Vol. 4, 603-609. July 1993 Cell Growth & Differentiation 603

Protein Tyrosine Phosphorylation and Activation ofPrimary Response Genes by Interleukin 11in B9-TY1 Cells’

Tinggui Yin and Yu-Chung Yang2

Department of Medicine (Hematology/Oncology) [T. Y., Y-C. Y.]

Department of Biochemistry and Molecular Biology [Y-C. Y.], andWalther Oncology Center, [Y-C. Y.], Indiana University School of

Medicine, Indianapolis, Indiana 46202

Abstract

Interleukin (IL) 1 1 is a multifunctional cytokine derivedfrom bone marrow stromal cells. To understand themechanisms by which IL-i 1 exerts its pleiotropicactions, we have analyzed IL-i 1-mediated signaltransduction pathways in IL-i i-dependent B9-TY1,which is a subclone of an IL-6-dependent B-cellhybridoma, B9. IL-i 1 stimulation of B9-TY1 cellsresulted in tyrosine phosphorylation of a 97/95kilodalton cellular protein in a dose-dependentmanner, and this effed was inhibited by tyrosine kinaseinhibitors genistein and herbimycin A, but not by aserine/threonine kinase inhibitor, H7. We nextexamined the early nuclear events in the IL-il-triggered intracellular signaling cascade. The datashowed that tisi 1, tis2l, and junB early response geneswere rapidly activated following IL-i 1 treatment. Thekinetic studies indicated that adivation of tisi 1 andjunB genes peaked at 30-60 mm and then declinedslowly afterward. The tis2l gene was constitutivelyexpressed, and the level of tis2l mRNA wassignificantly increased and maintained at the elevatedlevel following IL-i 1 stimulation. Inhibitor studies withgenistein, herbimycin A, and H7 revealed that tyrosinekinases and H7-sensitive serine/threonine kinases arerequired for the IL-i i-mediated adivation of tisi 1,tis2l, and junB genes. Using a variety of known proteinkinase inhibitors or activators, we have demonstratedthat H7-sensitive protein kinases activated by IL-i 1 aredistind from those of well-charaderized proteinkinase-second messenger systems. Taken together, theresults suggest that both tyrosine kinase adivity andH7-sensitive protein kinase adivity are critical in IL-ilsignal transdudion pathways leading to the adivationof tisi 1, tis2l, and junB early response genes.

lntrodudionlL3-1 1 is a stromal cell-derived cytokine originally discov-ered on the basis of its mitogenic activity on an IL-6-dependent mouse plasmacytoma cell line, 11165 (1). Ithas become increasingly evident that IL-i 1 is a multi-functional biofactor which plays essential roles in a van-ety of biological systems. Previous studies have suggestedthat IL-li, although lacking sequence homology with IL-6, is functionally related to IL-6. IL-il, like IL-6, canstimulate the proliferation of an IL-6-dependent mouseplasmacytoma cell line, Ti 165 (1), and an IL-6-dependentmouse B-cell hybnidoma cell line, B9 (see below); en-hance the lL-3-dependent development of mega-karyocyte colonies in both human and mouse bonemarrow cultures (1, 2); stimulate T-ceII-dependent, anti-gen-specific antibody production in vitro and in vivo innormal mice (3); accelerate the regeneration of antigen-specific antibody-forming cells in immunosuppressedmice following cyclophosphamide treatment (3); stimu-late plunipotent stem cells to come out of the G0 stage ofthe cell cycle to respond to intermediate or late actingfactors such as lL-3 or granulocyte-macrophage colonystimulating factor (4); induce acute phase protein synthe-sis in a rat hepatoma cell line, H-35, and in freshly isolatedrat hepatocytes (5); and inhibit lipoprotein lipase activityand preadipocyte differentiation (6, 7).

