs (5, 6, 8, 9). Invivo, the 9E3 gene is expressed at very low levels in connectivetissue, tendon, and bone, but it is highly expressed in theendothelial cells of young blood vessels (10–12). Furthermore,this gene is rapidly overexpressed upon wounding, remainsvery highly elevated during the inflammatory phase of healing,and 36 h after wounding declines to a plateau of elevatedexpression that remains constant throughout granulation tis-sue formation (10). The 9E3 protein is chemotactic for mono-cyte/macrophages and lymphocytes (12, 13); the high levels ofexpression shortly after wounding could be responsible for thisfunction. In addition, this protein is angiogenic in vivo (12),which might occur in response to the steady expression of the9E3 gene during granulation tissue formation. The observa-tions in vivo and in vitro combined with the biochemical prop-erties of the molecule (14) indicate that this gene could play animportant role in the inflammatory response and healing ofwounds.

We have found that of the growth factors, inflammatoryagents, and other molecules released upon wounding, thrombinis the most potent natural activator of 9E3 expression (14, 15).Thrombin is a serine protease that is generated at sites ofvascular injury and is known to regulate hemostasis andthrombosis via its serine protease activity by inducing plateletaggregation and clot formation. However, many other actionsof thrombin are independent of its thrombogenic activity andsuggest that this protease is also important directly or indi-rectly during the inflammatory response and in the prolifera-tive phase of wound healing (16–19). In these situations,thrombin is known to be chemotactic for monocytes and mito-genic for lymphocytes, fibroblasts, and smooth muscle cells(20–23). Furthermore, thrombin stimulates endothelial cells toexpress the neutrophil adhesion protein GMP-140 (24–26) andto produce platelet-derived growth factor that stimulatessmooth muscle cells to grow (27, 28).

Most of our understanding of signal transduction pathwaysturned on by thrombin comes from work with platelets (29, 30),Chinese hamster lung fibroblasts (31, 32), and endothelial cells(33). In these systems, upon binding to its seven transmem-brane domain receptor, thrombin cleaves part of the N termi-nus of the receptor, exposing a new peptide that then binds toone of the external loops of the receptor (34). This proteolyti-cally activated receptor for thrombin causes activation of Gi orGq,11 that turn on mitogenic and nonmitogenic events (18). Gi

inhibits adenylyl cyclase and cAMP and reduces the levels ofPKA (35), whereas Gq,11 activates phosphatidylinositol turn-over, PKC, Na1/H1 antiport, and induces c-fos and c-myc (19,36–38). In platelets, the Gq,11 can also activate phospholipase

*This work was supported in part by NIGMS, National Institutes ofHealth Grant GM48436 (to M. M.-G.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed. Tel.: 909-787-2585;Fax: 909-787-4509; E-mail: mmgreen@ucrac1.ucr.edu.

1 The abbreviations used are: MGSA, melanoma growth-stimulatingactivity; CEF, chicken embryo fibroblast; EGF, epidermal growth fac-tor; IP3, inositol trisphosphate; OAG, 1-oleoyl-2-acetyl-glycerol; PKA,protein kinase A; PKC, protein kinase C; qcCEF, quiescent confluentCEF; tCEF, transformed CEF; TGFa, transforming growth factor a;TRDP, thrombin receptor-derived peptide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 9, Issue of February 27, pp. 5226–5234, 1998© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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A2 with the subsequent generation of arachidonate-derivedmetabolites (29). These lipids, known as eicosanoids, includeprostaglandins, thromboxanes, and leukotrienes, which are of-ten involved in the inflammatory process. More recently, a newproteinase-activated receptor 2 was identified that has 30%homology to proteolytically activated receptor for thrombin butfor which the signaling events are not yet worked out (39).

The signaling events activated by thrombin, which are de-scribed above, are mediated by Ser/Thr kinases stimulatedshortly after receptor activation. However, more recently tyro-sine phosphorylation has also been implicated in the signalingevents triggered upon thrombin receptor activation. It has beenshown that tyrosine phosphorylation in platelets occurs inde-pendently of the activation of PKC (40) and that cells that donot undergo mitogenesis in response to thrombin stimulationrequire tyrosine kinase signals initiated by EGF and platelet-derived growth factor (41) to trigger cell division. In growth-responsive Chinese hamster fibroblasts, thrombin activates thec-src and fyn tyrosine kinases, which provide a link betweenthe thrombin receptor and the downstream events leading toactivation of ras and the mitogenic kinase cascade (42).

It is now abundantly clear that thrombin is a multifunctionalmolecule. The fact that thrombin and chemokines are impor-tant in the inflammatory response and granulation tissue for-mation, and the demonstration that thrombin stimulates theexpression of chemokines such as MGSA/groa (43, 44), 9E3 (14,15, 45), and interleukin-8 (46) may indicate that these chemo-kines could be the mediators of some of thrombin’s effects onhealing. Identification and understanding of the signal trans-duction pathways generated by thrombin leading to activationof chemokines can give insight into ways of manipulating thefunction of this latter class of molecules. Because the 9E3 geneis stimulated to high levels shortly after wounding and becausethrombin is the most potent natural activator of this gene, weare investigating the signal transduction mechanisms by whichthrombin activates 9E3 expression. To perform these studieswe use primary cell cultures of CEFs. This culture systemallows us to make inferences to the situation in vivo becausethe properties of the fibroblasts in wounded tissue and ingranulation tissue resemble those of embryonic fibroblasts (47).Our results show that the Ser/Thr kinases PKC and PKA arenot directly involved in the stimulation of this gene by throm-bin, but instead we find that tyrosine phosphorylation is a keyevent that occurs when CEFs are activated by thrombin toproduce 9E3. We show here that the EGF receptor and c-src aretwo of the tyrosine kinases importantly involved in thisprocess.

