A mutation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 that leads to ligand independence and tumorigenicity

The cytokines interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor bind with high affinity to a receptor complex that contains a ligand-specific alpha-chain and a common beta-chain, h beta c. We report here the isolation of a mutant form of h beta c, from growth factor-independent cells, that arose spontaneously after infection of a murine factor-dependent hematopoietic cell line (FDC-P1) with a retroviral h beta c expression construct. Analysis of this h beta c mutation shows that a small (37 amino acid) duplication of extracellular sequence that includes two conserved sequence motifs is sufficient to confer ligand-independent growth on these cells and lead to tumourigenicity. Because this is a conserved region in the cytokine receptor superfamily, our results suggest that the large family of cytokine receptors has the capacity to become oncogenically active.     

Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor beta subunit

Several activating mutations have recently been described in the common beta subunit for the human interleukin(IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (hbetac). Two of these, FIDelta and I374N, result, respectively, in a 37-amino acid duplication and an isoleucine-to-asparagine substitution in the extracellular domain. A third, V449E, leads to valine-to-glutamic acid substitution in the transmembrane domain. Previous studies have shown that when expressed in murine hemopoietic cells in vitro, the extracellular mutants can confer factor independence on only the granulocyte-macrophage lineage while the transmembrane mutant can do so to all cell types of the myeloid and erythroid compartments. To further study the signaling properties of the constitutively active hbetac mutants, we have used novel murine hemopoietic cell lines, which we describe in this report. These lines, FDB1 and FDB2, proliferate in murine IL-3 and undergo granulocyte-macrophage differentiation in response to murine GM-CSF. We find that while the transmembrane mutant, V449E, confers factor-independent proliferation on these cell lines, the extracellular hbetac mutants promote differentiation. Hence, in addition to their ability to confer factor independence on distinct cell types, transmembrane and extracellular activated hbetac mutants deliver distinct signals to the same cell type. Thus, the FDB cell lines, in combination with activated hbetac mutants, constitute a powerful new system to distinguish between signals that determine hemopoietic proliferation or differentiation. (Blood. 2000;95:120-127)     

Functional identification of secondary mutations inducing autonomous growth in synergy with a truncated interleukin-3 receptor: implications for multi-step oncogenesis

OBJECTIVE: A truncated common beta chain (Deltabeta(C)) of the interleukin-3 (IL-3) receptor complex was previously identified as a key factor in inducing autonomous growth of IL-3-independent mutants. Expression of Deltabeta(C) in IL-3-dependent hematopoietic cells does not result in immediate factor-independent growth, but increases the frequency of obtaining autonomous mutants by three to four orders of magnitude. This study was designed to delineate the mechanisms by which Deltabeta(C) increases the frequency to autonomous growth. DESIGN AND METHODS: Retroviral vectors were used to express Deltabeta(C) into IL-3-dependent myeloid cells, which were then tested for factor-independent growth. To determine if secondary genetic events were required for conversion to autonomous growth, elements of the Cre-loxP recombinant system were used to excise Deltabeta(C) in factor-independent clones. RESULTS: Excision of Deltabeta(C) in factor-independent clones revealed two types of phenotypes: reversion to factor-dependent growth (1/8) or continued IL-3-dependent growth (7/8). Analysis of cells that remained factor independent revealed constitutive activation of STAT5, not observed in factor-dependent revertants. Analysis of revertant cells demonstrated the presence of interacting secondary mutations that synergize with Deltabeta(C)-induced proliferation. A cysteine residue within the truncated extracellular domain of Deltabeta(C) was also found to be required for its oncogenic potential, supporting a model of dimerization for receptor activation. CONCLUSIONS: The high incidence of obtaining factor-independent mutants from cells expressing Deltabeta(C) results from the selection of mutations that either complement Deltabeta(C) expression to promote proliferation or that singly or in synergy with other secondary mutations negate the requirement of Deltabeta(C) expression for proliferation.     

Humoral and cell surface interactions during gamma-irradiation leukemogenesis in vitro.

