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Dissociation Between IFN--Induced Anti-Viral and Growth Signaling Pathways¹

Taruna Arora^*, Georgia Floyd-Smith, Mark J. Espy and Diane F. Jelinek^2,*

Departments of ^* Immunology and Microbiology, Mayo Clinic, Rochester, MN 55905; and Department of Biology and Molecular and Cellular Biology Program, Arizona State University, Tempe, AZ 85287

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ability of IFN- to induce an anti-viral state in a wide variety of cell types as well as to inhibit cellular growth has long been appreciated. It is less clear, however, whether both these effects lie downstream of a common signaling pathway. In this study we have taken advantage of an atypical human myeloma cell line (KAS-6/1) displaying a dramatic proliferative response to IFN- in an effort to resolve the signaling requirements for IFN--induced anti-viral and growth regulatory effects. Thus, we have analyzed the ability of IFN- to induce a number of known receptor-initiated events in this cell line and have compared these responses with those exhibited by a cell lineage- and maturation stage-matched myeloma cell line (ANBL-6) that displays typical IFN- responsiveness. Despite the widely contrasting effects of IFN- on cellular proliferation, IFN- was shown to be comparable in its ability to induce the expression of early response genes as well as induce resistance to viral infection in both cell lines. By contrast, the effects of IFN- on the activation of mitogen-activated protein kinase (MAPK) were strikingly distinct. Finally, although inhibition of MEK and MAPK activation had no effect on the induction of the anti-viral response, it completely blocked IFN--stimulated proliferation of the KAS-6/1 cells. In summary, our analysis of the role of the MAPK and anti-viral signaling pathways using these two cell lines suggests that the anti-viral and growth regulatory effects of IFN- display a differential requirement for activation of the MAPK pathway.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interferon- is commonly used as a therapeutic agent for the treatment of several malignancies, including multiple myeloma, because of its known anti-proliferative effects 1, 2, 3 . The effects of IFN- are mediated by ligand binding to the type I IFN receptor (IFNAR)³ complex. This receptor complex is composed of at least two subunits: the and ß subunits (reviewed in 4 . The ß subunit (IFNAR2) has been shown to bind ligand, and the subunit (IFNAR1) is required for the formation of high affinity receptors 5 . Both subunits undergo rapid ligand-dependent tyrosine phosphorylation, and the subunit itself acts as a species-specific transducer for type I IFN action 6 .

The lack of inherent kinase activity in these receptors is overcome through receptor-associated kinases of the Janus kinase (JAK) family. Thus, IFN- binding to its receptor triggers the rapid tyrosine phosphorylation of two members of the JAK kinase family, JAK1 and TYK2. These activated kinases subsequently mediate the tyrosine phosphorylation of both receptor subunits 7 . TYK2 and JAK1 kinases directly interact with the and ß subunits of the IFN- receptor, respectively. IFN- binding to its receptor also results in JAK kinase-mediated phosphorylation and activation of latent cytoplasmic transcription factors that function as STATs reviewed in Refs. 8, 9 . The phosphorylated factors, STAT1 and STAT2, along with a third component, p48, form a complex, IFN-stimulated gene factor-3, which activates transcription of IFN-stimulated genes (ISGs) through binding interactions with IFN-stimulated response elements (ISREs) in ISG promoters 10, 11, 12 . The end result of this pathway, therefore, is the expression of genes that mediate multiple biological activities.

At least 30 genes are known to be transcriptionally induced by type I IFNs including 2',5'-oligoadenylate (2'-5'A) synthetase, class I HLA molecules, and the IFN-responsive factor-1 (IRF-1) transcription factor (reviewed in 9 . For example, 2'-5'A synthetase is important for the anti-viral response, and its activity is required by cells to activate an endonuclease, RNase L, that degrades RNA 13, 14 . Indeed, lack of induction of 2'-5'A synthetase is observed to correlate with the inability of IFN- to generate antiviral responses in IFN--resistant cell lines 15, 16 .

Cell lines resistant to the anti-viral effects of IFN- are also usually resistant to the growth regulatory effects of IFN-, suggesting that these two responses are coordinately regulated downstream of a common signaling pathway. In this regard, the JAK-STAT signaling pathway has been implicated in mediating both the anti-proliferative and anti-viral effects of IFN-. The evidence for this conclusion is twofold. First, the induction of ISGs and an anti-viral response is defective in JAK1 and STAT1 knockout mice 17, 18 . Second, in studies using IFNAR1 mutant mice, JAK-STAT-dependent signaling appears to be required for both anti-proliferative and anti-viral responses 18 .

