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Groupe de Recherche Cytokines/Récepteurs/Transduction, Institut National de la Santé et de la Recherche Médicale, Unité 463, and Institut Féderatif de Recherche 26, Institut de Biologie, 44093 Nantes, France
Address all correspondence and requests for reprints to: Dr. Frédéric Blanchard, Institut National de la Santé et de la Recherche Médicale, Unité 463, Institut de Biologie, 9 Quai Moncousu, 44093 Nantes Cedex 01, France. E-mail: fblan{at}nantes.inserm.fr.
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Abstract |
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Introduction |
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Each IL-6-type cytokine is recognized by a specific receptor subunit (e.g. IL-6R
, LIFR, oncostatin M receptor ß, or IL-11R), and each complex subsequently interacts with the shared glycoprotein 130 (gp130) subunit (1, 2). Ligand-induced oligomerization of receptor subunits activates Janus protein tyrosine kinases, which, in turn, phosphorylate and activate STAT (signal transducer and activator of transcription) transcription factors (mainly STAT3) and linker proteins, such as Src homology protein tyrosine phosphatase-2 (SHP-2) or Src homology domain containing collagen-related protein (SHC), which propagate the signal to the ERK1/2 pathway. In Ba/F3 pro-B cells, the gp130/STAT3 pathway plays a key role in the G1 to S phase cell cycle transition and prevention of apoptosis, whereas the gp130/SHP2/p27ras(RAS)/ERK1/2 pathway is implicated in the S to G2/M transition and is essential for mitogenesis (10, 11).
We have recently identified a new LIFR, not related to LIFR or gp130, that binds LIF through its carbohydrate moieties, but does not interact with other members of the IL-6 cytokine family (12). This receptor was identified as the calcium-independent mannose 6-phosphate (Man-6-P)/IGF-II receptor (CIMPR), and preliminary data indicated that it mediates internalization and lysosomal degradation of LIF, but does not transduce proliferative signals (13). CIMPR is mainly expressed within endosomal compartments where it directs the sorting of Man-6-P-containing enzymes (glycosidases or proteases) to endosomes and lysosomes (14). This receptor is also present at the plasma membrane, where it mediates internalization and degradation of IGF-II (15) and activation of the precursor form of TGFß (latent TGFß) into biologically active TGFß (16). CIMPR-deficient mice have increased serum and tissue levels of IGF-II and Man-6-P-containing ligands and exhibit overgrowth, organomegaly, and perinatal death (17). In certain malignant cells such as gastrointestinal tumors, a defective expression of CIMPR (allelic loss and point mutations) correlates with enhanced concentration of the mitogenic and survival factor IGF-II and reduced concentration of active TGFß, a potent growth inhibitor (18). Furthermore, the CIMPR locus at 6q has been reported to be a hot spot for deletions in B cell non-Hodgkin lymphoma (19). Whereas the mitogenic and survival potencies of IGF-II rely on binding to IGF-I receptor or insulin receptor A isoform and subsequent activation of the Ras/ERK1/2 and the phosphoinositol 3-kinase (PI3K)/AKT/protein kinase B pathways (20, 21, 22, 23), the G1 growth arrest by TGFß is mediated by Smad 3 and 4 (24).
In mammals, a soluble form of CIMPR (sCIMPR) is naturally released by proteolytic cleavage in culture and in vivo circulates in the serum at concentrations up to 5 µg/ml (25, 26). sCIMPR binds IGF-II and blocks IGF-II-stimulated DNA synthesis in hepatocytes and fibroblasts (27). We investigated whether purified sCIMPR modulates LIF activities in a similar fashion. Our results indicate that sCIMPR effectively inhibits the LIF-dependent proliferation of various myeloid and lymphoid cell lines. However, this inhibition is also observed when using other IL-6-type cytokines that do not interact with sCIMPR. Indeed, neutralizing anti-IGF-II antibodies were as active as sCIMPR, suggesting that neutralization of IGF-II by sCIMPR plays a major role in the inhibition of IL-6-dependent cell proliferation. We propose a model where the specific transduction pathways activated by IGF-II (ERK1/2 and AKT) and IL-6 (STAT3) complement each other to sustain the proliferation and survival of myeloid and lymphoid cells.