MATERIALS AND METHODS

Reagents—Concentrations of activators and inhibitors of signaltransduction cited here were selected for efficacy from a wider range ofconcentrations initially tested. A23187 (1 mM), arachidonic acid (100mM), and bovine thrombin (9 units/ml) were obtained from Sigma.Calphostin C (200 nM), cholera toxin (100 ng/ml), genistein (1–25 mM),H-7 (200 nM), HA 1004-HCl (20 mM), H89 (100 nM), herbimycin A (1 mM),ionomycin (1 ng/ml), lavendustin A analogue RG 14355 (100–200 nM),pertussis toxin (100 ng/ml), staurosporine (200 nM), and tyrphostinAG1478 (500 nM) came from Calbiochem. 1-Oleoyl-2-acetyl-glycerol(300 mM) was obtained from Serdary Research Laboratories, London,Ontario, and human recombinant TGFa (10 ng/ml) and EGF (10 and100 ng/ml) were obtained from Intergen. 100% Me2SO was used as asolvent when appropriate. Anti-phosphotyrosine PY20 was purchasedfrom Transduction Labs and 4G10 from Upstate Biotechnology Inc.ECL reagents and antibodies conjugated to horseradish peroxidasewere obtained from Amersham. TRDP (200 mM) is a thrombin receptor-derived peptide with the following sequence: H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe-OH (Bachem).

Cell Culture—Primary cultures of CEFs were prepared from 10-day-old chicken embryos as described previously (48). On the fourth day,secondary cultures were prepared by trypsinizing and plating the pri-

mary cells in 199 medium containing 0.3% tryptose phosphate brothand 2% donor calf serum at a density of 1.2 3 106/60-mm plate. Trans-formed CEFs (tCEF) were obtained by infecting these cells with Roussarcoma virus XD-2 (an engineered form of Schmidt-Ruppin strain A)as described previously (49). To study the effects of thrombin on 9E3expression and the effects of known activators and inhibitors of signaltransduction, we used quiescent confluent CEF (qcCEF) cultures andincubated them in serum-free 199 medium containing the specific treat-ment at various concentrations and for varying times. After the initialexperiments, the inhibitors and activators were used at the dosesshown above and incubated for 1 h; thrombin was added for anotherhour and then the cells were washed and incubated in serum-freemedium for up to 18 h. At the end of the incubation period the super-natant was collected, protease inhibitors were added, and cell debriswas removed by centrifugation. Because qcCEFs are primary cellsrather than a cell line, there are small variations in the basal levels of9E3 expression from batch to batch of cells. Therefore, for each exper-iment and/or treatment of a different batch of cells we used internalpositive (thrombin-treated cells) and negative (untreated cells) controls.

Western Blotting—Volumes of the cell culture supernatant corre-sponding to equal amounts of protein in the cells extracts were loadedon 20% polyacrylamide-glycerol gels and electrophoresed at 16 mA forabout 4 h. The concentration of the protein was determined using theBio-Rad DC-protein kit. Transfer was performed using a semidry trans-fer apparatus (Millipore). After transfer, to check for evenness of pro-tein loading, the upper part of the gel was cut and stained with silverstaining for a protein that is ;35 kDa. This protein is present in thesupernatant of qcCEFs, and its concentration does not vary with treat-ment of the cells. The efficiency and evenness of transfer of the 9E3protein was monitored by silver staining the gel after the transfer. The9E3 protein was detected using polyclonal antibodies raised in rabbit(14) and enhanced chemiluminescence (ECL) reagents (Amersham).

Northern Blot Analysis—CEFs were homogenized and total RNA wasprepared using TRIzolTM reagent (Life Technologies, Inc.). RNA sam-ples (20 mg each) were denatured in formamide-formaldehyde buffercontaining ethidium bromide and separated on formaldehyde-agarosegel. After electrophoresis, the RNA was transferred to MagnaGraphnylon membrane (MSI Inc.) that were photographed to visualize thequality of the rRNA and confirm equal loading and even transfer. TheRNA was UV cross-linked to the membrane for 2 min and baked at60 °C for 2 h. Prehybridization was performed for 6–9 h, and hybrid-ization was carried out for 36 h following the procedures described inRef. 10. Microdensitometry analysis was performed by laser densito-metric scanning in a LKB microdensitometer.