Although the JAK-STAT pathway is clearly important, a number of recent studies suggest that additional signaling pathways may also be important for IFN--dependent biological responses. The Ras-Raf-MEK-MAPK signaling pathway has been shown to be activated downstream of a variety of growth factors and cytokine receptors and has frequently been associated with mitogenesis (reviewed in 19 . Given the typical anti-proliferative effects of IFNs, it is interesting, therefore, that both type I and type II IFNs have recently been shown to stimulate Raf-1 and MAPK activation in a JAK1-dependent, but Ras-independent, manner 20, 21 . The cross-talk between the JAK-STAT and MAPK pathways in IFN signaling is further demonstrated by observations that IFN-ß stimulation results in MAPK activation and its direct association with STAT1 as revealed in coimmunoprecipitation studies 22 . Furthermore, in the same study expression of a dominant negative form of MAPK inhibited induction of a STAT-regulated ISG15 ISRE reporter construct. In other reports IFN- has also been shown to activate phosphatidylinositol 3-kinase (PI-3K), and treatment of cells with wortmannin appears to inhibit type I IFN-regulated MAPK activation 23 . Thus, there is increasing evidence to suggest the existence of alternative signaling pathways downstream from the type I IFN receptor. The roles that these pathways play in IFN--stimulated anti-viral and growth regulatory effects is unclear.

Previous studies in our laboratory have described the establishment of a human myeloma cell line, KAS-6/1, which displays nonclassical IFN- responsiveness. Thus, the KAS-6/1 cell line displays a proliferative response to IFN-, whereas three additional cell lines are growth arrested by IFN- 24 . This system, therefore, provides a novel means to assess the potential link(s) between JAK-STAT activation and other signaling pathways as well as the roles of these pathways in IFN--regulated cellular growth and induction of anti-viral activity. Our results show that the MEK-MAPK signaling cascade plays a critical role in mediating the mitogenic effects of IFN- in KAS-6/1 cells, but not in mediating the anti-viral response. The implication of these results, therefore, is that there is a dissociation between the signaling requirements of the anti-viral vs the growth regulatory effects of IFN-.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell lines, culture medium, and reagents

The myeloma cell lines ANBL-6 and KAS-6/1 were derived from primary patient myeloma cells and have been previously described 24, 25 . The cell lines were maintained in RPMI 1640 supplemented with 5% heat-inactivated FCS, 100 U/ml penicillin G, 50 µg/ml gentamicin, 100 µg/ml streptomycin, 2 mmol/l glutamine, and 1 ng/ml IL-6 (provided by Immunex, Seattle, WA). Recombinant purified IFN-2b (Schering, Kenilworth, NJ) was used at the indicated concentrations. H7 and wortmannin were purchased from Sigma (St. Louis, MO). PD098059 was purchased from Research Biochemical International (Natick, MA). Dr. Oscar Colamonici (University of Tennessee, Memphis, TN) provided mouse mAbs to the IFNAR1 (designated IFNAR3) and IFNAR2 subunits (designated IFNAR-ß1) of the IFN- receptor 26 . Mouse mAbs for immunoprecipitation of IFNAR1 (AA3 and GB8) were gifts from Biogen (Cambridge, MA). The monomorphic anti-human class I Ab, w6/32, was a gift from Dr. Larry Pease (Mayo Clinic, Rochester, MN). FITC-conjugated goat anti-mouse F(ab')₂ was purchased from BioSource (Camarillo, CA).

Proliferation assays

Before each assay, cells were washed three times with saline, resuspended in medium containing 0.5% BSA, and cultured in 96-well flat-bottom microtiter plates at an initial density of 2.5 x 10⁴ cells/well in a final volume of 200 µl for 3 days. DNA synthesis was quantitated by [³H]thymidine uptake using previously described methodology 27 .

Immunofluorescence analysis

Cell surface staining was analyzed using previously described methodology 27 . Briefly, cells (5 x 10⁵) were incubated with mAbs to the IFN- receptor or HLA class I for 30 min on ice, washed, and then incubated with FITC-conjugated goat anti-mouse IgG for another 30 min on ice. The cells were then washed, fixed with 1% paraformaldehyde, and analyzed for immmunofluorescence on a FACS Vantage (Becton Dickinson, Mountain View, CA). Mouse IgG1 and IgG2b (Becton Dickinson) were used as control Abs. Collected data were analyzed using PCLYSIS software (Becton Dickinson).

