Dept of Immunology, Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, MN, USA

Correspondence to: D F Jelinek, Department of Immunology, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905; Fax: 507-266-0981

Interleukin 6 (IL-6) has been shown to be a key growth factor for myeloma cells. To study IL-6 signal transduction in multiple myeloma (MM), we employed chimeric receptors composed of the epidermal growth factor receptor (EGFR) extracellular domain, gp130 transmembrane domain, and full-length or truncated gp130 cytoplasmic domains lacking regions previously shown to be necessary for MAPK, STAT1, and STAT3 activation. The IL-6-dependent KAS-6/1 MM cell line was transfected with various chimeric receptor constructs and assayed for EGF responsiveness. EGF stimulation surprisingly stimulated DNA synthesis in all transfectants, regardless of receptor length. When cell proliferation was assayed instead, only transfectants capable of inducing high levels of STAT3 activation proliferated in response to EGF. Additional studies revealed that EGF stimulation resulted in tyrosine phosphorylation of endogenous gp130 in cells expressing the chimeric receptor. Replacing the gp130 transmembrane region with the EGFR transmembrane domain diminished but did not disrupt this interaction. This receptor interaction was also observed in the IL-6-dependent MM cell line ANBL-6. In summary, although our results suggest that STAT activation is crucial in gp130-mediated proliferation of myeloma cells, these results must be interpreted with caution given our demonstration of the interaction between chimeric and endogenous receptors in myeloma cells. Importantly, this interaction has not been noted in studies utilizing the same gp130 chimeric receptor system in non-MM cells.

Leukemia (2002) 16, 1189-1196. DOI: 10.1038/sj/leu/2402516

interleukin-6; gp130; multiple myeloma; chimeric receptor; lateral signal transduction

Introduction

Multiple myeloma (MM) accounts for approximately 1% of all malignant disease and over 12% of all hematological malignancies. This disease is characterized by the accumulation of malignant plasma cells in the bone marrow. There is substantial evidence that IL-6 plays an important role in multiple myeloma.¹ Although IL-6 stimulates differentiation (eg enhanced immunoglobulin secretion) of normal plasmablasts, IL-6 acts as a growth factor for many myeloma cell lines and fresh patient samples.^1,2,3 This altered response may result from the development of unique signaling pathways downstream of the IL-6 receptor. However, the mechanism(s) by which IL-6 stimulates growth rather than differentiation of myeloma cells remains to be elucidated.

The IL-6 receptor is a multi-subunit complex composed of the IL-6 binding subunit, IL-6R (gp80), and the signal transducing subunit, gp130. Although gp130 does not display intrinsic kinase activity, the membrane proximal Box 1 motif permits non-receptor tyrosine kinases, Janus kinases (JAKS), to constitutively associate with the receptor. Upon IL-6 binding to gp80, gp130 homodimerizes resulting in the juxtaposition of JAKs and cross-phosphorylation of each other. Activated JAKs subsequently phosphorylate tyrosine residues within the cytoplasmic tail of gp130, thereby providing docking sites for SH2 domain-containing molecules, such as SHP-2 and STAT factors.⁴ SHP-2 binding requires Y759 and leads to MAPK activation via a large complex of adaptor proteins including Gab1, Grb2, and Vav.^5,6,7,8 Y905 and Y915 are associated with STAT1 activation while Y767, 814, 905, and 915 facilitate STAT3 activation.^9,10