Phosphotyrosine Immunoblots—Cell extracts were prepared by lys-ing the cells in boiling 2 3 Laemmeli buffer and shearing by passingthrough a 26-gauge needle 10 times, followed by boiling in a water bathfor 5 min. The samples were electrophoresed on an 8% SDS-polyacryl-amide gel and transferred to the nitrocellulose membrane (Schleicher &Schuell) using a wet transfer system (Bio-Rad) and Towbin’s buffer for13 h at 30 V or 5 h at 60 V. The blots were blocked for 2 h at 37 °C inTris-buffered saline containing 0.1% Tween 20, 2% bovine serum albu-min, and 0.02% thimerasol (TBST-BSA) and then incubated with theanti-phosphotyrosine antibodies (PY20 at 1:2500 and 4G10 at 1:1000dilutions) in TBST containing 2% bovine serum albumin for 2 h at37 °C. The excess antibodies were washed 3 times for 5 min each, plusa longer 20-min wash with TBST. A second blocking step was done for1 h with TBST containing 5% nonfat milk. This was followed by incu-bation with goat anti-mouse antibodies conjugated to horseradish per-oxidase (1:5000) for 1 h at room temperature. The washings wereperformed as described for the immunoblots. The antibody detectionwas done using ECL reagents (Amersham).

RESULTS

Nonmitogenic Receptor-dependent Activation of 9E3 byThrombin—Addition of serum to CEFs in culture stimulates9E3 expression as cells enter the cell cycle (5, 6, 8, 9). Becausethrombin is mitogenic for fibroblasts, we expected that stimu-lation of the 9E3 protein by thrombin occurs via the samemitogenic pathway. To test this possibility, we plated CEFs inserum-free medium, added thrombin at 10 units/ml, and fedthem with serum-free medium plus 10 units/ml of thrombinevery 24 h for a period of 4 days. Parallel cultures were treatedwith medium containing 5% donor calf serum and 1% chickenserum. At the end of this period the numbers of cells werecounted and the 9E3 protein present in the supernatant for the

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final 24 h was analyzed by immunoblot with an antibody spe-cific for the 9E3 protein (14). The results showed that althoughthe total number of cells was approximately the same for bothtreatments (Fig. 1A), the amount of protein secreted into thesupernatant was at least 15-fold higher in the cells treatedwith thrombin (Fig. 1B). For the same period of time, the

amount of 9E3 protein produced by the CEFs treated withthrombin was similar to that produced by Rous sarcoma virustransformed CEFs (tCEFs), which express the 9E3 gene con-stitutively (Fig. 1B). Our observations at the protein level wereconfirmed by analysis of the mRNA under the same conditions(Fig. 1C); the level of the protein in cells with the varioustreatments correlates well with levels of mRNA under the sameconditions. To determine if the stimulation of 9E3 by thrombinis via the proteolytically activated receptor for thrombin recep-tor, we treated qcCEFs, which do not express 9E3, with thehuman thrombin receptor derived peptide (TRDP), which canactivate proteolytically activated receptor for thrombin muchlike thrombin (50). We found that this peptide could stimulateqcCEFs to express 9E3 to the same level as those stimulated bythrombin itself (Fig. 1D).

To further determine if the stimulation of 9E3 by thrombin isspecific, we performed a dose-dependent study by treatingqcCEFs with serum-free medium containing increasing dosesof thrombin for a period of 24 h. We found maximum accumu-lation of the protein in the supernatant with a treatment of 9units/ml or more of thrombin (Fig. 2A). Using a dose of 9 units,we determined that the levels of the 9E3 protein present in thesupernatant of treated cells increased with time up to 18 h withsaturated levels after this time (Fig. 2B).

The lack of detection of 9E3 early in the time course study(Fig. 2B) could simply be the result of the lack of time toaccumulate detectable levels of the protein at the early timepoint. To determine the time span required for thrombin acti-vation of 9E3, we treated qcCEFs with thrombin (9 units/ml)for shorter periods of time, then washed and incubated the cellswith serum-free medium for 18 h to allow accumulation of theprotein in the supernatant. We found that 1 min exposure ofthe cells to thrombin was sufficient to turn on the eventsleading to production of the 9E3 protein (Fig. 2C).

Additive Effect of v-src and Thrombin on 9E3 Expression—Our observation that activation of 9E3 by thrombin is inde-pendent of mitogenesis, coupled with a previous determinationthat activation of 9E3 by v-src is via a mitogenic pathway (51)raised the possibility that thrombin and the oncogene mightactivate 9E3 via different signal transduction pathways. If so,when added together, they should stimulate an additive effecton the expression of 9E3. To test this possibility, we treatedtCEFs with thrombin and analyzed them for both protein (Fig.3A) and mRNA (Fig. 3B) and observed that thrombin and v-srchave additive effects. These results suggest that thrombin ac-tivates 9E3 by a pathway independent of that turned on byv-src (which involves activation of PKC by the v-src oncogene)(51) but do not preclude the possibility that it could be asuperactivation of the same pathway involving PKC.