Immunoprecipitation and immunoblotting

Myeloma cells were cultured in IL-6-free medium for 48 h before IFN- addition. Cells (0.5–1.0 x 10⁷) were then lysed in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 10% glycerol, 0.1 mM EDTA, 200 mM NaCl, 0.5% Nonidet P-40, 1 mM Na₃VO₄, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 1 mM DTT, 0.2 mM PMSF, and 5 µg/ml aprotinin for 30 min on ice. For MAPK detection, 1% Triton was used as the detergent in the lysis buffer. The lysates were cleared of insoluble material by centrifugation for 10 min at 12,000 x g. For direct immunoblot analysis, cell lysates containing 50–75 µg of total protein were resolved by electrophoresis through 7.5% SDS-polyacrylamide gels and then transferred onto Immobilon-P membranes (Millipore, Bedford, MA). For immunoprecipitation of IFNAR1, cell lysates were first precleared with protein A-Sepharose (Pharmacia, Uppsala, Sweden). A total of 500 µg of precleared lysates were immunoprecipitated with 1 µg of AA3 anti-IFNAR1 Ab at 4°C for 2 h. The immune complexes were collected by adsorption to protein A-Sepharose beads by overnight agitation at 4°C. The immunoprecipitates were washed twice with lysis buffer and once with cold PBS. The complexes were then boiled in 30 µl of 2x SDS sample buffer and resolved on an SDS-PAGE gel followed by transfer to Immobilon-P membranes. The membranes were blocked in 25 mM Tris-HCl (pH 7.2), 150 mM NaCl, and 0.05% Tween-20 supplemented with 2% BSA and were subsequently probed with the indicated Abs. For reprobing the same blot, the membranes were stripped with 7 M guanidine and then renatured and blocked before immunoblotting with the indicated Abs. Immunoreactive proteins were detected using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL).

2'-5'A synthetase assay

ANBL-6 and KAS-6/1 cells were maintained as described above and then treated with diluent or 10, 100, or 1000 U of IFN- for 24 h. Cellular extracts were prepared as described previously 28 and stored at -70°C. Protein concentrations were determined using a protein assay kit (Bio-Rad, Richmond, CA) based on the method of Lowry. The 2'-5'A synthetase assay was performed essentially as described previously 28 with minor modifications. Poly(rI)-poly(rC) agarose (Pharmacia) was resuspended in 2'-5'A synthetase buffer (20 mm HEPES-KOH (pH 7.4), 120 mM KOAc, 25 mM Mg(OAc)₂, 2.5 mM DTT, and 0.2% Triton X-100) and washed twice in that same buffer. Binding reaction mixtures containing 4 µl of cellular extract and 20 µl of poly(rI)-poly(rC) agarose in a total volume of 40 µl were incubated at 4°C for 6 h and then centrifuged at 10,000 x g for 1 min. The supernatant was removed, and the pellet was washed two more times with 300 µl of 2'-5'A synthetase buffer. The reaction mixtures containing 3.3 mM [-³²P]ATP (sp. act. = 10 mCi/mmol) and 3.3 µM poly(I)-poly(C) in 2'-5'A synthetase buffer were incubated at 30°C for 18 h. The enzyme was heat inactivated, and the products of the reaction were analyzed by TLC as described previously. Autoradiography was used to identify the positions of reaction products, and the spots corresponding to 2'-5'A oligomers were excised and counted in a liquid scintillation counter.

Anti-viral assay

ANBL-6 and KAS-6/1 cells were treated with 500 U/ml of IFN- for 24 h and then challenged with 10¹⁰ particles of hCMV virus strain AD169 (American Type Culture Collection, Manassas, VA). After 72 h, virus-infected cells were quantitated by indirect fluorescence method using a CMV Brite kit (Biotest Diagnostics, Danville, NJ).

In vitro kinase assay

Cells were lysed on ice in lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM MgCl₂, 5 mM EGTA, 1% Triton X-100, 1 mM DTT, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 10 mM ß-glycerophosphate, and 0.1 mM sodium orthovanadate) and immunoprecipitated with MEK-1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and protein A-Sepharose for 2 h. Immunoprecipitates were washed twice with lysis buffer and twice with kinase buffer (20 mM HEPES (pH 7.5), 2.5 mM EGTA, 1 mM DTT, 10 mM ß-glycerophosphate, 0.1 mM Na₃VO₄, and 10 mM MgCl₂). The kinase reaction was conducted for 30 min at 30°C in a reaction buffer containing 1 µg/reaction GST-MAPK (kinase dead), 10 µCi of [-³²P]ATP/reaction, and 20 µM cold ATP. SDS-PAGE sample buffer was added, and proteins were eluted by boiling and resolved by 7.5% SDS-PAGE. The phosphorylated substrate was detected by autoradiography, and the counts per minute incorporated into substrate were quantitated by phosphorimage analysis.

RT-PCR

Total RNA was isolated from cells using the TRIZOL reagent and reverse transcribed using a first-strand DNA synthesis kit (Pharmacia). PCR to detect message for 6–16, IRF-1, 2'-5'A synthetase, ISG15, and ß-actin was performed in the presence of 1.5 mM MgCl₂, 1 mM dNTPs, 1 µM each of sense and antisense oligonucleotides, and 2.5 U of Taq DNA polymerase (Promega, Madison, WI). The primer sequences and their expected sizes are as follows: ß-actin: sense, 5'-GACTTCGAGCAAGAGATGGCCAC-3'; and antisense, 5'-CAATGCCAGGGTATGGTGGTG-3' (265 bp); 6–16: sense, 5'-CCTGCTGCTCTTCACTTGC-3'; and antisense, 5'-CCTCATCCTCCTCACTATCG-3' (352 bp); ISG15: sense, 5'-CCGTGAAGATGCTGGCG-3'; and antisense, 5'-CGAAGGTCAGCCAGAAC-3' (355 bp); IRF-1: sense, 5'-CGAAGTCCAGAGATG-3'; and antisense, 5'-CCTGGGCTGTCAATTTC-3' (461 bp); and 2'-5'A synthetase: sense, 5'-ATCAACAGTGCCAGA-3'; and antisense, 5'-GTCGTGAAGAGTGGTGC-3' (408 bp). Amplification of reverse-transcribed cDNA was performed for 30 cycles (denaturation at 95°C for 1 min, annealing at 60°C for 2 min, and extension at 72°C for 3 min). PCR products were visualized by electrophoresis on 2% agarose gels and staining with ethidium bromide.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
ANBL-6 and KAS-6/1 cell lines express functional IFNAR1 and IFNAR2 subunits