Although our current knowledge of IL-6 signal transduction largely derives from non-myeloma cell systems, there are numerous reports in the literature demonstrating the ability of IL-6 to stimulate tyrosine phosphorylation of JAK/STAT, MAPK, PTPID, Gab1, Grb2, SHC, Vav, and Src-family tyrosine kinases, and GTP-loading of p21^ras in myeloma cells.^{6,11,12,13,14,15,16,17} Despite the knowledge that an array of signaling molecules become phosphorylated in response to IL-6 stimulation, the role that these molecules play in the IL-6 proliferative response of myeloma cells remains largely unknown. There is evidence, however, using antisense oligonucleotides and constitutively active ras constructs,¹⁸ that the Ras/MAPK pathway may be important for the IL-6 proliferative response. More recently, De Vos et al¹⁹ reported that the JAK2 tyrosine kinase inhibitor, tyrphostin AG490, downregulated IL-6-induced ERK2 and STAT3 phosphorylation in a manner that paralleled inhibition of IL-6-induced growth. However, relatively high concentrations of AG490 were required to mediate complete growth inhibition, therefore, perhaps increasing the possibility of non-specific inhibition of other signaling molecules.

Chimeric receptor studies are an attractive alternative to using biochemical inhibitors, and such studies have contributed significantly to the mapping of receptor signaling pathways, including pathways downstream of the IL-6R complex.^{10,19,20,21,22,23} The altered extracellular domain of the chimera allows receptor activation via a ligand that normally induces no cellular response thereby allowing definition of the requisite signaling intermediates engaged by the cytoplasmic domain of the receptor of interest. The success of this approach is critically dependent on activation of the chimeric receptor in the absence of an interaction with endogenous wild-type receptor (ie gp130). Indeed, numerous studies have documented the feasibility of chimeric receptor systems in a variety of cell types.^10,21,24,25

Although a chimeric receptor system has not been previously used to directly study IL-6-stimulated myeloma cell growth, IL-3-dependent Ba/F3 murine pre-B cells transfected with human gp130 displayed a proliferative response upon stimulation with soluble IL-6R and IL-6. This system further demonstrated that Ba/F3 cells only required a 61-amino acid region of gp130 proximal to the transmembrane region in the proliferative response to IL-6 and soluble IL-6R.²⁶ Thus, the proliferation of these cells did not apparently require residues 703-918, the region of gp130 that houses tyrosine residues implicated in SHP-2, STAT1, and STAT3 binding. In contrast, other investigators used Ba/F3 cells transfected with a panel of granulocyte colony-stimulating factor receptor (G-CSFR)-gp130 chimeric receptors and found that both the JAK/STAT and Ras/MAPK pathways worked co-operatively to sustain cell survival.²⁴ Thus, even in this somewhat artificial model, the relative importance of the Ras/MAPK and JAK/STAT pathways in IL-6-induced cell proliferation is controversial.

To further investigate the signaling requirements for IL-6-induced myeloma cell proliferation, we employed a chimeric receptor approach and took advantage of our access to IL-6-dependent myeloma cell lines. Despite the widespread utility of this approach in other cell systems, our results revealed an unforeseen interaction between chimeric and endogenous receptors. Because this interaction has not been noted in studies utilizing the same gp130 chimeric receptor system in non-myeloma cells, our results have interesting implications and support the hypothesis that IL-6 receptor interactions may be altered in MM cells.

Materials and methods

Cell lines, culture medium, and reagents

The myeloma cell lines KAS-6/1³ and ANBL-6²⁷ were derived in our laboratory. Each cell line was maintained in RPMI 1640, 1 ng/ml IL-6 (generously provided by Novartis Pharma, Basel, Switzerland), and 10% FCS. Recombinant EGF was purchased from Biosource (Camarillo, CA, USA). The phycoerythrin-conjugated anti-EGFR monoclonal antibody (mAb) as well as the isotype-matched control was purchased from Pharmingen (San Diego, CA, USA). The B-R3 anti-gp130 antibody (Biosource) was used for immunoprecipitation, and an anti-c-myc antibody (Calbiochem, San Diego, CA, USA) was used for both immunoprecipitation and Western blotting. Both the anti-gp130 and anti-phosphotyrosine (pY) antibodies used for Western blotting were purchased from Upstate Biotechnology (Lake Placid, NY, USA). The anti-phospho-ERK1/2 (E10), anti-phospho-STAT3 (Y705), and anti-STAT3 antibodies were purchased from New England Biolabs (Beverly, MA, USA). The anti-ERK1/2 antibody was purchased from Transduction Laboratories (Lexington, KY, USA). HRP-linked anti-mouse or rabbit IgG secondary antibodies and an enhanced chemiluminescence (ECL) detection system were purchased from Amersham (Arlington Heights, IL, USA).