Gamma-irradiation of plateau phase cultures of the clonal murine bone marrow stromal cell line D2XRII followed by cocultivation of a clonal interleukin 3 (IL-3) (granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent hematopoietic progenitor cell line FDC-P1JL26 results in a significant increase in "cobblestone islands" of attachment and emergence of subclonal factor-independent malignant sublines. Biochemical purification of conditioned medium from irradiated D2XRII cells yielded a 75,000-dalton glycoprotein termed leukemogenic stromal factor (LSF) that was neutralized by a polyclonal antiserum to murine macrophage colony-stimulating factor (M-CSF). A monoclonal antibody to the murine M-CSF receptor (c-fms) neutralized the biological activity of this molecule in a manner comparable to its effect on recombinant human or murine M-CSF. FDC-P1JL26 parent cells were positive for Ly5, MEL-14, mGR, VLA-4, PGP-1 (CD44), and Thy1.2. After culture in LSF, Thy1.2, MEL-14, and mGR became undetectable; however, significant cell surface MAC-1 antigen and c-fms (M-CSF receptor) were expressed. Neither line was positive for Ly6, Ly22, I-CAM-1, or B220 antigen. LSF-precultured FDC-P1JL26 cells transferred as single cells to microwell culture with 5000-cGy-irradiated D2XRII cells revealed a 60-fold increase in frequency of cobblestone island formation and evolution of factor-independent subclones compared to the parent line. Both parent and LSF-precultured cells became factor independent at a 100-fold lower frequency if kept in suspension in LSF in the absence of stromal cells. Antiserum to M-CSF or monoclonal antibody to the murine M-CSF receptor (c-fms) did not inhibit or displace cobblestone island formation by either clone of FDC-P1 on irradiated stromal cells indicating a mechanism of binding not involving the M-CSF receptor. However, anti-serum to the M-CSF receptor inhibited growth of one factor-independent subclone. In separate studies, a subclone of IL-3-dependent 32Dc13 cells, expressing the transfected murine c-fms protooncogene but not the parent 32Dc13 cell line or another subclone expressing the transfected gene for the human M-CSF receptor, showed adherence and became factor independent when cocultivated with irradiated D2XRII stromal cells. Thus, irradiated stromal cells bind M-CSF receptor-positive hematopoietic progenitor cells and induce c-fms-dependent factor-independent tumorigenic subclones. The cellular interactions in this model may be relevant to gamma-irradiation leukemogenesis in vivo.     

A constitutively activated chimeric cytokine receptor confers factor- independent growth in hematopoietic cell lines

The high-affinity receptor for granulocyte-macrophage colony- stimulating factor (GMR) comprises at least 2 distinct subunits, alpha and beta common (beta c), whereas the normal erythropoietin receptor (nEpoR) comprises only one known subunit. An arginine to cysteine (R129C) mutation of the extracytoplasmic domain of the murine EpoR leads to Epo-independent growth in transduced cells (cEpoR). To investigate the proliferative functions of the cytoplasmic regions of each GMR subunit separately and the potential of the R129C EpoR mutation to induce factor-independent growth through heterologous receptor regions, we constructed four hybrid receptors: the extracellular region of either murine nEpoR or cEpoR linked to the transmembrane and cytoplasmic regions of either the human GMR alpha or beta c subunit (nE alpha, nE beta, cE alpha, and cE beta). We then expressed them in an interleukin-3-dependent murine cell line, Ba/F3. Expression of nE beta led to Epo-dependent growth, whereas expression of cE beta conferred factor-independent growth. Surprisingly, expression of cE alpha also resulted in factor-independent cell growth, whereas nE alpha did not respond to Epo. Furthermore, the functional hybrid receptors showed Epo-dependent (nE beta) or constitutive (cE alpha and cE beta) tyrosine phosphorylation of the cytoplasmic kinases JAK1 and JAK2. We reasoned that the proliferative signal of cE alpha was transduced either through the alpha tail itself or through an accessory protein such as the endogenous murine beta common subunit (mu beta c). To distinguish these possibilities, the chimeric receptor cE alpha was expressed in the interleukin-2-dependent murine cell line, CTLL-2, that does not express mu beta c. cE alpha did not induce cell growth in CTLL-2; however, when mu beta c was coexpressed with cE alpha in CTLL-2, factor-independent growth was reconstituted. In conclusion, the cytoplasmic domain of the GMR alpha subunit requires a beta chain for transduction of a proliferative signal. Furthermore, the R129C EpoR mutation can constitutively activate heterologous receptors to mediate factor-independent proliferation.     

Identification of primary structural features that define the differential actions of IL-3 and GM-CSF receptors