Initial experiments examined the cell surface expression levels of the IFN type I receptor components using mAbs specific for each component. As shown in Fig. 1A, both ANBL-6 and KAS-6/1 cell lines exhibited high expression levels of both and ß subunits of IFN type I receptor. No apparent differences in the density of surface expression of either subunit could be observed using these Abs.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. A, Cell surface expression of and ß subunits of the IFN- receptor. Expression levels of receptor subunits on ANBL-6 and KAS-6/1 cells were analyzed by flow cytometry using Abs specific for and ß subunits. Staining with the isotype-matched control mAb is indicated by the solid lines, and staining for the -chain (upper panels) and ß-chain (lower panels) is indicated by the dotted lines. B, IFN- binding leads to receptor activation. Lysates from unstimulated and IFN--stimulated ANBL-6 and KAS-6/1 cells were immunoprecipitated using Ab specific for IFNAR1 ( subunit), resolved by SDS-PAGE, and Western blotted with anti-ptyr (4G10) Ab (upper panel). The membrane was then probed for IFNAR1 (bottom panel).

IFN- induces similar expression of known ISGs in both cell lines

Using gel shift assays, IFN--stimulated activation of STAT1, -2, and -3 in both cell lines was previously demonstrated 24 . To assess the functional role of IFN--dependent JAK kinase activation in vivo, we next analyzed the induction of a variety of known ISGs. As shown in Fig. 2A, a 2-h treatment of both ANBL-6 and KAS-6/1 cells with IFN- induced expression of mRNA for 6–16, ISG15, and IRF-1 ISGs. However, no changes in ß-actin mRNA expression were observed under the same conditions. We also assayed the effects of IFN- on HLA class I induction by flow cytometry using a monomorphic HLA class I Ab, w6/32. As shown in Fig. 2B, IFN- treatment of both ANBL-6 and KAS-6/1 cells for 24 h resulted in an increase in HLA class I expression. Moreover, IFN- treatment resulted in a comparable increase in class I expression in both cell lines (2.5- vs 2.0-fold induction in ANBL-6 and KAS-6/1 cells, respectively).

View larger version (42K): [in this window] [in a new window]   FIGURE 2. IFN- stimulates ISG induction in ANBL-6 and KAS-6/1 cells. A, Cells were left unstimulated or were stimulated for 2 h with IFN-. Total RNA was prepared, and the expression of 6–16, IRF-1, and ISG15 was assessed by RT-PCR. As a control, ß-actin was also amplified from the same cDNA. B, Cells were cultured with or without IFN- for 24 h. Cell surface expression of HLA class I was examined by flow cytometry using polyclonal Ab w6/32. Dashed lines indicate staining with control Ab, dotted lines indicate class I expression on unstimulated cells, and solid lines indicate class I expression following IFN- stimulation. The results are representative of multiple experiments.

2'-5'A synthetase activity is strongly induced by IFN- in both growth-inhibited and growth-stimulated cell lines

Anti-proliferative responses to IFN- are frequently accompanied by the induction of anti-viral responses. These responses correlate with enhanced expression of another ISG, 2'-5'A synthetase 13, 16 . Therefore, we next examined the ability of IFN- to induce the enzymatic activity of 2'-5'A synthetase in IFN- growth-inhibited ANBL-6 and growth-stimulated KAS-6/1 cells. Both cell lines displayed an increase in the activity of 2'-5'A synthetase upon IFN- stimulation (Fig. 3). However, the ANBL-6 cells showed a greater induction (10-fold) of 2'-5'A synthetase activity than the KAS-6/1 cells (5-fold) at low doses of IFN- (10 U/ml).

View larger version (63K): [in this window] [in a new window]   FIGURE 3. Induction of 2'-5'A synthetase activity by IFN- stimulation. Cells were cultured without (open bars) or with 10 (striped bars), 100 (dotted bars), or 1000 (solid bars) U/ml IFN- for 24 h, and cellular extracts were assayed for enzyme activity of 2'-5'A synthetase using a reaction mix containing [-32P]ATP and poly(I)-poly(C). Products were separated by TLC, and radioactivity incorporated into 2'-5'A oligomers was quantified by scintillation counting. The data are representative of results obtained from two independent experiments.