EGFR/gp130 constructs and generation of stable transfectants

The EGFR/gp130 chimeric receptor constructs (cloned into the SR expression vector) were generously provided by Dr Neil Stahl (Regeneron Pharmaceuticals).¹⁰ The chimeric receptor constructs were first subcloned into the pCI-Neo expression vector (Promega, Madison, WI, USA). The EGFRTM chimeric receptor construct was generated by PCR SOEing²⁸ using a full-length EGFR cDNA (provided by Dr Nita Maihle, Mayo Clinic) and the EGFR/gp130 chimeric receptor as template. Sequence encoding the gp130 transmembrane domain (residues 620-641; IVVPVCLAFLLTTLLGVLFCF) was replaced with the EGFR transmembrane domain (residues 622-644; IATGMVGALLLLLVVALGIGLFM) and the construct was verified by nucleotide sequence analysis. Myeloma cell lines were transfected with 40 g of DNA using a square wave electroporator (BTX electroporation system; Genetronics, San Diego, CA, USA) set at 20 ms and 250 V. Bulk transfectants were selected in 250 g/ml G418 (Calbiochem) and sorted for high expression of the EGFR/gp130 chimera by fluorescence-activated cell sorting (FACS). Stable transfectants were maintained in growth medium supplemented with 250 g/ml G418.

Immunofluorescence analysis

Chimeric receptor expression levels were determined by flow cytometry. Briefly, cells were incubated with a PE-conjugated antibody to the EGF receptor or an isotype-matched control for 30 min on ice. The cells were then washed, fixed with 1% paraformaldehyde, and analyzed for immunofluorescence on a FACS Vantage (Becton Dickinson, Mountain View, CA, USA). Collected data were analyzed using WINMDI version 2.5 software.

DNA synthesis and cell proliferation assays

The myeloma cell transfectants were washed twice to remove IL-6 present in the culture media and resuspended in RPMI plus 0.5% BSA overnight. Cells were cultured in 96-well flat-bottom microtiter plates (Costar, Cambridge, MA, USA) at a density of 2.5 ´ 10⁴ cells/well and in a final volume of 200 l. Cultures were conducted in triplicate in the presence of 1 ng/ml IL-6 or 10 ng/ml EGF for 3 days at 37°C in the presence of 5% CO₂. Cultures were pulsed with 1 Ci tritiated thymidine (³H-TdR; 5.0 Ci/mmol, Amersham) after 48 h. Cells were harvested 18 h later and ³H-TdR incorporation levels determined using a Beckman scintillation counter. Cell proliferation was determined by counting the number of viable cells per well as determined by trypan blue exclusion. Cells were plated at 1 ´ 10⁶ cells/ml and analyzed after a 5-day incubation with 1 ng/ml IL-6 or 10 ng/ml EGF.