Although the biochemical nature of IL-i 1 receptor hasbeen partially characterized (6), the signal transductionmechanisms generating the broad range of IL-i 1 biolog-ical activities are not known. Initial studies showed thatIL-i 1 stimulation results in the induction of protein ty-rosine phosphorylation in 3T3-Ll preadipocytes (6).These results suggested that protein tyrosine kinases mayplay important roles in IL-i 1-mediated biological func-tions. However, more downstream signals induced by IL-1 1 following the protein tyrosine phosphorylation stepare not clear. In this investigation, we report that IL-i 1can support the growth of B9-TY1 cells, which are asubclone of lL-6-dependent mouse B-cell hybnidoma, B9cells. Using B9-TY1 cells, we have demonstrated that IL-1 1 stimulation rapidly induced protein tyrosine phos-phorylation and subsequently activated transcription ofseveral primary response genes which may play impor-tant roles in cell growth and differentiation (8-13). Fur-thermore, we have tested the effects of various proteinkinase inhibitors or activators on IL-i 1-induced tyrosine

Received 1/8/93; revised 4/27/93; accepted 4/30/93.1This work was supported by USPHS Grants ROl DK43105 and ROl

HL48819 to Y-C. Y. Y-C. Y. is a Scholar of the Leukemia Society ofAmerica. T. Y. is supported in part by a Fellowship from the AmericanHeart Association, Indiana Affiliated, Inc.2 To whom requests for reprints should be addressed, at Walther Oncol-ogy Center, Indiana University, School of Medicine, 975 West Walnut

Street, Room 501, Indianapolis, IN 46202.

3 The abbreviations used are: IL, interleukin; KDa, kilodalton(s); PKC,protein kinase C; PKA, cyclic AMP-dependent protein kinase; TPA, 12-O-tetradecanoylphorbol-1 3-acetate; dBcAMP, N-6,2-O-dibutynyl cyclicAMP; 8BncGMP, 8-bromo-cyclic GMP; SP, sphingosine; H7, 1-(5-isoquin-olinesulfonyl)-2-methylpipenazine; W7, N-(6-aminohexyl)-5-chloro-1.

naphthalenesulfonamide; GS, genistein; SOS, sodium dodecyl sulfate;PAGE, polyacrylamide gel electrophoresis; SSC, standard saline citrate;B2-M, B2.microglobulin.

IL-i 1 concentration (ng/mI)

604 Signal Transduction Mediated by IL-i 1

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Fig. 1. IL-il-induced B9-TY1 cell proliferation. B9-TY1 cells (2 X iO�

cells/well in 96-well plates in tniplicates) were cultured in the presenceof different concentrations of IL-i 1 at 37’C, 5% CO2 for 48 h. Cells werepulsed with 0.5 �zCi/welI of tritiated thymidmne for 4 h. Cells wereharvested, and radioactivity was determined with a Beckman scintillationcounter. The results are expressed as counts/mm. SD is less than 10%.

phosphorylation and primary response gene expressionin B9-TY1 cells.

Results

Establishment and Growth of IL-i 1-dependent B9-TYiCells. B9 cells were originally derived from an lL-6-dependent mouse B-cell hybridorna (14). The growth ofestablished B9 cells is dependent on either human ormouse lL-6. Since IL-i i was discovered as a mitogenicfactor for an lL-6-dependent mouse plasmacytoma cellline, T1165 (1), we examined whether IL-li can alsostimulate the proliferation of IL-6-dependent mouse B9

cells. Our initial studies showed that only a small per-centage of B9 cells are responsive to IL-i 1, based on thecell viability in the proliferation assay. An IL-i i-depend-ent cell line designated as B9-TY1 was subsequentlyestablished from B9 cells by semisolid cell cloning tech-niques in the presence of IL-il. The established B9-TY1cells are dependent on either IL-i 1 or lL-6 for growth.As shown in Fig. 1, proliferation of B9-TY1 cells stimu-