IFN- treatment results in resistance to hCMV infection despite the differential growth responsiveness of ANBL-6 and KAS-6/1 cells

In an effort to extend the results shown in Fig. 3, we next assayed the ability of IFN- to induce an anti-viral response in ANBL-6 and KAS-6/1 cells. For these experiments, the ability of IFN- to provide protection against hCMV infection of these myeloma cell lines was studied. As shown in Table I, both ANBL-6 and KAS-6/1 cell lines were protected against hCMV virus infection by IFN-. In contrast, cells without IFN- pretreatment showed no protection against hCMV infection. These results suggest that there is a dissociation between the anti-viral effects of IFN- and its effects on growth regulation.

View this table: [in this window] [in a new window]   Table I. Effects of IFN- on the induction of anti-viral responses in myeloma cells1

IFN- stimulates MAPK activation in KAS-6/1 cells

In addition to activation of the JAK-STAT signaling cascade, several recent reports have demonstrated that IFN- stimulation causes activation of MAPK, and this may occur in a JAK1-dependent and Ras-independent manner 19, 20 . To determine whether this signaling pathway was also activated in the ANBL-6 and KAS-6/1 cells, we next analyzed MAPK activation using Abs that detect the active phosphorylated forms of both ERK1 and ERK2. As shown in Fig. 4, the two cell lines differed dramatically when IFN- was assessed for its ability to induce MAPK activation. Thus, whereas IFN- rapidly induced the appearance of both phospho-ERK1 and phospho-ERK2 in the KAS-6/1 cells (Fig. 4A), IFN- had virtually no effect on MAPK activation in the ANBL-6 cells (Fig. 4B). By contrast, IL-6, a known growth factor for these cells, was fully capable of stimulating MAPK activation in the ANBL-6 cells (Fig. 4C). Reprobing for ERK1 and ERK2 showed that both cell lines express the ERK2 isoform predominantly. In experiments not shown, when MEK activation was assessed in vitro using a kinase-dead GST-MAPK fusion protein, similar results were obtained. Thus, IFN- stimulated an eightfold induction in MEK activity over unstimulated cells, whereas it was essentially without effect on the ANBL-6 cells.

View larger version (58K): [in this window] [in a new window]   FIGURE 4. Kinetics of MAPK activation in ANBL-6 and KAS-6/1 cells. ANBL-6 cells were stimulated with 1 x 104 U/ml of IFN- (B) or 33 ng/ml of IL-6 (C) for the indicated lengths of time. KAS-6/1 cells were stimulated with IFN- under similar conditions (A). For the positive control, cells were stimulated with 10 ng/ml PMA for 5 min. Cellular extracts were prepared, resolved on 10% SDS-PAGE, transferred to Immobilon-P membranes, and immunoblotted with Ab specific for threonine-tyrosine-phosphorylated p44 ERK1 and p42 ERK2. The membranes were stripped and reprobed for total MAPK using Ab specific for p44 ERK1 and p42 ERK2. The results are representative of multiple experiments.

Pretreatment with the MEK inhibitor, PD098059, abrogates IFN--dependent proliferation of the KAS-6/1 cell line