Whole cell lysate preparation, immunoprecipitation and Western blot analysis

For assay of STAT3 and MAPK activation, transfectants were starved overnight in RPMI plus 0.5% BSA. Cells (10 ´ 10⁶) were stimulated with 50 ng/ml IL-6 or 100 ng/ml EGF for 10 min at 37°C and lysed in cold lysis buffer (20 mM Tris-HCl; 137 mM NaCl; 1 mM MgCl₂ 1 mM CaCl₂; 1% Nonidet P-40; 10% glycerol; 2 mM EDTA; 10 mM Na pyrophosphate; 10 mM NaF; 2 mM Na₃VO₄ 2 mM PMSF; 8 g/ml leupeptin, aprotinin, pepstatin; and 4 g/ml DTT). Lysates were cleared of insoluble material by centrifugation for 10 min at 14 000 r.p.m. Fifty g of protein were denatured with an equal volume of 2´ SDS sample buffer (125 mM Tris, pH 6.8; 5% glycerol; 0.02% SDS (wt/vol); 0.02% 2-mercaptoethanol; 10 g/ml bromophenol blue; solution pH 6.6), boiled for 10 min, and electrophoresed on a 7.5% SDS-PAGE gel. Proteins were transferred to an Immobilon P membrane (Millipore, Bedford, MA, USA). Membranes were blocked in 25 mM Tris-Cl (pH 7.2), 150 mM NaCl, and 0.2% (wt/vol) Tween 20 (TBST) supplemented with either 5% non-fat dried milk or 2% BSA for 1 h. Following incubation with the designated primary antibody, HRP-linked anti-mouse or rabbit IgG secondary antibody was added and immunoreactive proteins were detected using enhanced chemiluminescence detection and autoradiography. For immunoprecipitation of gp 130 and the chimeric receptor, cells (30 ´ 10⁶) were stimulated as described above. Lysates were incubated with 4 g anti-gpl130 or 1 g anti-c-myc antibody pre-bound to Gamma-bind G sepharose beads (Amersham) for a minimum of 2 h. The immunoprecipitates were washed three times in lysis buffer. The complexes were then boiled in 30 l of 2´ SDS sample buffer and resolved on a 7.5% SDS-PAGE gel followed by transfer to Immobilin P membranes. Immunoblotting was performed as described above.

Results

Establishment of stable transfectants expressing the EGFR/gp130 chimeric receptor

A panel of EGFR/gp130 chimeric receptors was utilized to study gp130 signal transduction in multiple myeloma (MM). These receptors are composed of the EGFR extracellular domain, gp130 or EGFR transmembrane domain, and full-length or truncated gp130 cytoplasmic domain. Successive truncations of the EGFR/gp130 cytoplasmic tail schematically eliminated regions of gp130 shown previously to be necessary for MAPK, STAT1, and STAT3 activation in COS7 cells¹⁰ (Figure 1). Truncation of the EGFR/gp130 chimera targeted the five carboxy-terminal tyrosine residues of gp130. Whereas the full-length chimeric receptor maintains all five tyrosine residues, Y4-5 lacks two STAT1/3 docking sites (Y905, 915), Y2-5 lacks all STAT1/3 binding sites (Y767, 814, 905, 915), and Y1-5 lacks all STAT binding sites and the tyrosine residue (Y759) shown to be necessary for MAPK activation in other cell systems (Figure 1). To map the regions of gp130 necessary for IL-6-induced MM cell proliferation, the IL-6-dependent KAS-6/1 cell line was transfected with the various chimeric receptor constructs. Importantly, the parental KAS-6/1 cell line does not express EGFR, as measured by flow cytometry (Figure 2, pCI-Neo) and RT-PCR (data not shown). Stable transfectants were obtained after selection in G418 and fluorescence-activated cell sorting (FACS) of EGFR/gp130-positive cells. As can be seen in Figure 2, all KAS-6/1 transfectants expressed comparable levels of the full-length and truncated EGFR/gp130 chimeric receptors.

gp130 amino acid residues 767-918 are required for EGF-induced myeloma cell proliferation

We next wished to determine the effects of gp130 truncation on myeloma cell proliferation. To accomplish this, the transfectants were stimulated with either IL-6 or EGF, and DNA synthesis was assessed by ³H thymidine incorporation and cell proliferation was determined by counting viable cells. As shown in Figure 3, all transfectants maintained the ability to synthesize DNA in response to IL-6. As expected, KAS-6/1 cells transfected with empty vector (pCI-Neo) showed no response to EGF. Surprisingly, EGF induced DNA synthesis in all transfectants, regardless of receptor length. Although the overall magnitude of the EGF response of the Y1-5 chimera was attenuated by comparison with the other chimeric receptors, EGF still stimulated a four-fold increase in ³H-TdR incorporation over that observed in media alone. These data suggested that the five carboxy-terminal tyrosine residues are not required for EGF-induced DNA synthesis by the KAS-6/1 transfectants. We next assessed proliferation by counting the number of viable cells present after a 5-day incubation with IL-6 or EGF (Figure 4). By contrast to the DNA synthesis results, transfectants expressing the Y2-5 chimeric receptor which lacks all four STAT binding sites but maintains Y759, the residue previously described to be key in activation of MAPK, showed only minimal proliferation in response to EGF. Finally, cells expressing the Y1-5 chimeric receptor failed to exhibit a proliferative response to EGF. These data suggest that although each chimeric receptor could support some level of DNA synthesis, EGF-induced cell proliferation appears to require at least one STAT3-binding motif.