Stimulation of 9E3 by Thrombin Is Enhanced by Inhibitionof Ser/Thr Kinases—Thrombin is known to stimulate severalsignal transduction pathways resulting in activation of a vari-ety of genes and leading to many different biological responses.The pathway stimulated by thrombin that activates PKC in-volves activation of Gq,11 which activates phospholipase Cb

that, in turn, cleaves phosphatidylinositol phosphate into IP3

and diacylglycerol. The latter activates PKC by interactingdirectly with this kinase, whereas IP3 interacts with its recep-tor, present in the endoplasmic reticulum resulting in intracel-lular Ca21 release. To determine if PKC is involved in thestimulation of 9E3 by thrombin, we treated qcCEFs with OAG(oleoyl-2-acetyl-glycerol), an analogue of diacylglycerol that ac-tivates PKC directly (52), and found that OAG did not stimu-late the production of the 9E3 protein (Fig. 4A). To furtherthese studies, we treated qcCEFs with calphostin C (Fig. 4B), aspecific inhibitor of PKC (53) and found that this treatment did

FIG. 1. Thrombin stimulation of 9E3 is receptor-mediated butis not due to its mitogenic properties. A, effects of thrombin andserum on cell division. Cells were treated daily for 4 days with 10units/ml of thrombin or 5% donor calf serum plus 1% chicken serum.Thrombin stimulated cell division to the same degree as serum. B,Western blot analysis of the levels of the 9E3 protein produced by CEFsstimulated with serum (5% donor calf 1 1% chicken), 10 units/mlthrombin, and by v-src (tCEFs). Thrombin and v-src stimulate theproduction of similar amounts of 9E3 protein; microdensitometry anal-ysis showed 15 times more protein for the cells treated with thrombin ortransformed with src than in cells treated with serum. C, same as in Bbut with comparison of the levels of 9E3 mRNA. D, stimulation of 9E3by TRDP (thrombin receptor-derived peptide). Untreated cells (qcCEF),qcCEFs treated with TRDP (200 mM), and qcCEFs treated with 10units/ml of thrombin are shown in panel D. TRDP is as efficient asthrombin in stimulating 9E3 expression.

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not inhibit stimulation of 9E3 by thrombin but instead it po-tentiated thrombin stimulation. Furthermore, when we treatedqcCEFs with the broad spectrum Ser/Thr kinase inhibitors H7and staurosporine (53, 54), we found that both inhibitors po-tentiated the stimulation of 9E3 by thrombin and that stauro-sporine by itself stimulated 9E3 expression (Fig. 4B). In addi-tion, we also found that an increase in cytosolic Ca21 bytreatment with the ionophores ionomycin and A23187 (Fig. 4C)had little effect on the levels of the 9E3 protein and did not

enhance that stimulated by thrombin. These results suggestthat the signaling pathways activated by Gq,11, and in partic-ular the activation of PKC, do not play a major role in stimu-lation of 9E3 by thrombin.

Thrombin is also known to interact with its receptor andactivate Gi or Gs, and more recently it has also been shown toactivate tyrosine phosphorylation (55). We treated qcCEFswith thrombin in the presence of inhibitors or activators of thekey players involved in these signaling pathways. Activation ofthe Gi pathway leads to inhibition of adenylyl cyclase, decreasein cAMP production, and therefore lack of activation of PKA. Totest if Gi is involved, we used pertussis toxin, which ADP-ribosylates this G-protein, inhibiting it permanently (56, 57).We found that inhibition of Gi by pertussis toxin failed toinhibit, and in fact potentiated, the stimulation of 9E3 expres-sion by thrombin (Fig. 5A). Treatment of cells with HA1004 andH89, inhibitors of PKA and other Ser/Thr kinases (58, 59), alsopotentiated the stimulation of 9E3 expression by thrombin(Fig. 5A), suggesting that high levels of PKA might inhibit 9E3expression. We addressed this possibility by treating qcCEFswith cholera toxin, which specifically activates Gs and in turnleads to the activation of PKA. This treatment had no affect onthe stimulation of 9E3 by thrombin (Fig. 5B), indicating thatPKA is not critical for the expression of this gene.

Tyrosine Kinases Are Involved in the Activation of 9E3 Ex-pression by Thrombin—Our findings that Ser/Thr kinases arenot significantly involved in 9E3 stimulation by thrombin, led

FIG. 2. Dose and time dependence of thrombin-stimulated 9E3expression. A, dose dependence. CEFs were incubated with increasingconcentrations of thrombin and the levels of 9E3 protein in the super-natants were analyzed by immunoblot and microdensitometry. 9E3levels saturate at 9 units/ml thrombin. B, time dependence. CEFs wereincubated with 9 units/ml thrombin for increasing time intervals, andthe levels of 9E3 protein in the supernatants were also analyzed byimmunoblot and microdensitometry. 9E3 levels saturate at 18 h ofexposure. C, qcCEFs were exposed to thrombin (9 units/ml) for varyingperiods of time. At the end of each exposure, the medium containingthrombin was removed, and cells were washed with Tris-glucose bufferand further incubated for 18 h in serum-free medium before collectionof the protein. One minute exposure to thrombin was sufficient totrigger the signals leading to detectable 9E3 expression.