The results described above indicated that IFN- increased phosphorylation of ERK1 and ERK2 in KAS-6/1 cells but not in ANBL-6 cells. These results suggested that the divergent IFN- responses displayed by the two cell lines may, therefore, result from differential activation of the MAPK signaling pathway. Because the data shown in Table I also suggested that the cell lines are comparably rendered resistant to viral infection by IFN-, the results collectively suggest a dissociation between the anti-viral and growth signaling pathways. To begin to address this possibility in greater detail, we next studied the effect of specific inhibitors of signaling pathways on IFN-stimulated thymidine uptake in KAS-6/1 cells. As shown in Fig. 5A, the serine-threonine kinase inhibitor H7, had no effect on IFN--dependent thymidine uptake in KAS-6/1 and ANBL-6 cells. This inhibitor has been shown by other investigators to disrupt the JAK-STAT signaling pathway downstream of the IFNAR and gp130-linked receptors 29, 30 . In Fig. 5B, it may also be seen that wortmannin, when used at concentrations that result in specific inhibition of PI-3K, did not alter the effects of IFN- on the proliferation of either cell line. However, pretreatment of KAS-6/1 cells with a specific inhibitor of MEK, PD098059, resulted in complete inhibition of IFN--mediated proliferation in a dose-dependent manner (Fig. 5C). By contrast, this inhibitor was without any effect on IFN--mediated growth inhibitory responses in the ANBL-6 cell line (Fig. 5C). To verify that PD098059 was indeed inhibiting MEK activity, two experimental approaches were employed. First, we determined the ability of PD098059 to block MEK activation in IFN--stimulated KAS-6/1 cells. As shown in Fig. 6A, IFN- stimulated a dramatic increase in MEK activity when assayed after a 15-min stimulation. Of note, PD098059 completely inhibited this induction. Second, we assessed the ability of PD098059 to block the appearance of IFN-stimulated phospho-ERK1 and phospho-ERK2 (Fig. 6B). Again, IFN- stimulated MAPK activation in the KAS-6/1 cells. Although H7 was ineffective in inhibiting MAPK activation, PD098059 was completely effective in this regard. In experiments not shown, wortmannin was similarly found to be without effect on MAPK activation. These results suggest, therefore, that activation of MAPK plays a critical role in IFN--stimulated KAS-6/1 proliferation.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Effects of inhibitors on IFN--mediated thymidine uptake. ANBL-6 and KAS-6/1 cells were cultured at a density of 2.5 x 104 cells/well in triplicate in medium containing 0.5% BSA and were pretreated with DMSO or the indicated concentrations of H7 (A), wortmannin (B), and PD098059 (C) for 1 h before addition of 1 x 103 U/ml IFN-. Cells were pulsed with [3H]thymidine for the last 18 h of 3-day culture, and thymidine incorporation was measured using a liquid scintillation counter. The data are representative of multiple experiments. Error bars not shown are within the dimensions of the symbols.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Effects of inhibitors on IFN--stimulated MEK and MAPK activity in KAS-6/1 cells. A, KAS-6/1 cells were pretreated with DMSO or 25 µM PD098059 for 1 h before addition of 1 x 104 U/ml IFN- for the indicated periods of time. For a positive control, cells were also stimulated with 10 ng/ml of PMA for 15 min. MEK1 was immunoprecipitated from the lysates, and its activity was assessed by an in vitro kinase assay using [-32P]ATP and GST-MAPK as the substrate. As an additional control, immune complexes isolated using a nonspecific Ab were incubated with [-32P]ATP and substrate. The data indicate the fold increase in GST-MAPK phosphorylation over that in unstimulated cells. B, KAS-6/1 cells were pretreated with DMSO, 50 µM H7, or 25 µM PD098059 for 1 h followed by addition of 1 x 104 U/ml of IFN- for the indicated time periods. Whole cell lysates were resolved by SDS-PAGE, transferred to membranes, and immunoblotted with phophoERK1/ERK2-specific Ab (upper panel). Membranes were stripped and reprobed for total MAPK (bottom panel). The results are representative of multiple experiments.

Inhibition of the MAPK pathway does not affect IFN--stimulated ISG induction or anti-viral responses

Because the results presented above demonstrated that IFN- triggers an anti-viral response and a MAPK-dependent proliferative response in the KAS-6/1 cells, we next wanted to determine whether IFN--stimulated MAPK activation was required in the anti-viral response. To address this question, we examined the effects of PD098059 and H7 on JAK-STAT signaling-dependent induction of ISGs, which are known to be expressed during anti-viral responses. As shown in Fig. 7, IFN- stimulated expression of IRF-1 and 2'-5'A synthetase mRNA was inhibited by pretreatment of ANBL-6 and KAS-6/1 cells with H7. By contrast, addition of PD098059 was largely without effect on IFN--mediated induction of these genes in both cell lines. Further analysis of IFN--stimulated anti-viral responses showed that although H7 pretreatment did not interfere with IFN--stimulated MAPK activation (Fig. 6), H7 pretreatment significantly blocked the IFN--mediated protective responses against hCMV infection of both myeloma cell lines (Fig. 8). Of interest, PD098059, which inhibited IFN--stimulated MEK and MAPK activities in KAS-6/1 cells (Fig. 6, A and B), had no effect on IFN--dependent resistance against hCMV infection in either cell line. These results suggest that although IFN- stimulates strong activation of MAPK in the KAS-6/1 cells, this signaling pathway is not essential for the anti-viral response.

View larger version (56K): [in this window] [in a new window]   FIGURE 7. Effects of H7 and PD098059 on 2'-5'A S and IRF-1 mRNA expression. ANBL-6 (left panel) and KAS-6/1 (right panel) cells were pretreated with 50 µM H7 and 25 µM PD098059 for 1 h followed by stimulation with IFN- for 2 h. Total RNA was prepared, reverse transcribed, and PCR amplified using primers for 2'-5'A synthetase, IRF-1, and ß-actin. The results are representative of multiple independent experiments.