Both STAT and MAPK signal transduction pathways are activated by all EGFR/gp130 truncation mutants upon EGF stimulation

To determine the signaling pathways responsible for EGF-induced DNA synthesis and proliferation, whole cell lysates were prepared from each transfectant after stimulation with either IL-6 or EGF and analyzed by Western blot (Figure 5). As expected, STAT3 and MAPK were phosphorylated in response to IL-6 in all transfectants. EGF stimulation did not induce activation of these signaling molecules in the KAS-6/1 pCI-Neo control cells. Surprisingly, however, both MAPK and STAT3 were phosphorylated in response to EGF stimulation in all chimeric receptor transfectants. Importantly, STAT3 phosphorylation levels were markedly decreased in cells expressing the two shortest chimeric receptors (Figure 5). Of note, the decrease in EGF-induced STAT3 activation by cells expressing Y1-5 or Y2-5 correlated with diminished EGF-induced proliferation by these transfectants. These results support the conclusion that MAPK activation alone does not support gp130-mediated proliferative response of myeloma cells. Instead, there also appears to be a requirement for STAT3 activation.

EGF stimulates tyrosine phosphorylation of endogenous gp130 in EGFR/gp130 transfectants

We were intrigued by the ability of the Y1-5 chimeric receptor to activate MAPK and by the low-level STAT3 activation that was supported by Y2-5 and Y1-5 mutant receptors. These unexpected observations could reflect two possible scenarios: (1) IL-6-induced activation of MAPK and STAT3 by myeloma cells may not require any of the five carboxy-terminal tyrosine residues; or (2) the EGFR/gp130 chimeric receptor may be capable of interacting in an unexpected manner with endogenous gp130 in KAS-6/1 cells (ie wild-type gp130 confers function to the truncated chimeric receptors). Focusing first on the latter possibility, we next analyzed the phosphorylation status of endogenous gp130 after stimulation with either IL-6 or EGF (Figure 6). gp130 was immunoprecipitated from cell lysates using a mAb (B-R3) directed against the extracellular domain of the receptor, allowing direct isolation of endogenous gp130 but not the EGFR/gp130 chimera. The EGFR/gp130 chimeric receptors were immunoprecipitated using a c-myc mAb. As expected, IL-6 stimulated phosphorylation of endogenous gp130 and EGF induced phosphorylation of the chimeric receptor. Although KAS-6/1 cells expressing empty vector (pCI-Neo) showed no response to EGF, EGFR/gp130 transfectants exhibited substantial tyrosine phosphorylation of endogenous gp130 upon EGF stimulation (Figure 6). Thus, in the KAS-6/1 myeloma cell line, the EGFR/gp130 chimeric receptor communicates and/or interacts with endogenous receptor. Of interest, this interaction and/or communication is not reciprocal since IL-6 stimulation did not result in tyrosine phosphorylation of the chimeric receptor (Figure 6). Notably, Y1-5 transfectants also supported phosphorylation of endogenous gp130 in response to EGF (Figure 6) suggesting that this receptor cross-talk requires only 116 transmembrane proximal residues of the gp130 cytoplasmic tail.