lated by IL-i 1 is dose dependent. Receptor studies withradiolabeled IL-i 1 revealed the existence ofa single classofIL-1 1 receptor with a K,1 of9.9 x 10l� M and a receptordensity of 75 sites/cell on B9-TY1 cells, demonstratingthat IL-li-induced B9-TY1 cell growth is accomplishedmost likely through interactions with its specific receptors

on these cells.Effect of IL-i 1 on Protein Tyrosine Phosphorylation in

B9-TY1 Cells. It has become increasingly evident thatprotein tyrosine kinases play important roles in the signaltransduction pathways mediated by growth factors in

different systems (8, 9, 15). In our experiments, we haveexamined the possible role of protein tyrosine phos-phorylation in IL-i i-supported growth of B9-TY1 cells.The 97/95 KDa tyrosine-phosphorylated protein was theonly band which consistently showed an increase intyrosine phosphorylation following IL-i 1 stimulationwhen compared with unstimulated cells in over 20 mdi-

Time 0 1 5 15 30 60 90120150(mm)

A

IL-Il(M)

B

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KDa

- 205

- 116.5

80

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Fig. 2. Protein tyrosine phosphorylation induced by IL-i 1 in B9-TY1cells. After IL-il starvation for 14 h, B9-TY1 cells (1 x iO’/0.i ml) werestimulated with IL-i 1 (500 ng/mI) for the indicated times (A) or with theindicated concentrations of IL-i 1 for 20 mm (B). The same amounts ofproteins (equivalent to 1 x i0� cells/lane( were separated by 7.5% SDS-

PAGE, and the tyrosine-phosphorylated proteins were determined by

antiphosphotyrosine immunoblotting, as described in “Materials and

Methods.”

vidual experiments. This tyrosine-phosphorylated pro-tein appeared within 1 mm after stimulation with IL-i 1and peaked by 15-30 mm (Fig. 2A). Fig. 2B shows thatthe induction of the 97 KDa tyrosmne-phosphorylatedprotein by IL-i 1 was dose dependent.

Effects of Genistein, Herbimycin A, and H7 on IL-il-induced Protein Tyrosine Phosphorylation. To show thatthe induction of protein tyrosine phosphorylation by IL-

B

TPA(48h)

TPA(2Omin)

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Fig. 3. Effects ofGS, herbimycin A, H7, and TPA treatment on IL-li-induced tyrosine phosphonylation. After IL-li starvation for 14 h, B9-TY1 cells werepreincubated with the indicated concentrations of GS or H7 for 40 mm (A) or with indicated concentrations of herbimycin A for 14 h (C), cells were then

stimulated with 500 ng/ml of IL-li for 20 mm. B, after B9-TY1 cells were incubated in the presence or absence of TPA (300 ng/mI) for 48 h, cells werestarved for 14 h and then stimulated with 500 ng/ml of IL-i 1 or with 300 ng/ml of TPA for 20 mm. The amounts of proteins equivalent to 2 x i05 cells/lane ti and B( or 1 X i0� cells/lane (C) were separated by 7.5% SDS-PAGE. Protein tyrosine phosphorylation was determined as described in ‘Materials

and Methods.” DMSO, dimethyl sulfoxide.

Cell Growth & Differentiation 605

A KDa

-205

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- + + + + + + + IL-li

50 100 H7(lzM)

- - - - 50 100 - - GS(�zg/mI)- - 005 01 006 01 - - DMSO(%)

1 i is due to the activation of protein tyrosine kinase, we

tested the effects of tyrosine kinase inhibitors genisteinand herbimycin A as well as a serine/threonmne kinaseinhibitor, H7, on protein tyrosine phosphorylation, andthe results are shown in Fig. 3, A and C. Induction ofprotein tyrosine phosphorylation by IL-i i was inhibitedby genistein or herbimycin A in a dose-dependent man-ner, but not by H7, which is a strong inhibitor for PKC,cGMP, and PKA. Furthermore, we have demonstratedthat PKC is not involved in IL-li-induced tyrosine phos-phorylation in B9-TYi cells, as shown in Fig. 3B. In theseexperiments, depletion of PKC with TPA treatment for48 h did notalter IL-u-induced tyrosine phosphorylationbut completely abolished TPA-induced tyrosinephosphorylation.