View larger version (54K): [in this window] [in a new window]   FIGURE 8. Effects of H7 and PD098059 on IFN--induced anti-viral effects. ANBL-6 and KAS-6/1 cells were pretreated with 50 µM H7 or 25 µM PD098059 for 1 h followed by addition of 1 x 103 U/ml of IFN-. Twenty-four hours later, cells were infected with hCMV. Immunohistochemistry was performed 48 h postinfection to detect hCMV early Ag. The data are representative of results obtained from multiple experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our studies first focused on the possibility that the differential IFN- responsiveness resulted from differences in IFN receptor expression or activation of the proximal kinases. However, our results demonstrated that ligand binding to the IFN- receptor resulted in similar ligand-induced tyrosine phosphorylation of the IFN- receptor as well as that of the receptor-associated kinases JAK1 and Tyk2. The IFN type I receptor has been demonstrated to be a multichain complex in which the subunit can complex with either a truncated, short form or a long form of the ß subunit 4 . The long form of the ß subunit is required for the activation of both JAK1 and Tyk2 4, 5 . Although our experiments have not directly addressed what form of the ß subunit is expressed in these two cell lines, our observations that both JAK1 and Tyk2 were activated following IFN- stimulation suggest that both cell lines express a functional long form of the ß subunit of the type I IFN receptor.

We also analyzed the ability of IFN- to induce a number of known ISGs, particularly those known to be involved in the anti-viral response. However, we again failed to detect any differences in the induction of 6–16, ISG15, IRF-1, MHC class I, and 2'-5'A synthetase. In another study 24 we have shown that IFN- also stimulates the DNA binding activity of IRF-1, a transcription factor with growth inhibitory potential, in both growth-inhibited ANBL-6 and proliferating KAS-6/1 cells. IFN- also induces expression of an ISG that encodes for protein kinase R, which has been shown to play a role in the growth inhibition by down-regulating c-Myc expression 31 . However, our analysis of c-Myc expression did not indicate any alteration in its expression by IFN- in the ANBL-6 and KAS-6/1 cell lines 32 . The possibility remains, however, that other as yet to be identified ISGs may be differentially induced in these cell lines. Induction of these ISGs is mediated by binding of IFN-stimulated gene factor-3 to the ISREs in the promoters of these genes. In this regard, we have previously shown that STAT1 and STAT2 are activated in similar fashion in both cell lines 24 . Also, treatment with H7 inhibited IFN--dependent induction of IRF-1 and 2'-5'A synthetase as well as increased the sensitivity to hCMV viral infection. H7, a serine-threonine kinase inhibitor, has previously been shown to inhibit IFN--dependent ISG induction and anti-viral responses 33 . It is proposed to act by inhibiting serine phosphorylation of STAT1 and STAT3 factors 30, 34 . All these observations further support our conclusions that the JAK-STAT pathway is activated by IFN- in KAS-6/1 cells in a similar manner as in the growth-inhibited ANBL-6 cells and that it may support anti-viral responses in both cell lines.

Results from mutant cell lines and receptor knockout models have led to the understanding that a common receptor-initiated signaling pathway is required for both anti-viral and anti-proliferative responses 6, 16, 35 . Thus, because IFN- did not induce an anti-proliferative response in the KAS-6/1 cells, it was possible that IFN- would similarly be ineffective at inducing an anti-viral response in these cells. However, our analysis surprisingly revealed that anti-viral responses, as assayed by measuring 2'-5'A synthetase activity and by the ability of IFN- to protect against hCMV viral infection, were induced in KAS-6/1 cells. Thus, despite the contrasting growth responsiveness to IFN-, anti-viral responses were comparably induced in the ANBL-6 and KAS-6/1 cells. Ghislain et al. 5 have suggested that low affinity interactions of IFN- with its receptor subunits are sufficient for anti-viral responses. By contrast, high affinity interactions are required for anti-proliferative responses, and these interactions depend upon expression and function of the subunit of IFN receptor. Therefore, it is possible that the absence of an anti-proliferative response in the KAS-6/1 cells could result from the lack of expression of a functional subunit of IFNAR. However, our analysis of IFNAR expression revealed that the subunit was expressed by both ANBL-6 (growth-arrested) and KAS-6/1 (growth-stimulated) cell lines and was tyrosine phosphorylated following IFN- stimulation. Collectively, our data demonstrating comparable receptor expression, JAK/STAT activation (this study and 24 , ISG induction, and induction of anti-viral responses in two cell lines that differ dramatically with respect to the growth regulatory effects of IFN- strongly suggest that the anti-viral and growth regulatory effects are differentially regulated.

Several independent observations have also suggested that more than one pathway may operate to transduce IFN- signals. For example, the differential responses of HeLa M and other HeLa cell lines to IFN- suggest that alternative signaling pathways may exist for IFN--mediated biological responses 36 . Our observations are consistent with these studies and further suggest that within the same cell, IFN- binding may trigger more than one signaling pathway to mediate different biological effects. Previously, it has been shown by Petricoin et al. 37 that the anti-proliferative effects of IFN- in T cells require CD45 and ZAP-70. Consistent with the knowledge that plasma cells typically lack CD45 expression, both the myeloma cell lines studied in this report similarly lack expression of CD45. This observation suggests that different mechanisms may be operative in myeloma cells vs T cells. In addition, our analysis of Syk, a member of the ZAP-70 family expressed by B-lineage cells, failed to reveal an effect of IFN- on Syk activity in either growth-stimulated KAS-6/1 or growth-inhibited ANBL-6 cells (T. Arora and D. F. Jelinek, unpublished observations). PI-3 kinase has also been suggested to play a role in the stimulation of the JAK-STAT signaling pathway by IFN- 23 . However, IFN- did not stimulate PI-3K activation in ANBL-6 and KAS-6/1 cells as measured by p85 tyrosine phosphorylation (T. Arora and D. F. Jelinek, unpublished observations), nor did wortmannin alter IFN--regulated growth responses. Finally, although an H7-sensitive kinase has been implicated as being important in IFN responsiveness, H7 did not alter the growth signaling properties of IFN-. H7 was, however, effective at blocking the anti-viral effects of IFN- on KAS-6/1 cells and ANBL-6 cells, albeit to a lesser degree.