Activation of human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors, ectopically expressed in FDCP-mix multipotent cells, stimulates self-renewal or myeloid differentiation, respectively. These receptors are composed of unique alpha subunits that interact with common beta(c) subunits. A chimeric receptor (hGM/beta(c)), comprising the extracellular domain of the hGM-CSF receptor alpha subunit (hGM Ralpha) fused to the intracellular domain of hbeta(c), was generated to determine whether hbeta(c) activation is alone sufficient to promote differentiation. hGM-CSF activation of hGM/beta(c), expressed in the presence and absence of the hbeta(c) subunit, promoted maintenance of primitive phenotype. This indicates that the cytosolic domain of the hGM Ralpha chain is required for differentiation mediated by activation of the hGM Ralpha, beta(c) receptor complex. We have previously demonstrated that the alpha cytosolic domain confers signal specificity for IL-3 and GM-CSF receptors. Bioinformatic analysis of the IL-3 Ralpha and GM Ralpha subunits identified a tripeptide sequence, adjacent to the conserved proline-rich domain, which was potentially a key difference between them. Cross-exchange of the equivalent tripeptides between the alpha subunits altered receptor function compared to the wild-type receptors. Both the mutant and the corresponding wild-type receptors promoted survival and proliferation in the short-term but had distinct effects on developmental outcome. The mutated hGM Ralpha promoted long-term proliferation and maintenance of primitive cell morphology, whereas cytokine activation of the corresponding hIL-3 Ralpha mutant promoted myeloid differentiation. We have thus identified a region of the alpha cytosolic domain that is of critical importance for defining receptor specificity.     

Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.

The interleukin 2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two Janus family protein tyrosine kinases (PTKs), Jak1 and Jak3, were shown to associate with IL-2R beta c and IL-2R gamma c, respectively, and their PTK activities are increased after IL-2 stimulation. A Jak3 mutant with truncation of the C-terminal PTK domain lacks its intrinsic kinase activity but can still associate with IL-2R gamma c. In a hematopoietic cell line, F7, that responds to either IL-2 or IL-3, overexpression of this Jak3 mutant results in selective inhibition of the IL-2-induced activation of Jak1/Jak3 PTKs and of cell proliferation. Of the three target nuclear protooncogenes of the IL-2 signaling, c-fos and c-myc genes, but not the bcl-2 gene, were found to be impaired. On the other hand, overexpression of the dominant negative form of the IL-2R gamma c chain, which lacks most of its cytoplasmic domain, in F7 cells resulted in the inhibition of all three protooncogenes. These results provide a further molecular basis for the critical role of Jak3 in IL-2 signaling and also suggest a Jak PTK-independent signaling pathway(s) for the bcl-2 gene induction by IL-2R.     

The phosphoserine-585-dependent pathway of the GM-CSF/IL-3/IL-5 receptors mediates hematopoietic cell survival through activation of NF-kappaB and induction of bcl-2

We have recently identified a novel mechanism of hematopoietic cell survival that involves site-specific serine phosphorylation of the common beta subunit (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors. However, the downstream components of this pathway are not known, nor is its relationship to survival signals triggered by tyrosine phosphorylation of the receptor clear. We have now found that phosphorylation of Ser585 of beta(c) in response to GM-CSF recruited 14-3-3 and phosphatidyl inositol 3-OH kinase (PI 3-kinase) to the receptor, while phosphorylation of the neighboring Tyr577 within this "viability domain" promoted the activation of both Src homology and collagen (Shc) and Ras. These are independent processes as demonstrated by the intact reactivity of phosphospecific anti-Ser585 and anti-Tyr577 antibodies on the cytotoxic T-lymphocyte-ecotrophic retroviral receptor neomycin (CTL-EN) mutants beta(c)Tyr577Phe and beta(c)Ser585Gly, respectively. Importantly, while mutants in which either Ser585 (beta(c)Ser585Gly) or all tyrosines (beta(c)F8) were substituted showed a defect in Akt phosphorylation, nuclear factor kappaB (NF-kappaB) activation, bcl-2 induction, and cell survival, the mutant beta(c)Tyr577Phe was defective in Shc, Ras, and extracellular signal-related kinase (ERK) activation, but supported CTL-EN cell survival in response to GM-CSF. These results demonstrate that both serine and tyrosine phosphorylation pathways play a role in hematopoietic cell survival, are initially independent of each other, and converge on NF-kappaB to promote bcl-2 expression.     

Monoclonal antibody against the common beta subunit (beta c) of the human interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor receptors shows upregulation of beta c by IL-1 and tumor necrosis factor-alpha.

High-affinity receptors for human granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are composed of two distinct subunits, alpha and beta. Each receptor has its own ligand-specific alpha subunit, and the three receptors share the common beta subunit, beta c. Using a transfectant of NIH3T3 cells expressing the high-affinity human GM-CSF receptor, monoclonal antibodies (MoAbs) against beta c were generated. These MoAbs specifically bound to cells bearing beta c and immunoprecipitated the beta c protein of 120 Kd. Using these MoAbs, expression of beta c was examined. It is known that IL-1 augments the proliferative response of a human factor-dependent hematopoietic cell line TF-1 to either GM-CSF, IL-3, or IL-5, and that it upregulates the high-affinity receptors for GM-CSF, IL-3, and IL-5. Antibody binding and immunoprecipitation demonstrated that IL-1 increased cell surface expression of beta c. This enhancement by IL-1 was accompanied by an increased level of beta c mRNA. In addition, we found that tumor necrosis factor-alpha (TNF-alpha) also increased the expression of beta c, although it did not augment the proliferative response of TF-1 to GM-CSF, IL-3, and IL-5.     