EGFR/gp130 interaction with endogenous receptor is not facilitated by the transmembrane domain of gp130

Binding of IL-6 to gp130 requires the immunoglobulin-like domain and the cytokine-binding domain in the extracellular region of gp130.^29,30 Furthermore, gp130 signaling is dependent upon the juxtamembrane fibronectin modules in the extracellular domain of the receptor.³¹ Because the role of the transmembrane domain in receptor activation has not been fully characterized, we wished to determine what role this domain might play, if any, in the unexpected cross-talk between chimeric receptors and endogenous gp130. As noted above, the EGFR/gp130 chimeric receptors utilize the transmembrane domain of gp130. We therefore created an alternative EGFR/gp130 chimeric receptor expressing the EGFR transmembrane region in place of the gp130 transmembrane domain (EGFRTM; Figure 1). KAS-6/1 cells expressing the EGFRTM chimera gained responsiveness to EGF, as measured by ³H-TdR incorporation (Figure 7a) and viable cell counts (data not shown), in a fashion similar to cells expressing the EGFR/gp130 chimeric receptor. Immunoprecipitation of gp130 from these transfectants revealed that exchange of the gp130 transmembrane region for the transmembrane domain of EGFR did not disrupt the communication between the chimeric receptor and endogenous gp130 (Figure 7b). However, the level of EGF-induced gp130 phosphorylation in the EGFRTM transfectants was somewhat decreased compared to that seen in the cells expressing the EGFR/gp130 chimeric receptor.

Chimeric receptor interaction with endogenous receptor is not unique to the KAS-6/1 myeloma cell line

To address the possibility that this phenomenon was unique to the KAS-6/1 cell line, we expressed the full-length EGFRTM chimeric receptor in the IL-6-dependent myeloma cell line, ANBL-6. Stable transfectants were selected and sorted for uniform chimeric receptor expression (data not shown). Analysis of gp130 revealed that EGF also induced phosphorylation of endogenous gp130 in the ANBL-6 transfectants (Figure 8). Thus, the EGFR/gp130 chimera is capable of activating endogenous gp130 in two distinct myeloma cell lines. Because this interaction has not been noted in studies utilizing the same gp130 chimeric receptor system in non-MM cells,²⁵ it is striking that this cross-talk is observed in two independent IL-6-dependent human myeloma cell lines.

Discussion

In the present study, we have employed a chimeric receptor system to analyze in greater detail the role(s) of various signaling pathways in the proliferative response of myeloma cells to IL-6. Using the IL-6-dependent KAS-6/1 myeloma cell line, our data demonstrate an important role for STAT3 activation in IL-6-stimulated myeloma cell growth. Thus, MAPK activation alone was unable to support IL-6-stimulated cell proliferation. In addition, this study describes for the first time the ability of an EGFR/gp130 chimeric receptor to cross-communicate with endogenous gp130 as revealed by the tyrosine phosphorylation of endogenous gp130 following stimulation with EGF.

Although a chimeric receptor system has not been previously utilized to examine IL-6-driven growth of myeloma cells, other investigators have used chimeric receptors and Ba/F3 cells to elucidate the relative importance of each of the five carboxy-terminal tyrosine residues in gp130. As discussed above, however, the relative importance of the Ras/MAPK and JAK/STAT pathways in IL-6-induced Ba/F3 proliferation remains controversial. In the present study, discordant results were obtained when two different assays of proliferation were used. Thus, activation of STAT3 was not an absolute requirement for IL-6-stimulated DNA synthesis, whereas cell proliferation assays clearly indicated the importance of this pathway. Our observations that STAT3 activation is not essential for DNA synthesis are consistent with results published by Murakami et al;²⁶ however, our conclusion that STAT3 activation is important for proliferation is consistent with work published by Fukada et al.²⁴ Although DNA synthesis in the absence of cell division appears paradoxical, there is precedence for this in the literature.³² Importantly, STAT3 has been shown to be constitutively activated in bone marrow mononuclear cells from multiple myeloma patients.¹³