FIG. 3. Additive effect of thrombin and v-src on 9E3 expres-sion. A, Western blot analysis of the 9E3 protein produced by cellstreated with thrombin (9 units/ml), transformed with Rous sarcomavirus (tCEFs) or tCEFs treated with thrombin (thr1tCEFs). The graphrepresents quantitation by microdensitometry of the amount of proteinpresent in each band. Addition of thrombin (9 units/ml) to tCEFsessentially doubled production of the 9E3 protein. B, Northern blotanalysis of the 9E3 mRNA for samples treated as in A.

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us to investigate the role of tyrosine kinases in this process(40–42, 60). We found that genistein, a general inhibitor oftyrosine kinases (61), abolished the expression of 9E3 inducedby thrombin both at the protein (Fig. 6A) and mRNA levels(Fig. 6B). To investigate the nature of the proteins phosphoryl-ated on tyrosines, we performed Western blot analysis of ex-tracts prepared from cells treated with thrombin, using anti-phosphotyrosine antibodies. A time course study showed thatproteins of molecular masses 220, 170, 60, 44, and 42 kDa werephosphorylated on tyrosines in a biphasic manner upon throm-bin stimulation of qcCEFs with the first peak at 5–7 min andthe second at 3–6 h after activation (Fig. 7). By reprobing thesame blot or identical blots with specific antibodies, we foundthat the 170-kDa band was labeled by an antibody to the EGFreceptor, the 60-kDa band was labeled by an antibody to thec-src protein and the 44- and 42-kDa bands were labeled byantibodies to ERK1 and ERK2, respectively. In this study we

focus on the potential involvement of the c-src and the EGFreceptor tyrosine kinases.

To determine if c-src is involved in the stimulation of 9E3 bythrombin we used the specific inhibitor herbimycin A. Treat-ment of qcCEFs with herbimycin A at doses that inhibit pp60c-

src only (IC50 5 900 nM) (62) shows that this inhibitor is capableof eliminating 9E3 expression stimulated by thrombin (Fig.

FIG. 4. Thrombin stimulation of 9E3 does not involve the Gq,11pathway. Western blot analysis of the 9E3 protein produced by cellswith various treatments. In all cases the concentration of thrombin was9 units/ml, and Me2SO was used as the solvent for most activators andinhibitors. A, cells were treated with an analogue of diacylglycerol(OAG; 300 mM), which activates PKC, and were not stimulated toexpress 9E3. B, cells were treated with the specific inhibitor of PKC,calphostin C (200 nM), and with the broad spectrum inhibitors of Ser/Thr kinases, H7-dihydrochloride (200 nM), and staurosporine (200 nM).Calphostin C potentiated the stimulation of 9E3 by thrombin and so didthe broader spectrum Ser/Thr kinase inhibitors, indicating that Ser/Thrkinase inhibitors in general potentiate the stimulation of 9E3 by throm-bin. Although the other inhibitors by themselves had no effect on 9E3expression, staurosporine stimulated it. C, cells treated with the iono-phores, ionomycin (1 ng/ml) and A23187 (1 mM), in the presence orabsence of thrombin (9 units/ml). The increase in Ca21 levels triggeredby ionomycin or A23187 did not significantly stimulate 9E3.

FIG. 5. Thrombin stimulation of 9E3 does not involve the Gi orGs pathways. Effects of Ga inhibitors or activators on 9E3 expression,through Gi (A) and through Gs (B). In all cases the concentration ofthrombin was 9 units/ml. 100% Me2SO was used as the solvent for mostactivators and inhibitors; pertussis toxin (ptx) and cholera toxin (ctx)were used at 100 ng/ml; HA1004-HCl at 20 mM; control consisted ofuntreated qcCEFs. Inhibition of Gi by pertussis toxin and inhibition ofPKA by HA1004-HCl and H89 potentiated the expression of 9E3 bythrombin (A), whereas stimulation of Gs by cholera toxin had no effect(B).

FIG. 6. Thrombin stimulation of 9E3 involves the activation oftyrosine kinases. The broad spectrum tyrosine kinase inhibitor genis-tein (25 mM) inhibits the stimulation of 9E3 by thrombin (9 units/ml)both at the protein (A) and mRNA (B) levels. Controls consisted ofuntreated qcCEFs.

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8A), indicating that c-src is importantly involved in the signaltransduction cascade initiated by thrombin.

To investigate if the EGF receptor is involved in activation of9E3 by thrombin, we inhibited its tyrosine kinase by using twoselective inhibitors, lavendustin A RG14355 (62) and the highlyselective and strong inhibitor, tyrphostin AG1478 (62–64).Lavendustin A RG14355 virtually eliminated the enhancementof production of the 9E3 protein stimulated by thrombin (Fig.8B), and tyrphostin AG1478 completely inhibited enhancedstimulation by thrombin of both the 9E3 mRNA and protein(Fig. 8, C and D). To further investigate the potential involve-ment of the EGF receptor, we treated qcCEFs with hrEGF inthe presence and in the absence of tyrphostin AG1478 andperformed similar experiments with hrTGFa, which is knownto utilize the EGF receptor (65). We found that hrTGFa stim-ulates the production of the 9E3 protein in qcCEFs, confirmingour previous findings (8), and that this effect is inhibited bytyrphostin AG1478 (Fig. 8E). hrEGF, which binds less well tothis receptor in chickens than does hrTGFa (66), also stimu-lated production of the 9E3 protein but required more than 10times as much for an effect comparable to that of TGFa; tyr-phostin AG1478 similarly abolished this stimulation (Fig. 8E).These results taken together show that the EGF receptor isinvolved in the activation of 9E3 via direct interactions with itsligands, EGF and TGFa, and indirectly by interaction of throm-bin with the thrombin receptor.