Activation of tisl 1, tis2l, and junB Early ResponseGenes by IL-li. It is believed that early response genesplay critical roles in growth factor-mediated signal trans-duction (8-13). We therefore examined the expressionof i 1 early response genes (tisi, tis7, tis8, tislO, tisi 1,tis2l, Ira-i, NGFJ-B, c-myc, c-los, and junB) in B9-TYicells treated with IL-i 1 . Northern blot analysis showedthat tisi 1, tis2l, and junB mRNAs were significantly in-creased after stimulation with IL-i 1 . The kinetic studiesshowed that both tisi 1 and junB genes were activatedwithin 15 mm after the addition of IL-i 1 (data not shown),

peaked at 30-60 mm, and then declined slowly thereafter(Fig. 4A). The elevated levels of tisi 1 and junB messageswere still observed 6 h following IL-i 1 stimulation. Thetis2l gene was constitutively expressed in B9-TY1 cellsand can be further induced by IL-li. The level of tis2lmRNA was significantly augmented following stimulationwith IL-i 1 for 60 mm and continued to increase for atleast 6 h. The roles of Us! 1, tis2l, and junB gene expres-sion in IL-li-induced growth signaling require furtherinvestigation.

- + ++ + + + - IL-Il

- - 01 1 3 . - - H.rblmycln A (,iglml)

.-- H76gM)- . 0.01 0.1 0.3 - 0.3 0.3 DMSO(%)

We also examined the molecular mechanisms respon-sible for the activation of tisi 1, tis2l, and junB genes byIL-i i . In these experiments, nuclear run-on transcriptionassays were used to assess whether the activation ofthese three early response genes by IL-i 1 is at the tran-scriptional level. IL-i 1 stimulation of B9-TY1 cells for 60mm significantly induced tisi 1, tis2l, and junB genetranscription in the isolated nuclei (Fig. 48). Pretreatmentof cells with cycloheximide for 30 mm did not affect thetranscription of these three primary response genes, in-dicating that posttranscriptional modifications may ac-count for the IL-i i-stimulated transcription of tisi 1, tis2l,and junB. Since the message levels of tisi 1, tis2l, andjunB are still much higher than the basal level 6 h afterthe addition of IL-il, we further examined the stabilityof these transcripts. The results showed that half-lives for(is! !, tis2!, and junB mRNAs are approximately 30, 30,and 60 mm, respectively (data not shown), indicating thathigh levels oftisl 1, tis2!, and junB mRNA expression arenot due to message stabilization. Taken together, theseresults suggest that transcriptional activation is the majormechanism oflL-ii-induced increases in Us! !, is2!, andjunB mRNAs.

Effects of Protein Kinase Inhibitors and Activators onIL-i i-induced tisi 1, tis2l, and junB Gene Expression. Asdemonstrated by immunoblotting, IL-i 1 rapidly inducedtyrosine phosphorylation of a 97/95 KDa protein. Tofurther understand whether tyrosine kinases are involvedin IL-il-induced (is! !, (is2!, and junB gene activation,we tested the effect oftyrosmne kinase inhibitors genisteinand herbimycin A on IL-il-induced expression of is!!,(is2!, and junB. As shown in Fig. SA, both genistein andherbimycin A significantly inhibited IL-li-triggered tis!!,(is2!, and junB gene expression. The inhibition of is!!,(is2!, and junB gene expression by genistein or herbi-mycin A is not due to nonspecific cytotoxicity, because

(mln)