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Materials and Methods |
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(Ba/F3 gp130/IL-11R) (28, 29) or gp130, LIFR, and IL-6R (Ba/F3 gp130/LIFR/IL-6R; gift from Dr. Jean-Luc Taupin, Bordeaux, France) and the mouse myeloid DA 2 cell line (30) were cultured in RPMI 1640 supplemented with 2% FCS and 5 ng/ml IL-11 or CHO-LIF, respectively. The murine pro-B 32D cell line expressing endogenous IL-2R
and transfected with human IL-2Rß and IL-15R was maintained in RPMI 1640 supplemented with 10% FCS and 0.4 ng/ml recombinant human IL-15 (Bernard, J., and Y. Jacques, manuscript in preparation). The human myeloma XG2 cell line and the mouse hybridoma B9 and 7TD1 cell lines were cultured in RPMI 1640 supplemented with 5% FCS and 1 and 0.1 ng/ml recombinant human IL-6, respectively. The human T lymphoma Kit 225 cell line was cultured in RPMI 1640 supplemented with 6% FCS and 1 ng/ml recombinant human IL-2. These culture conditions are summarized in Table 1
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produced in CHO cells was a gift from Jean Luc Taupin.
Antibodies.
Antihuman IGF-II antibody and pan-specific TGFß antibody were obtained from R&D Systems. Antiphospho-Akt (Ser473), antiphospho-ERK1/2 (Thr202, Tyr204), and antiphospho-STAT 3 (Tyr 705) antibodies were obtained from Cell Signaling Technologies (Beverly, MA); antiphospho-STAT5 (Tyr694) antibody was purchased from Zymed Laboratories, Inc. (San Francisco, CA); antihuman gp130 mAb B-R3 was obtained from Diaclone Research (Besançon, France); and phycoerythrin-goat antimouse antibody was puchased from Immunotech (Marseilles, France).
sCIMPR was purified from fetal bovine serum in two steps: affinity chromatography on a phosphomannan column (32) and gel filtration on Superdex200 (HR 10/30, Amersham Pharmacia Biotech, Orsay, France). The purity of the sCIMPR, as evaluated by SDS-PAGE and silver staining, was greater than 90%.
UO126 and LY294002 were obtained from Calbiochem (La Jolla, CA).
Surface plasmon resonance studies
These experiments were performed with the BIACore 2000 optical biosensor (BIACore, Uppsala, Sweden). sCIMPR was covalently coupled through its primary amino groups to a carboxymethyl dextran flow cell (CM5, BIACore) as recommended by the manufacturer. All binding experiments were performed at a flow rate of 10 µl/min. Binding of soluble human gp130 (147 nM) to sCIMPR was carried out in the absence or presence of 10 mM Man-6-P. The resonance signal measured on the control flow cell was subtracted from the signal measured on the experimental flow cell. The resulting sensorgrams were analyzed using BIAEvaluation (BIACore) software.
Flow cytometric analysis
For cell surface expression of gp130, gp130- and IL-11R-expressing Ba/F3 cells or parental BaF3 cells were incubated for 1 h at 4 C with 1 µg/ml of the anti-gp130 B-R3 mAb, washed, and further incubated (30 min at 4 C) with PE-goat antimouse Ab. Cell-associated fluorescence was analyzed by flow cytometry (FACScan, BD Biosciences, Mountain View, CA) using CellQuest software.