Our results also demonstrated that MAPK activation alone is insufficient to support gp130-mediated myeloma cell growth. This result is consistent with previous observations that activation of the MAPK pathway in Ba/F3 cells in the absence of STAT3 activation may promote G₁ to S phase transition, but not an S to G₂/M transition.^24,26 Of additional interest, the Y1-5 chimeric receptor supported robust MAPK activation, equivalent to the level obtained by all other transfectants, despite the absence of Y759. Although this spurious MAPK activation may be secondary to chimeric receptor interactions with endogenous gp130 (discussed in further detail below), it is possible that gp130 signaling in the KAS-6/1 cell line may utilize a non-classical mechanism of MAPK activation allowing MAPK activation in the absence of Y759. In support of this hypothesis, Schiemann et al³³ showed that mutation of Y759 in a G-CSFR/gp130 chimeric receptor inhibited MAPK activity by only 55%. Additionally, Chauhan et al³⁴ found that oncostatin M stimulation of the U266 myeloma cell line induced a direct association of JAK2 with Grb2 and Sos, key members of the MAPK activation cascade.

This study also provides evidence for an unprecedented interaction between this chimeric receptor system and endogenous gp130. This same chimeric receptor has been expressed and studied in COS7 cells and the SKW6.4 B cell line.^10,25 SKW6.4 cells naturally express human gp130; however, this study did not note an interaction between chimeric receptor and endogenous gp130. Studies are currently underway to determine the mechanisms of receptor interaction and whether this phenomenon is cell-type specific. Results shown in this study, however, have revealed that the unexpected interaction also occurs even when the gp130 transmembrane region was replaced with the EGFR transmembrane region. In addition, preliminary results using a chimeric receptor with a defective Box 1 region suggests that JAK activation is crucial for the receptor cross-talk.

Although the mode of interaction between the EGFR/gp130 and endogenous gp130 remains undefined, this unexpected cross-talk may provide insight into the mechanism of IL-6-induced myeloma cell proliferation. It is possible that our chimeric receptor system may have uncovered a novel natural mechanism of intracellular gp130 interaction in myeloma cells. Thus, in response to IL-6, two molecules of gp130 will contribute to the formation of each hexameric complex. Additional monomeric gp130 may be recruited and, consequently, activated by a complex-associated tyrosine kinase such as JAK1. Subsequently, this signal may be laterally transduced to additional monomeric gp130 recruits. This method of receptor recruitment is hypothesized to facilitate amplification of the initial ligand-induced receptor activation signal, such that the threshold level required for cellular responses is more rapidly reached. Furthermore, the altered growth response of myeloma cells to IL-6 may be attributed to this mechanism of signal amplification (ie the strength of signal may lead to the altered proliferative response to IL-6³⁵). In support of this theory, the ErbB1 receptor tyrosine kinase has very recently been shown to be capable of propagating ligand-independent lateral signal transduction thereby providing a mechanism whereby low levels of growth factor can fully activate ErbB1-linked growth regulatory signals.³⁶

The mechanisms underlying IL-6 growth responsiveness of myeloma cells remain incompletely characterized. Our panel of IL-6-dependent myeloma cell lines provides an excellent model system in which to analyze IL-6-induced signal transduction that leads to cell proliferation. Chimeric receptor systems are commonly utilized to study signal transduction in various cell lines independent of endogenous receptor. Although the EGFR/gp130 chimeric receptor signaled with no apparent interference by endogenous wild-type gp130 in the SKW6.4 B lymphoblastoid cell line,²⁵ EGFR/gp130 interacts with endogenous gp130 in both the KAS-6/1 and ANBL-6 myeloma cell lines. The observation that this cross-talk may be unique to myeloma cells is intriguing. An investigation into underlying mechanisms may provide us with unexpected insight into the mechanism of IL-6 receptor interactions and signaling in myeloma cells. Furthermore, despite the unexpected interaction of the EGFR/gp130 chimeric receptor with endogenous gp130, this study suggests that STAT factors may play an important role in gp130-mediated myeloma cell proliferation.

Acknowledgements

We thank Dr Anne Novak for critical reading of the manuscript. This work was supported by National Institutes of Health grant CA62242.