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Fig. 4. Induction of primary response genes by IL-li. A, after IL-il andserum starvation for 16 h, B9-TY1 cells (5 x l0’/ml) were stimulated withIL-i 1 (500 ng/ml) for the indicated times, and the total RNAs were isolated

and analyzed by Northern blot (iO gg/lane) with tisl 1, tis2l, and junBgene probes. The B2-M probe was used as an internal control. B, nuclear

run-on transcription assays were performed with nuclei isolated from IL-il-deprived B9-TYi cells following (left lane) no stimulation; (middlelane) stimulation with 500 ng/ml of IL-i 1 for 60 mm; (right lane) pretreat-

ment with iO gg/ml of cycloheximide for 30 mm followed by stimulationwith 500 ng/ml of IL-i i for 60 mm. Labeled RNA probes were isolated

and hybridized to filters immobilized with 5 gg each of plasmid DNAscontaining tisll, tis2l, junB, B2-M DNA, or plasmid DNA without insert

as the negative control.

treatment of cells with 100 �g/ml of genistein for 120mm or with 3 j.zg/mI of herbimycmn A for 14 h did notaffect cell viability (data not shown), indicating that ty-rosine kinase(s) is indeed involved in IL-li-mediatedactivation of early response genes. We have also exam-ned the effect of the serine/threonine kinase inhibitor

H7 on early response gene expression. Surprisingly, ac-

tivation of tis! !, tis2!, and junB genes by IL-i 1 wascompletely blocked by pretreatment of cells with 25 �M

H7 for 30 mm (Fig. SA), and cell viability was not changedby H7 even at SO �tM concentration. These results sug-gested that H7-sensitive protein kinase(s) is also involvedin IL-i i-triggered activation of early response genes.

Since H7 is a general inhibitor for protein kinases, weexamined whether known protein kinase activators suchas TPA (PKC activator), dBcAMP (PKA activator),8BrcGMP (cGMP-dependent protein kinase activator),cholera toxin (GTP-bmnding protein activator), and cal-cium ionophore A23i87 (calcium/calmodulin-depend-ent protein kinase activator) can induce the same primaryresponse genes as those induced by IL-il. Among theactivators tested, only TPA can induce low levels of Usl!,(is2!, and junB gene expression when compared with IL-1 1 (Fig. 5, B and C, lel�. Further experiments showedthat depletion of PKC by 48-h treatment with TPA com-pletely abrogated the ability of TPA to induce (is ! !, (is2!,and junB gene expression but had no effect on IL-il-induced expression of these genes (Fig. SC, righ�. Theseresults implicate that the mechanisms for activation of(is! 1, (is2!, and junB genes by IL-i 1 are distinct fromthose of PKC, PKA, cGMP-dependent kinase, Ca2� cal-modulin-dependent kinases, or GTP-binding proteins. Asexpected, sphingosmne, a PKC inhibitor, did not inhibitthe expression of (is! !, (is2!, and junB genes triggeredby IL-i 1 . In addition, a calmodulin antagonist, W7, hadno effect on IL-li-induced tis! !, tis2!, and junB mRNAexpression (Fig. SD). These results, together with kinaseactivator studies, strongly suggest that tyrosine kinase(s)and H7-sensitive kinase(s), which are distinct from thoseof known kinase-second messenger systems, are requiredfor IL-il-triggered is! !, (is2!, and junB gene activation.