For cell cycle analysis, cells were washed and lysed in PBS containing 0.12% Triton X-100, 0.12 mM EDTA, 50 µg/ml ribonuclease A, and 50 µg/ml propidium iodide. The intensity of propidium iodide labeling was measured by flow cytometry (FACScan, BD Biosciences) using CellQuest software.
sCIMPR binding and competition assay
sCIMPR was radioiodinated at a specific radioactivity of approximately 2700 µCi/nmol as previously described (31). Radioiodinated sCIMPR was as active as nonlabeled sCIMPR in a cell proliferation inhibitory assay using Ba/F3 gp130/IL-11R cells. For the binding assay, Ba/F3 or Ba/F3 gp130/IL-11R cells (1 x 106/well in 96-well, round-bottomed plates) were incubated with increasing concentrations of labeled sCIMPR (up to 17 nM) for 60 min at 4 C under agitation (equilibrium conditions). Nonspecific binding was determined by including a 100-fold excess of unlabeled sCIMPR. The final reaction volume was 50 µl/well. Separation and measurement of cell bound and unbound fractions were performed as previously described (31).
Cell proliferation and viability assays
Cells were starved of cytokine for 4 h at 37 C in the presence of serum and incubated at 2 x 105 cell/ml in a final volume of 100 µl with cytokines or inhibitors. After incubation at 37 C for 72 h, cellular proliferation and viability were assessed by a sodium 3'-[1-phenylaminocarbonyl]-3,4-tetrazolium (XTT)-based assay (Roche, Mannheim, Germany). In Fig. 6, cell viability was estimated by trypan blue exclusion.
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cells were starved of IL-11 for 4 h at 37 C in RPMI 1640 culture medium without serum and stimulated 15 min with IL-11, IL-3, IGF-II, or their combinations. When indicated, cytokines were preincubated for 90 min with or without 10 µg/ml sCIMPR before addition to the cells. Cells were lysed in radioimmune precipitation buffer containing antiproteases as described previously (33). Protein concentration was determined by the bicinchoninic acid kit (Pierce Chemical Co., Rockford, IL).
Western blotting.
Twenty micrograms of total cell lysate proteins were run on 10% SDS-PAGE and electrophoretically transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA). The membrane was then blotted with antibodies in PBS, 0.05% Tween 20, and 3% BSA for 2–16 h; washed; and probed with the secondary antibody coupled to horseradish peroxidase for 45 min. Antibody binding was visualized with the enhanced chemiluminescence system (ECL kit, Roche). After scanning, the intensity of each lane was measured by pixel densitometry with NIH Image software.
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Results |
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, a natural, soluble form of CIMPR purified from fetal bovine serum reduced the proliferation and/or survival of DA2 cells in the presence of a subsaturating concentration of LIF (5 ng/ml). With 10 µg/ml sCIMPR, maximal inhibition of proliferation (63.9 ± 1.4%) was attained in the presence of LIF, whereas only 24.3% inhibition of proliferation was observed in the presence of IL-3.
To determine whether the growth inhibitory effect of sCIMPR was restricted to LIF-dependent proliferation, we first used pro-B Ba/F3 cells, which stably expressed 1) gp130, LIFR and IL-6R and depends on LIF, IL-6, or IL-3 for cellular expansion (not shown), or 2) gp130 and IL-11R and depends on IL-11 or IL-3 for growth (Fig. 1A). Unexpectedly, sCIMPR inhibited the proliferation (77–87% inhibition) driven by all IL-6-type cytokines tested (LIF, IL-6, and IL-11; Fig. 1B and Table 1) despite the fact that IL-6 and IL-11 do not harbor Man-6-P and do not interact with CIMPR (13). A 35% inhibition of proliferation was observed with two human myeloma cell lines that depend for proliferation on IL-6 either added in the culture medium (XG2) or produced endogenously and acting in an autocrine fashion (U266; Table 1) (34). Interestingly, sCIMPR totally prevented proliferation of the mouse 7TD1 hybridoma cell line driven by IL-6, but had no effect on IL-6- or IL-11-dependent proliferation of the mouse B9 hybridoma cell line. The inhibitory effect of sCIMPR was restricted to IL-6-type cytokines, because neither 32D-IL-2Rß/IL-15R cells (that proliferate in the presence of IL-3, IL-2, or IL-15) nor Kit 225 T lymphoma cells (that proliferate in the presence of IL-2) were sensitive to sCIMPR.