Our data clearly demonstrated that IFN- stimulation resulted in differential induction of the MAPK signaling pathway. Thus, a strong induction of MEK and MAPK activity correlated with IFN--stimulated proliferative responses in KAS-6/1 cells. Further, our results demonstrated that this mitogenic signaling pathway does not appear to be required for IFN--induced anti-viral responses in KAS-6/1 cells. Overall, our data suggest that in the KAS-6/1 myeloma cell line, strong activation of the MAPK signaling pathway may be the predominant mechanism that underlies the atypical IFN--induced growth response.

Our results differ from those of other studies that have suggested a role for MAPK activation in IFN-mediated classical biological responses. Thus, David et al. 22 have demonstrated that IFN-ß binding to its receptor resulted in MAPK activation and coimmunoprecipitation with STAT1 in cell lines that are growth inhibited by IFN. Other reports have also suggested that serine phosphorylation of STAT1 by MAPK results in enhanced expression of ISGs by IFN 34 . Our results show that IFN- is ineffective in stimulating measurable MEK or MAPK activity in the growth-inhibited ANBL-6 cells. These results suggest that MAPK activation can lead to two different outcomes in gene transcription. Thus, strong activation of the Ras-MAPK pathway may result in the activation of transcription factors that play a role in mitogenic signaling. By contrast, weak MAPK activation may be sufficient in mediating phosphorylation of STAT factors that may be important in the anti-proliferative response but may fail to trigger events necessary for cell cycle progression. In this regard, we have previously shown that IFN- induces expression of the p19ink4D CDK inhibitor in ANBL-6 cells 38 . By contrast, IFN- treatment of KAS-6/1 cells resulted in the induction of cyclin D2 expression within 6 h of stimulation in the absence of an effect on p19ink4D expression. Therefore, it is possible that the inability to strongly activate MAPK coupled with the expression of cell cycle inhibitory proteins leads to an anti-proliferative response in these cells, whereas strong activation of MAPK coupled with the induction of cell cycle-promoting proteins result in growth stimulation by IFN-. It remains possible, therefore, that the strength and duration of MAPK activation critically govern the differential induction of cell cycle regulatory proteins by IFN- and hence growth responses in ANBL-6 and KAS-6/1 cells.

In conclusion, our results demonstrate that in type I IFN signaling in KAS-6/1 cells, an independent MEK-MAPK pathway is activated and is required for IFN--stimulated cellular proliferation. The mechanism by which MAPK is strongly activated in KAS-6/1 cells but not in ANBL-6 cells, however, remains unclear but is currently under investigation. Our analysis of the JAK-STAT pathway, ISG expression, and anti-viral responses shows that this pathway is intact in both cell lines and does not require MAPK activation. Therefore, these results suggest that an additional signaling pathway(s) downstream of the IFN- receptor may affect the growth regulatory properties of IFN-. Thus, our observations suggest that IFN--stimulated classical anti-viral and atypical proliferative responses within the same cell line are mediated independently by JAK-STAT and MAPK pathways, respectively. Our studies have not ruled out the possibility that a common receptor-proximal signaling pathway may diverge at a later point to mediate different biological responses. Therefore, it remains feasible that IFN- may trigger one signaling pathway (JAK-STAT) in the proximity of the receptor, which may later branch off to mediate growth responses via strong activation of the MEK-MAPK pathway and anti-viral responses by activation of alternative pathways.

	Acknowledgments

 
We thank Bonnie K. Arendt and Renee C. Tschumper for their excellent technical support.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants CA64442 and CA62228.

² Address correspondence and reprint requests to Dr. Diane F. Jelinek, Department of Immunology, Rochester, MN 55905. E-mail address:

³ Abbreviations used in this paper: IFNAR, type I IFN receptor; JAK, Janus kinase; TYK, tyrosine kinase; ISG, IFN-stimulated gene; ISRE, IFN-stimulated response element; 2'-5'A, 2',5'-oligoadenylate; IRF-1, IFN-responsive factor-1; PI-3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; h, human; GST, glutathione-S-transferase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase.

Received for publication October 29, 1998. Accepted for publication December 16, 1998.

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