DISCUSSION

Previous work has shown that serum factors stimulate 9E3expression (5, 6) and that expression under these conditions isinitiated during the G0/G1 transition of the cell cycle and de-clines in S-phase (8). The results presented here show that forthe same number of cells, stimulation of 9E3 by thrombin is15-fold higher than that stimulated by serum. Furthermore, inall of the other experiments performed here, we used qcCEFsthat are contact inhibited; under these conditions only a smallnumber of cells will undergo cell division during 18 h of expo-sure to serum or thrombin. The reduced amounts of 9E3 in thesupernatant of qcCEFs treated with serum is reflective of thesmall number of cells entering G1 (8), whereas the increasedamounts of 9E3 after thrombin treatment must reflect expres-

sion by many of the preexisting quiescent cells. Therefore,thrombin stimulation of the 9E3 gene occurs by a pathway thatdoes not involve mitogenesis.

It has been reported previously that when cells are infectedwith viruses that carry the oncogenes v-fps or v-src, they ex-press the 9E3 gene constitutively; these oncogenes activatePKC, which in turn is responsible for triggering mitogenicevents that lead to 9E3 expression (51, 67, 68). Our observationthat stimulation of 9E3 by thrombin occurs through a nonmi-togenic pathway, coupled with the additive effect with v-srcstimulation, suggests that v-src and thrombin operate primar-ily through different independent pathways. Indeed, thrombinactivation of 9E3 does not involve PKC and is potentiated by avariety of inhibitors of Ser/Thr kinases, indicating that inhibi-tion of these kinases in general potentiates the stimulation of9E3 by thrombin. In support of this proposal are the findings ofIshii et al. (69) who showed that the receptor for thrombin isturned off by Ser/Thr kinases that phosphorylate these aminoacids present in the C terminus of the receptor. Thus, inhibi-tion of these kinases might allow the thrombin receptor to actfor longer periods and produce greater effects.

We show here that tyrosine kinases are importantly involvedin stimulation of the 9E3 gene by thrombin. Five specific pro-teins (four that co-migrate with the c-src, the EGF receptor,and the ERK 1/ERK 2 kinases) are phosphorylated on tyrosineswithin 5–7 min of stimulation by thrombin, and this stimula-tion is synchronous (Fig. 7). This observation, coupled with therapid activation of the gene (15), suggests direct involvement ofthese proteins in 9E3 stimulation. Their possible roles in 9E3activation are the subject of ongoing research in our laboratory.

Our observation that herbimycin A, which preferentially in-hibits kinases of the c-src family (62, 70), inhibits 9E3 expres-sion stimulated by thrombin, suggests involvement of c-src inthis activation. In support of involvement of c-src is the obser-vation that when chinese hamster fibroblasts are treated withthrombin, the c-src tyrosine kinase is activated (42). Also, stau-rosporine, a broad spectrum Ser/Thr kinase inhibitor, not onlypotentiated the action of thrombin but by itself stimulated lowlevels of 9E3 expression. It has recently been found that stau-rosporine induces spreading of a human colon cancer cell line(70, 71) and that this effect is inhibited by herbimycin A. Oneof the proteins phosphorylated on tyrosines upon staurosporinetreatment was the c-src tyrosine kinase (70, 71). Therefore, it ispossible that the stimulation of 9E3 by staurosporine alone isdue to the activation of this kinase by this inhibitor, furthersupporting the involvement of c-src in the expression of 9E3. Itwas surprising that c-src is involved in this tyrosine-kinase-de-pendent pathway of 9E3 activation by thrombin, given thatactivation of this gene by v-src occurs via a PKC-dependentpathway (51). However, we note that it has been shown (72)that v-src can turn on both PKC-dependent and PKC-independ-ent pathways leading to gene expression, showing that src canparticipate in multiple signaling pathways.

We have used inhibitors of the tyrosine kinase activity of theEGF receptor to address the involvement of this receptor in thestimulation of 9E3 expression by EGF, TGFa, and thrombin.The inhibitors we used have varying specificity for the EGFreceptor tyrosine kinase. Genistein inhibits tyrosine kinaseactivity by competing with these enzymes for the ATP bindingsite and therefore is a broad spectrum inhibitor. Genisteineliminated thrombin stimulation of 9E3 (Fig. 6). Lavendustin ARG14355 is a cell-permeable selective inhibitor for the EGFreceptor tyrosine kinase (IC50 5 11 nM) and for pp60src (IC50 5500 nM) (62). At the concentrations used in our study (100 nM)it is selective for the EGF receptor tyrosine kinase. We foundthat lavendustin A RG14355 virtually inhibited the stimula-

FIG. 7. Tyrosine phosphorylation of cellular proteins afterthrombin stimulation of qcCEFs. A, phosphotyrosine immunoblotafter incubation of qcCEFs with thrombin for increasing amounts oftime. At least five proteins are differentially phosphorylated showing amaximum at 5–7 min, a minimum at 30 min, and a second maximum at3–9 h. Using specific antibodies, four of the five bands in the same gelwere identified as co-migrating with the EGF receptor (170 kDa), c-src(60 kDa), ERK1 (44 kDa), and ERK2 (42 kDa) proteins. The essentiallyconstant intensity of the bands at 110–120 kDa demonstrate evenloading of the gel. Note that ERK1 and ERK2 appear as very wellseparated bands because we ran 7% SDS-polyacrylamide gel electro-phoresis gels rather than 10–12%.