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IGF-II, in the presence of a low dose of IL-11 (0.1 ng/ml) and 2% fetal serum, dose-dependently increased the proliferation of Ba/F3 gp130/IL-11R cells with a half-maximal stimulation of approximately 100 ng/ml (Fig. 1A). In the absence of IL-11, only trace stimulation of proliferation was observed with IGF-II or increasing concentrations of serum (Fig. 1A and data not shown), confirming the IL-11 dependency of this cell line. An anti-IGF-II antibody that does not cross-react with IGF-I neutralized the proliferation driven by IL-11 and 2% fetal calf serum to the same extent (80%) as did sCIMPR (Fig. 1C). Anti-IGF-II antibodies or sCIMPR also inhibited, by 45%, the proliferation of Ba/F3 gp130/IL-11R cells driven by IL-11 and 2% adult human serum (data not shown). In all myeloid and lymphoid cell lines tested that are sensitive to sCIMPR inhibition (DA2, Ba/F3, and U266 cells), anti-IGF-II antibodies reduced proliferation to the same extent as did sCIMPR (data not shown).
As shown in Fig. 2, human TGFß added exogenously inhibited the IL-11-dependent proliferation of Ba/F3 cells expressing gp130 and IL-11R. A pan-specific TGFß antibody recognizing TGFß1, -2, -3, and -5 of various origins completely prevented the inhibitory effect of TGFß, but had no action on the inhibitory effect of sCIMPR.
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) or in CHO mammalian cells (data not shown), but not soluble glycosylated LIFR (data not shown), binds to immobilized sCIMPR. Analysis of the kinetic association and dissociation curves gave an equilibrium dissociation constant (Kd) of 12.3 nM for the shgp130-sCIMPR interaction. We then tested whether this interaction involved Man-6-P moieties, as previously shown for glycosylated LIF binding to CIMPR (12, 13). This turned out to be the case, as Man-6-P (Fig. 3A), but not Glc 6-P (data not shown), reduced shgp130 binding to sCIMPR.
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were first shown to express a high level of gp130 at the cell surface compared with parental Ba/F3 cells (Fig. 3B). Iodinated sCIMPR, which retained a full inhibitory effect in proliferation assays, bound equally well to parental or gp130-expressing Ba/F3 cells (Fig. 3C). sCIMPR binding was prevented by excess Man-6-P, suggesting the presence of endogenous Man-6-P-containing glycoproteins on the surface of Ba/F3 cells. Given the fact that parental Ba/F3 cells are strictly negative for gp130 and IL-11R, and that gp130-plus IL-11R-transfected Ba/F3 cells express several thousands of gp130 and IL-11R molecules at the plasma membrane (29), these results suggested that sCIMPR did not interact with the membrane form of gp130 or IL-11R and therefore was unlikely to neutralize membrane gp130 or IL-11R. Indeed, sCIMPR did not modify IL-6-type cytokine binding to membrane gp130 (e.g. high affinity receptors; not shown) or gp130 signal transduction (Fig. 4).
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cells (Fig. 4A). No effect on STAT5 or AKT could be detected, and trace activation of ERK1/2 was detected only at high IL-11 concentrations (100 ng/ml; Figs. 4 and 5A). In contrast, IL-3 (10 ng/ml) activated STAT3, STAT5, ERK1/2, and AKT. IGF-II activated only ERK1/2 and AKT, but not STAT3 or STAT5. Interestingly a combination of IL-11 and IGF-II was able to activate STAT3, ERK1/2, and AKT (Fig. 5A). It should be noted that there was a more than additive effect on ERK1/2 and AKT, whereas STAT3 activation was slightly reduced. The latter result could be due to a negative regulation of STAT3 by ERK1/2, as reported previously in other cell systems (2, 33, 35). Similar results were obtained in U266 cells: IL-6 activated STAT3, whereas IGF-II activated AKT (Fig. 4C). In contrast to Ba/F3 gp130/IL-11R cells, U266 cells constitutively expressed high levels of activated ERK1/2, and IL-6 or IGF-II did not increase the phosphorylation of these kinases (Fig. 4C).