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Figure 1 Schematic of EGFR/gp130 chimeric receptor constructs. The receptors are composed of the extracellular domain of the EGFR and the transmembrane and various lengths of the cytoplasmic domain of gp130 (residues 620-918). The EGFRTM chimeric receptor contains the transmembrane region of EGFR (residues 620-644).

Figure 2 KAS-6/1 transfectants express comparable levels of the EGFR/gp130 chimeric receptors. Transfectants were stained with a PE-conjugated EGFR mAb (bold line) or an isotype-matched control (light line) and analyzed by flow cytometry.

Figure 3 All transfectants, regardless of chimeric receptor length, synthesize DNA in response to EGF. KAS-6/1 transfectants were cultured in media alone (open bars), or were stimulated with IL-6 (hatched bars) or EGF (solid bars) for 3 days before assaying DNA synthesis. Values represent the mean c.p.m. of three independent experiments, each experimental point performed in triplicate.

Figure 4 Y2-5 and Y1-5 truncated chimeric receptors do not support EGF-induced proliferation. KAS-6/1 transfectants were cultured in media alone (open bars), or were stimulated with IL-6 (hatched bars) or EGF (solid bars) for 5 days before assaying proliferation. The number of viable cells per well was determined by Trypan Blue staining and cell counts using a hemacytometer. Values represent the mean of two independent experiments.

Figure 5 STAT3 and MAPK activation by chimeric receptor transfectants. KAS-6/1 transfectants were stimulated with IL-6 or EGF for 10 min before preparation of total cellular lysates. 100 ng of protein was resolved by SDS-PAGE, transferred to an Immobilon-P membrane, and immunoblotted with anti-phospho-STAT3 and anti-phospho-MAPK antibodies. To ensure equal protein loading, membranes were stripped and sequentially blotted with antibodies against either MAPK or STAT3.

Figure 6 EGF stimulation induces phosphorylation of endogenous gp130 in KAS-6/1 cells expressing the full-length (EGFR/gp130) or shortest (Y1-5) chimeric receptor. KAS-6/1 transfectants expressing the wild-type chimeric receptor were stimulated for 10 min with IL-6 or EGF. Cell lysates were incubated with anti-gp130 (B-R3) or anti-c-myc antibodies to immunoprecipitate endogenous gp130 or the chimeric receptor, respectively. Precipitated proteins were resolved by SDS-PAGE, transferred to an Immobilon-P membrane, and immunoblotted with the designated antibodies.

Figure 7 Effects of EGFR transmembrane substitution on EGF-induced phosphorylation of endogenous gp130. (a) KAS-6/1 transfectants were cultured in media alone (open bars), or were stimulated with of IL-6 (hatched bars) or EGF (solid bars) for 3 days before assaying DNA synthesis. Two representative experiments are shown. Values represent the mean c.p.m. of three points per condition. (b) KAS-6/1 transfectants expressing EGFRTM were stimulated for 10 min with IL-6 or EGF. Cell lysates were incubated with anti-gp130 (B-R3) or anti-c-myc antibodies to immunoprecipitate endogenous gp130 or the chimeric receptor, respectively. Precipitated proteins were resolved by SDS-PAGE, transferred to an Immobilon-P membrane, and immunoblotted with the designated antibodies.

Figure 8 ANBL-6 cells expressing the EGFRTM chimeric receptor phosphorylate endogenous gp130 in response to EGF. The ANBL-6 myeloma cell line was stably transfected with empty vector (pCI) or the EGFRTM chimeric receptor construct. ANBL-6 transfectants expressing the EGFRTM chimeric receptor were stimulated for 10 min with IL-6 or EGF. Cell lysates were incubated with anti-gp130 (B-R3) or anti-c-myc antibodies to immunoprecipitate endogenous gp130 or the chimeric receptor, respectively. Precipitated proteins were resolved by SDS-PAGE, transferred to an Immobilon-P membrane, and immunoblotted with the designated antibodies.