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tion of 9E3 by thrombin (Fig. 8A). We also used tyrphostinAG1478, a more potent and selective inhibitor for this receptortyrosine kinase (62–64). The evidence that tyrphostin AG1478is highly specific for the EGF receptor tyrosine kinase is nowextensive. Fry et al. (63), in a paper directed specifically toselectivity of inhibition for the EGF receptor, showed that aquinazoline identical to tyrphostin AG1478 except for substi-tution of bromine for chlorine on the free benzene ring of themolecule is a highly selective inhibitor for the EGF receptor atconcentrations from a few nanomolar to a few micromolar.Furthermore, in a comprehensive review of tyrosine kinaseinhibition, Levitzki and Gazit (62) showed that tyrphostinAG1478 inhibits the EGF receptor tyrosine kinase in vitro withan IC50 5 3 nM, whereas the corresponding IC50 values forinhibition of other tyrosine kinases by this molecule were allgreater than 50 mM (HER2-neu IC50 . 100 mM; platelet-derivedgrowth factor IC50 . 100 mM). In the present study in vivo,concentrations of 500 nM tyrphostin AG1478 completely oblit-erated stimulation of 9E3 by thrombin, hrTGFa, and hrEGF.Finally, Daub et al. (64) also showed specificity of tyrphostinAG1478 when studying the stimulation of the fos gene by

thrombin via transactivation of the EGF receptor tyrosine ki-nase in Rat-1 fibroblasts. They confirmed their results by use ofa dominant negative mutant of the receptor that is capable ofinhibiting events downstream from the receptor by formingsignaling defective heterodimers with the wild-type receptor.Both tyrphostin AG1478 and this mutation eliminated the EGFreceptor phosphorylation stimulated by thrombin and abol-ished activation of the fos gene. Experiments using a dominantnegative mutant of the EGF receptor, similar to those of Daubet al. (64), are not possible in our system because we are usingprimary cell cultures, and there are no chicken cell lines toperform such studies. However, use of such a mutant by theseauthors confirmed the specificity of tyrphostin AG1478, ena-bling us to infer that the stimulation of 9E3 by thrombininvolves analogous transactivation of the EGF receptor as animportant step in the 9E3 gene activation cascade triggered bythrombin.

Transactivation of a growth factor receptor by another cell-surface receptor is only a recently recognized phenomenon (64,73). These two previous studies also involve transactivation ofthe EGF receptor; the former via the thrombin receptor (dis-

FIG. 8. Involvement of the EGF/TGFa receptor and c-src tyrosine kinases in 9E3 expression. A, cells treated with thrombin in thepresence of 1 mM herbimycin A (an inhibitor specific for the c-src family of kinases) showed little or no 9E3 expression. B, lavendustin A RG14355,a selective inhibitor for the EGF/TGFa receptor tyrosine kinase, greatly reduced the level of stimulation of the 9E3 protein by thrombin in qcCEFs.C and D, qcCEFs incubated with thrombin in the presence of 500 nM tyrphostin AG1478 (a highly selective and strong inhibitor of the EGF/TGFareceptor tyrosine kinase) showed lower levels of both the 9E3 mRNA (C) and protein (D) than were present in control qcCEFs, showing completeelimination of the enhancement of expression produced by thrombin. E, both EGF and TGFa stimulated production of the 9E3 protein and botheffects were inhibited by tryphostin AG1478. Neither of the growth factor-stimulated levels were comparable to that of thrombin, and EGF requiredmuch higher doses for the same result as TGFa (see “Discussion”).

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cussed in the previous paragraph) and the latter via the TNFareceptor. In both cases, demonstration of transactivation in-volved detection of fos gene expression. fos is an immediateearly response gene that is activated by phosphorylation of theElk1 transcription factor by mitogen-activated protein kinases(74). Here we have shown activation of another immediateearly response gene, 9E3 (5), which codes for a secreted protein,a chemokine. The promoter of this gene also contains an Elk1binding site (75) and mitogen-activated protein kinase is alsoinvolved in 9E3 gene expression stimulated by thrombin.2 Fur-ther work will be required to determine if transactivation of theEGF receptor could represent a general mechanism for turningon immediate early response genes by receptors that do nothave an intrinsic tyrosine kinase activity.