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) or neutralizing anti-IGF-II antibodies (data not shown), signal transduction by IL-3 or IL-11 was unchanged in Ba/F3 gp130/IL-11R cells, but ERK1/2 or AKT activation by IGF-II (either alone or in combination with IL-11) was reduced by more than 90%. Similar results were obtained when using 4% serum instead of IGF-II (data not shown).
The implication of STAT3, ERK1/2, and AKT in Ba/F3 cell proliferation and survival was further evaluated. As shown in Fig. 6A, the absence of IL-11 in serum-containing culture medium totally prevented cellular expansion over the 48-h incubation period. At that time, the majority of the cells were blocked in the G1 phase of the cell cycle (Fig. 6E). Cell viability decreased from 90–95% to 60–70% by 24 h, as assessed by trypan blue staining (Fig. 6B), and was associated with increased apoptosis, as confirmed microscopically after DNA labeling with the Hoechst dye (nuclear blebbing) and by annexin V staining (data not shown). sCIMPR completely prevented cellular expansion triggered by IL-11 (Fig. 6A), reduced cell viability to 40–50% within 24 h (Fig. 6B), and induced a strong apoptotic reaction, as attested by a majority of the cells with a sub-G1 DNA content (Fig. 6E). UO126 and LY294002, specific inhibitors of the MAPK kinase/ERK1/2 and PI3K/AKT pathways, respectively, both totally inhibited cellular expansion triggered by IL-11 and serum (Fig. 6C) and induced cell death within 24 h (Fig. 6D). Together these results indicated that STAT3, ERK1/2, and AKT are necessary to support Ba/F3 cell cycle progression and maintain cell viability.
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Discussion |
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First, at 10 ng/ml, a concentration sufficient to trigger strong cellular expansion, IL-6-type cytokines activates STAT3, but not the signal transduction pathways (e.g. ERK1/2 and AKT) necessary for proliferation and/or survival. In hemopoietic cells, STAT3 has been shown to play a key role in the G1 to S phase cell cycle transition and resistance to apoptosis through the up-regulation of Bcl-2; c-Myc; cyclins D2, D3, and A; and cdc25A and the concomitant down-regulation of p21 and p27 (10, 11). In contrast, ERK1/2 activation leads to induction of the immediate-early genes necessary for mitosis, such as c-fos, c-jun, and egr-1 (10). The antiapoptotic molecule AKT phosphorylates Bcl-xL/Bcl-2-associated death promoter (BAD), which results in liberation of BAD from bcl-2 and/or bcl-xL, thus liberating these latter antiapoptotic proteins and allowing them to bind and inactivate BAX. Other reports have shown that IL-6-type cytokines can stimulate the insulin receptor substrate/PI3K/AKT and SHP-2/growth factor receptor-bound protein 2 (GRB2)/ERK1/2 pathway in myeloma or other cell lines (1, 2, 35, 36). However, high concentrations of cytokines were used (100 ng/ml), and stimulation of these pathways were always low compared with that achieved with serum or IGFs. Together with the fact that serum-free culture medium containing IL-6-type cytokines was unable to sustain the proliferation of Ba/F3 cells, these results suggest that additional factors present in serum are needed to complement signal transduction by low doses of IL-6-type cytokines.
Second, antibodies against IGF-II are as active as sCIMPR for inhibition of IL-6-, LIF-, or IL-11-dependent growth as well as prevention of ERK1/2 and AKT activation by IGF-II or serum. It is of particular interest that IL-6-type cytokines and IGF-II have additive effects on STAT3 and reproducibly synergize for ERK1/2 and AKT activation, suggesting a particularly effective complementation between these two types of factors. These observations help explain the relative insensitivity of IL-3 to sCIMPR action, because IL-3 on its own fully activates STATs, ERK1/2, and AKT. Similar conclusions can be made with others cytokines, such as IL-2, that are insensitive to sCIMPR. Together these results strongly suggest a key and specific role for IGF-II in the proliferation of cells driven by IL-6-type cytokines.