Discussion

Our initial observation that a small population of lL-6-dependent mouse B-cell hybridoma B9 cells are respon-sive to IL-li has allowed us to develop an IL-li-depend-ent cell line designated as B9-TYi . The growth of estab-lished B9-TY1 cells is dependent on either IL-i 1 or IL-6.Using IL-il-dependent B9-TY1 cells, we have investi-gated the possible intracellular signaling pathways lead-ing to cell growth mediated by IL-i i . The results dem-onstrate that IL-i 1 binding to its specific receptors re-suIted in the rapid tyrosine phosphorylation of a 97/95KDa protein. Interestingly, both 97 and 95 KDa tyrosine-phosphorylated proteins appeared at 1 mm, and theintensity of the 95 KDa tyrosmne-phosphorylated proteinis stronger than that of the 97 KDa protein. The 95 KDaband disappeared by 15 mm, whereas the intensity ofthe 97 KDa protein became much stronger when com-pared with that at 1 mm. We speculated that the 95 and97 KDa tyrosmne-phosphorylated proteins are the sameprotein with different levels of tyrosine phosphorylation.The behavior of 97/95 KDa tyrosmne-phosphorylated pro-teins induced by IL-li is also seen in other IL-li-respon-sive cell lines such as human TF-i leukemic cells, mouse313-Li preadipocytes, and rat H-35 hepatoma cells (datanot shown). The results with protein kinase inhibitors andactivators demonstrated that tyrosine phosphorylation ofthe 97/95 KDa protein by IL-i 1 is most likely through theactivation of tyrosine kinases other than PKC, PKA, andcGMP-dependent kinase.

We also examined the expression of primary responsegenes in IL-li-stimulated B9-TY1 cells. It was found that

606 Signal Transduction Mediated by IL-i 1

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three ((is! !, (is2!, and junB) of ii early response genestested were strongly activated by IL-li. (is2! is one ofthe immediate early response genes induced by a varietyof growth factors (16, 17). The available (is2! sequencesuggested that it may encode a transcriptional regulatoryprotein (i6), although the exact role of (is2l is not clearat the present time. (is! 1 gene encodes a novel proline-, serine-, and glycine-rich nuclear protein (i8-20) whichhas a novel zinc finger structure and binds to zinc. tis!!

gene expression can be induced by growth factors ormitogens in a variety of tissues, especially in fetal tissues

or growing liver cells, suggesting that the product of the(is! ! gene may be involved in cellular growth or differ-

entiation. The gene product of junB is a well-character-ized transcriptional regulator (2i, 22) which belongs toone of the jun/AP-i family of nuclear protooncogeneproteins. In fact, the protein encoded by the junB gene

can form a heterodimer with fos protein, which is in-volved in transcriptional regulation and binds with in-

creased affinity to AP-l sites or closely related sequences(2 1 ). Further studies need to be carried out to determinethe role of#{252}s!!, tis2!, and junB gene activation in IL-li-mediated signaling.

Studies with a variety of protein kinase inhibitors andactivators suggested that tyrosine kinases and H7-sensi-tive protein kinase(s) are essential for activation of (is!!,tis2!, and junB gene transcription by IL-il. Although

several lines of evidence have demonstrated that H7-

sensitive protein kinase(s) is distinct from any well-char-

acterized protein kinases such as PKC, PKA, cGMP, orcalcium/calmodulmn-dependent protein kinases (see “Re-suIts”), the mechanisms by which H7-sensitive proteinkinase(s) may function in IL-i 1 -mediated signal transduc-tion leading to the activation of is! !, (is2!, and junBgene expression remain to be clarified.

Recent investigations revealed that many growth fac-

tors such as IL-6 (23), leukemia-inhibitory factor (12, 24,25), oncostatin M (25, 26), and ciliary neurotrophic factor

(24), which have some overlapping biological activities,share common signal transduction mechanisms primarily

by using the same signal transducer, gpi3o. Since mostbiological activities of IL-i 1 are similar to those of IL-6,we compared possible signal transduction pathways uti-lized by IL-i 1 or IL-6. Our results indicate that the more

upstream signal, such as protein tyrosine phosphorylationtriggered by IL-i 1, is different from that of lL-6. In addi-tion to the 97/95 KDa tyrosine-phosphorylated protein,IL-6 stimulated tyrosine phosphorylation of three addi-tional proteins with molecular masses of 130, 150, and165 KDa in B9-TYl cells (data not shown). Studies with

a variety of protein kinase inhibitors revealed that IL-i 1and IL-6 induced the same primary response geneexpression (data not shown), and the mechanisms of(is! !, (is2!, and junB gene activation induced by IL-il orIL-6 are indistinguishable (data not shown). These data

are consistent with previous studies (12, 13) that tyrosinekinase(s) and H7-sensitive kinase(s) are involved in IL-6-mediated signal transduction. Our results suggest that IL-