The greater stimulation of 9E3 by TGFa than by EGF in thisstudy is explained by differences in affinity for the chicken EGFreceptor. Although hrEGF and hrTGFa bind with equal affinityto the receptor in humans, hrTGFa binds with 100-fold greateraffinity than does hrEGF to the chicken receptor (66), henceEGF requires much higher doses to cause the same effect. Ourobservation that EGF and TGFa are less efficient than throm-bin in stimulating 9E3 expression is consistent with the factthat these growth factors are mitogenic for CEFs (76) andstimulate 9E3 via entry into the cell cycle (8). We previouslyshowed using in situ hybridization that only some cells inqcCEF cultures can be stimulated to enter cell division andthat in experiments involving growth factors, only the dividingcells express 9E3 (8). On the other hand, thrombin stimulationdoes not require cell division, hence it will stimulate 9E3 ex-pression in a large fraction of cells resulting in greater inte-grated expression. An additional factor that could contribute tothis difference between these growth factors and thrombin isthat growth factors interact with their receptors and down-regulate them, whereas the activation of the EGF receptor bythrombin is intracellular and therefore may stay on for longerperiods of time.

At first thought, it might seem surprising that the EGFreceptor can function as the entry point to a signal-transduc-tion pathway leading to gene expression through a mitogenicpathway (e.g. for EGF and TGFa) and also serve as a majorearly step in a nonmitogenic pathway (e.g. for thrombin). How-ever, it is already known that after activation by EGF andconsequent dimerization, the EGF receptor autophosphoryl-ates on several tyrosine residues that serve as docking sites fora variety of molecular mediators for different signal-transduc-tion pathways that emanate from this receptor (77). The detailsof the role played by this receptor in the stimulation of 9E3 bythrombin presumably are different from that for either EGF orTGFa because thrombin activates the EGF receptor intracel-lularly. Therefore, our results suggest the possibility of stillgreater complexity of behavior of the EGF receptor than pre-viously recognized.

Our results do not yet clarify whether c-src activation pre-cedes or succeeds EGF receptor activation. However, uponthrombin interaction with its receptor, it has been shown thatc-src is rapidly activated by both pertussis toxin-sensitive and-insensitive G-proteins (78). In addition, in Rat-1 fibroblasts,v-src phosphorylates Gaq,11 on tyrosines and increases its ac-tivity (79, 80). This is also true for several other Ga-proteins(81). Therefore, it appears that upon thrombin activation of itsreceptor, there is mutual interaction between a Ga protein andc-src (82, 83). Furthermore, the association of c-src with theplasma membrane (84) and its known ability to bind to the

EGF receptor (85) suggests that it may be involved in thetransactivation event. These and other aspects of the signalingpathways stimulated by thrombin and leading to 9E3 expres-sion are currently being pursued in our laboratory.

In summary, thrombin stimulation of the 9E3 chemokinegene in qcCEFs occurs via a nonmitogenic pathway and isaccompanied by synchronous, biphasic, phosphorylation of sev-eral proteins including c-src and the EGF receptor. Similarstimulation of 9E3 by EGF and TGFa verify that the EGFreceptor is functional in these cells, and a series of inhibitorsthat are highly selective for c-src and the EGF receptor tyrosinekinases eliminate 9E3 stimulation by thrombin. This study isnow the third to show stimulation of an immediate early re-sponse gene via transactivation of the EGF receptor after ini-tial binding of a ligand to a receptor that does not have anintrinsic tyrosine kinase activity, suggesting that this could bea general mechanism for activation of immediate early re-sponse genes by the latter class of receptors.

On a broader scale, the stimulation of the 9E3 gene via entryinto the cell cycle may be akin to that occurring in normaltissues where expression is very low and probably representsthe result of a small number of cells that are undergoing celldivision (8, 10). In contrast, the high levels of 9E3 expressiontriggered by thrombin could represent a response from all cellsto an emergency situation such as wounding, tumorigenesis orviral entry into cells. The signal transduction pathway bywhich this is accomplished, involving transactivation of theEGF receptor and other tyrosine kinases, offers the opportu-nity for amplification of the signals and a prolonged response,much like the activation of adenylcyclase by the epinephrinereceptor. This type of activation could provide an advantagewhen a rapid and strong tissue response is needed.

Acknowledgments—We thank H. W. Green, D. Strauss, and R.Zidovetzki for critical reading of the manuscript.

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Additions and Corrections

Vol. 273 (1998) 5447–5450

Conantokin-G precursor and its role in g-carboxyla-tion by a vitamin K-dependent carboxylase from aConus snail.

Pradip K. Bandyopadhyay, Clark J. Colledge, Craig S.Walker, Li-Ming Zhou, David R. Hillyard, and Baldomero M.Olivera

Page 5449, Table I: the Km for FLEEL should read 230.1 6 6mM instead of 2301 6 6 mM.

Vol. 273 (1998) 5226–5234

Thrombin activation of the 9E3/CEF4 chemokine in-volves tyrosine kinases including c-src and the epi-dermal growth factor receptor.

Sucheta M. Vaingankar and Manuela Martins-Green

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14658

Sucheta M. Vaingankar and Manuela Martins-Green and the Epidermal Growth Factor Receptor srcIncluding c-

Thrombin Aivation of the 9E3/CEF4 Chemokine Involves Tyrosine Kinases

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