In this study surface plasmon resonance experiments clearly demonstrate that sCIMPR is able to bind with nanomolar affinity to soluble human gp130 and that this interaction involves the binding to Man-6-P residues harbored by glycosylated side-chains on gp130. In contrast to soluble gp130, we were unable to detect an interaction between sCIMPR and the plasma membrane form of gp130. One explanation is that interaction between the membrane forms of CIMPR and gp130 in the Golgi apparatus results in specific lysosomal targeting and degradation, precluding its routing to the cell surface. The expression of gp130 at the cell surface, as detected by flow cytometry, indicates that not all gp130 molecules acquire Man-6-P, as we previously described for LIF (13). Indeed, CIMPR-deficient fibroblasts express higher levels of membrane high affinity LIFRs, indicating increased levels of gp130 and/or LIFR at the cell surface (our unpublished results). Therefore, it seems that the primary role of CIMPR is not to neutralize plasma membrane gp130, but, rather, to control its expression and location within the cell. This regulatory action might also participate in the antitumor effect of CIMPR.
An intriguing result is the total resistance of the B9 hybridoma cell line, which strictly depends on IL-6 or IL-11 for proliferation, whereas myeloid DA2 cells, pro-B cells, multiple myeloma cell lines (blast-B cells), and another hybridoma cell line, the 7TD1 cell line, were sensitive to growth suppression by sCIMPR. These results suggest that B9 cells, a hybridoma of plasma blasts and myeloma cells, do not depend on IGF-II for proliferation and that other growth factors or cytokines, perhaps IGF-I or IL-4, may replace IGF-II (6, 23). Another possibility is that IL-6-type cytokines on their own are able to activate ERK1/2 and AKT and therefore to sustain proliferation in these cells (37). Current experiments are underway to discriminate between these hypotheses.
It is interesting to note that proliferation of bone marrow CD34+ hemopoietic stem cells is increased by multiple factors, including stem cell factor, IL-6-type cytokines, as well as IGF-II (3, 4, 5, 21, 23, 38). Whether sCIMPR, through neutralization of IGF-II, controls early stages of hemopoiesis, especially of the B lineage, will be the goal of future investigations. sCIMPR thus is a candidate regulator of B and plasma cell growth, as previously suggested for hepatocytes and fibroblasts (27). How this receptor is shed from the cell surface is not clearly determined. It has been shown that disruption of late endosomes increases the release of soluble CIMPR (39). Serum levels of sCIMPR are very high in the fetus (up to 5 µg/ml) and decreased in infants (1 µg/ml) and adults (0.7 µg/ml) (26). Heart and muscle appear to be major sources in fetal rats, whereas the liver is the major one in adults (25). sCIMPR has been found in complexes with IGF-II in the blood, but free IGF-II or IGF-II carried by other IGF-binding proteins is assumed to be predominant, explaining the IGF-II activity present in serum (23, 40, 41). In this study we show that physiological concentrations of sCIMPR (1–10 µg/ml) reduce myeloid and lymphoid cell proliferation triggered by IL-6-type cytokines and 2–5% fetal serum (providing <0.25 µg/ml sCIMPR). Additional studies are needed to determine the physiopathological conditions in which sCIMPR could totally neutralize IGF-II, but our results indicate that sCIMPR and IGF-II are two opposing circulating factors that control cell proliferation induced by IL-6-type cytokines, and their roles/actions should be taken into account whenever studies involve IL-6-type cytokines.