Cell Growth & [)itterentiation 607

fig �. Ettects ot known proteinkinase inhibitors and activatorson to! 1, tis.!?, and junB gene

expression. A, after IL-i 1 andserum starvation for 16 h, B9-

TYt cells were preincuhatedwith indicated concentrations of

CS (�g/ml) or H7 hAM) ton 40 mmor with herhimvcin A Herh( (�.ig/mli or i4 h. and cells were then

stimulated vsith 500 ng/ml of IL-ii for 60 mm. B, after IL-li andserum starxation for t6 h, B9-TY1 cells were stimulated withIL-il (500 ng/mb; 1 ms�dBcAMP; 1 m� 8BrcCMP; 2 m�a

calcium onophore A23187; or 1

�ig/ml of cholera toxin for 60 mm.C. B9-TYi (ells were cultured in

the presence (right) or absence((eli) of 300 ng/ml of TPA for 48

h. then incubated in the absence0? IL-i i (or 14 h, followed bystimulation with IL-i i 1500 ng/

mb or TPA (300 ng/mh br 60

mm. 0. after IL-i i and serumstarvation for i6 h, B9-TYi cellswere pretreated with or without

10 CM SP or 40 �LM � for 30mm. cells were then stimulatedwith 500 ng/mI of lL�ii for 60mm. Total RNAs were isolated

and analyzed h� slot blot withI,’, i I . fis2 I . unB, or B2-M (inter-

nal controli probe.

608 Signal Transduction Mediated by IL-i 1

1 1 and IL-6 may share common downstream signalingevents with the possibility of using the same signal trans-ducer, gpi3o. Direct evidence for the involvement ofgpi3o in IL-li-mediated signal transduction remains tobe identified.

In conclusion, we demonstrated that IL-i i-inducedtyrosine phosphorylation is the first essential step toinitiate intracellular signaling leading to cell growth in B9-TY1 cells. The findings that IL-i 1 activated tis! !, tis2!,and junB gene transcription indicate that these threegenes may play some roles in controlling IL-i i-inducedcell growth. The studies with genistein, henbimycmn A,and H7 suggest that tyrosine kinases and H7-sensitiveprotein kinases are essential in IL-il-induced (is!!, tis2!,and junB gene expression and these H7-sensitive proteinkinases are distinct from those of PKC, PKA, cGMP, orcalcium/calmodulin-dependent protein kinases on GTP-binding proteins. Further identification and characteniza-tion of protein tyrosine kinases and H7-sensitive proteinkinases will be essential to elucidate the molecular mech-anisms of signal transduction mediated by lL-1 1.

Materials and Methods

Materials. Recombinant human IL-i 1 (specific activity,2.5 x 106 units/mg) was kindly provided by GeneticsInstitute (Cambridge, MA). TPA, dBcAMP, 8BrcGMP,cholera toxin, calcium ionophore A23i87, SP, W7, andalkaline phosphatase-conjugated goat anti-mouse IgG (Fcspecific) antibody were from Sigma Chemical Co. (St.Louis, MO); H7 was purchased from Seikagaku America(St. Petersburg, FL); GS and herbimycin A were fromGIBCO; antiphosphotyrosine monoclonal antibody wasfrom Upstate Biotechnology (Lake Placid, NY).