As IGF-I and IGF-II both activate the IGF-I receptor, IGF-I would be expected to substitute for IGF-II when using sCIMPR or neutralizing anti-IGF-II antibodies. We observed that sCIMPR is a less potent growth inhibitor when using adult serum (45% inhibition) instead of fetal serum (80% inhibition), suggesting that IGF-I substitutes for IGF-II more effectively in adults than in the fetus. These observations are in good agreement with the growth of IGF-I-, IGF-II-, and IGF-IR-deficient mice, which indicates that between embryonic d 11 and 12.5, IGF-I receptor serves only the mitogenic signaling of IGF-II, whereas from embryonic d 13.5 onward, IGF-I receptor interacts with both IGF-I and IGF-II (17, 42). Another possibility is that IGF-I cannot totally substitute for IGF-II because IGF-II uses not the IGF-I receptor, but another, still unknown, specific receptor, as suggested previously (17, 42).
Defective CIMPR gene expression in CIMPR-deficient mice or in peculiar tumor cells, such as hepatocarcinoma, gastrointestinal tumors, or lymphoma, is invariably associated with overgrowth or high proliferative potential, respectively (17, 18, 19, 43). CIMPR, whether cell associated or soluble, is considered an embryo growth regulator and a potential tumor suppressor. It is therefore of particular interest to determine whether any defect in sCIMPR expression occurs in hemopoietic disorders where IL-6-type cytokines and IGFs may play a role, such as multiple myeloma (6, 7), primary effusion lymphoma, Castleman disease (44), and polycythemia vera (45, 46). Various mechanisms are involved in tumor suppression by CIMPR. First, this receptor limits the level of secreted lysosomal enzymes responsible for extracellular matrix degradation and tumor dissemination (14). Second, it is necessary for the binding and uptake of granzyme B and therefore essential for T cell-mediated apoptosis of target cells (47). Third, it binds to and increases activation of the growth inhibitor TGFß through a complex pathway involving urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator, and plasmin (16). Fourth, it induces internalization and degradation of various growth-promoting factors such as IGF-II (15), glycosylated LIF, and other Man-6-P-containing cytokines such as macrophage colony-stimulating factor (13). Finally, it also interacts with receptors such as the epidermal growth factor receptor (48) and gp130 (the present study).
In summary, this study shows 1) that the IL-6 type cytokine/gp130/STAT3 pathway is not sufficient by itself to sustain the proliferation and survival of myeloid and lymphoid cells; 2) that the IGF-II/IGF-IR/ERK1/2 and AKT pathways are both necessary to support IL-6-type cytokines-dependent growth; and 3) that sCIMPR is a candidate natural molecule, presumably as other inhibitors of the IGF-I pathway, for the development of novel therapeutic strategies in the treatment of hemopoietic disorders in which IL-6-type cytokines play a role, among them multiple myeloma.
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Acknowledgments |
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/IL-6R cells, Jean Content for 7TD1 cells, Martine Amiot for XG2 cells, François Traincard for Dictyostelium preparations, and Sylvie Hermouet for critical comments on the manuscript. |
Footnotes |
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L.D. and B.C.D. contributed equally to this work.
Present address of L.D.: Touchstone Center for Diabetes Research, University of Texas Southwestern Medical Center, Dallas, TX 75390.
Abbreviations: CIMPR, Calcium-independent mannose 6-phosphate receptor; FCS, fetal calf serum; gp130, glycoprotein 130; IL-6R, IL-6 receptor; LIF, leukemia inhibitory factor; LIFR, leukemia inhibitory factor receptor; Man-6-P, mannose-6-phosphate; PI3K, phosphoinositol 3-kinase; sCIMPR, soluble form of calcium-independent mannose 6-phosphate receptor; SHP, Src homology protein tyrosine phosphatase; STAT, signal transducer and activator of transcription; XTT, sodium 3'-[1-phenylaminocarbonyl]-3,4-tetrazolium.
Received May 19, 2003.
Accepted for publication August 21, 2003.
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References |
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complex in the pathogenesis of multiple myeloma. Eur Cytokine Network 11:546–551[Medline]
, and oncostatin M receptor ß by distinct mechanisms. J Biol Chem 276:47038–47045
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