Cell Culture. B9 cells, an IL-6-dependent mouse B-cellhybnidoma cell line, were grown in RPMI 1640 mediumplus iO% fetal bovine serum and 40 units/mI of recom-binant human IL-6. B9-TY1 cells were subcloned fromB9 cells in the presence of 100 units/mI of human IL-i 1with semisolid cell culture system (27) and were grownin RPMI 1640 medium plus iO% fetal bovine serum inthe presence of 50 units/mI of human IL-i 1 . The growthof established B9-TY1 cells is dependent on either IL-i 1or IL-6. The viability of B9-TY1 cells after deprivation ofIL-il for 14-16 h is more than 95%.

Determination of Protein Tyrosine Phosphorylation byImmunoblotting. For protein tyrosine phosphorylation,B9-TYi cells were washed three times with serum-freeRPMI 1640 medium and incubated for i4-i6 h at 37#{176}C,5% CO2 in serum-free medium without any growth fac-tons. After factor starvation, cells (106/0.1 ml) were stim-ulated with IL-i 1 at 37#{176}Cfor different periods of time asindicated in “Results” and washed once with cold phos-phate-buffered saline containing 2 m�i sodium orthov-anadate. Then cells were lysed with 100 zl of lysis buffer(6), and the same amounts of solubilized proteins (equiv-alent to 1-2 x i0� cells/lane) were analyzed by 7.5%SDS-PAGE. Immunoblotting for determination of tyro-sine-phosphorylated proteins was performed as previ-ously described (6).

RNA Preparation and Analysis. IL-i i-deprived B9-TY1cells were stimulated with IL-i 1 for various times at 37#{176}C.Total RNAs were isolated by the method of Chomczynskiand Sacchi (28) with acid guanidmnium-thiocyanate-phenol-chloroform extraction. RNAs were either sepa-

rated by 1% aganose-formaldehyde gel (29) and trans-ferred to nitrocellulose membranes or directly applied tonitrocellulose membranes with the minifold II slot blotapparatus (Bio-Rad), according to manufacturer’sinstructions.

DNA Probe Preparation and Hybridization. DNAprobes were labeled by the random oligonucleotideprimer labeling method. Primary response gene probesused in these studies were is!, tis7, tis8, tislO, (is! !, tis2!(17), Ira-i (30), NCF!-B (31), junB, c-myc, and c-los (fromAmerican Type Culture Collection). B2-M probe (32) wasused as the internal control for the normalization of theamounts of RNA samples analyzed. Hybridizations wereperformed at 42#{176}Covernight (29). Membranes werewashed twice at 22#{176}Cin lx SSC (29) pIus 0.1% SDS andthen washed once at 65#{176}Cin O.2x SSC plus 0.1% SDS.Membranes were exposed on X-ray film, and relativeintensities of hybridization signals were quantitated witha Bio-Rad densitometer according to manufacturer’sinstructions.

Nuclear Run-on Assays. Preparation of nuclei and tnan-scription reactions were performed as described (22).Labeled RNA probes were purified and hybridized for44-48 h at 42#{176}Cin 10 m�i Tnis-HCI (pH 7.5), 4x SSC,0.1% SDS, i mM EDTA, 2x Denhardt’s solution, 40%formamide, and 100 �zg/mI tRNA to denatured plasmidDNAs (5 tag/slot) immobilized on nitrocellulose mem-branes. After hybridization, membranes were washedonce with 2X SSC-0.i% SDS at 37#{176}Cfor 30 mm, thentreated with RNase A in 2x SSC at 22#{176}Cfor S mm, andfinally washed once with 0.ix SSC-0.i% SDS at 42#{176}Cfor30 mm.

Acknowledgments

We would like to thank Dr. Paul Schendel at Genetics Institute, Cam-bridge, MA, for providing purified recombinant human IL-li; Dr. H.Henschman for tisi ,tis7, tis8, tisi 0, tisi 1,and tis2 1 plasmids; Dr. T. Curranfor fna-i plasmid; and Dr. J. Milbnandt for NGFI-B plasmid.

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