Identification of a 14-3-3 binding sequence in the common beta chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors that is serine-phosphorylated by GM-CSF.

The common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of beta(c). However, the contribution of serine phosphorylation in beta(c) to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of beta(c) that interacts with the adaptor protein 14-3-3zeta. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3zeta fusion protein showed that 14-3-3 directly associates with beta(c) but not the GM-CSF receptor alpha chain. C-terminal truncation mutants of beta(c) further showed that a region between amino acids 544 and 626 in beta(c) was required for its association with 14-3-3zeta. This region contains the sequence (582)HSRSLP(587), which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of (582)HSRSLP(587) for EFAAAA completely abolished interaction of beta(c) with GST-14-3-3zeta. Furthermore, the interaction of beta(c) with GST-14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when (585)Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated (585)Ser bound 14-3-3zeta with an affinity of 150 nmol/L. To study the regulation of (585)S phosphorylation in vivo, we raised antibodies that specifically recognized (585)Ser-phosphorylated beta(c). Using these antibodies, we showed that GM-CSF stimulation strongly upregulated (585)Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site ((577)Tyr) to the 14-3-3-binding site ((582)HSRSLP(587)) and their conservation between mouse, rat, and human beta(c) but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.     

ETK2 receptor tyrosine kinase promotes survival of factor-dependent FDC-P1 progenitor cells

By virtue of its high expression in both developing hematopoietic tissues and many myeloid leukemia cells lines, the embryonic tyrosine kinase receptor ETK2 (also known as Tyro3, Sky, and Rse) has been postulated to play a role in early hematopoiesis. To investigate this role, we expressed murine ETK2 in the interleukin 3 (IL-3) dependent myeloid progenitor cell line FDC-P1 and examined its effect on growth factor dependence.ETK2 cDNAs encoding full-length or kinase domain-deleted receptor were retrovirally transduced into murine FDC-P1 cells. Survival, cell cycle status, and proliferative responses of ETK2 expressing clones were studied at normal and reduced growth factor concentrations. ETK2 was expressed as a functional tyrosine kinase of 110 and 150 kDa. This proto-oncogene altered the growth of FDC-P1 cells, allowing survival at reduced growth factor concentrations and delaying apoptosis after IL-3 withdrawal. ETK2-expressing clones contained a higher fraction of cells in the S/G2/M phases of the cell cycle, both after cytokine withdrawal and in the presence of IL-3. Furthermore, these cells had a modestly enhanced proliferative response to IL-3 and granulocyte-macrophage colony-stimulating factor, suggesting that ETK2 intracellular signaling may converge with that of hematopoietic growth factors. The effects of ETK2 expression on viability and proliferation were largely dependent on a functional intracellular tyrosine kinase domain. These results support a role for ETK2 in the survival and/or expansion of primitive hematopoietic cells and suggest that this tyrosine kinase may be implicated in myeloid leukemogenesis as well.     

Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines

The c-kit receptor tyrosine kinase (KIT) is activated upon ligand binding, thereby leading to a variety of signaling events that play a fundamental role in hematopoiesis. In addition to ligand-dependent activation, we have previously shown that KIT is constitutively activated in a ligand-independent manner by two point mutations, Val- 559-->Gly (G559) mutation in the juxtamembrane domain and Asp-814-->Val (V814) mutation in the phosphotransferase domain. To investigate the biochemical consequence and biologic significance of these mutations, retroviral vectors encoding KITG559 or KITV814 were introduced into murine pro-B-type Ba/F3 cells and myeloid FDC-P1 cells, both of which require interleukin-3 (IL-3) for their growth and survival. In the cells, KITG559 or KITV814 were found to be constitutively phophorylated on tyrosine in the absence of stem cell factor (SCF) that is a ligand for KIT. Chemical cross-linking analysis showed that a substantial fraction of the phosphorylated KITG559 underwent dimerization even in the absence of SCF, whereas the phosphorylated KITV814 did not, suggesting the distinct mechanisms underlying constitutive activation of KIT by G559 and V814 mutations. Furthermore, the cells expressing either KITG559 or KITV814 were found to show a factor-independent growth, whereas the cells expressing wild-type KIT (KITWT) proliferated in response to SCF as well as IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing KITG559 or KITV814 into nude mice resulted in production of large tumors at all sites of the injection within 2 weeks, and all nude mice quickly succumbed to leukemia and died. These results suggest that, although the mechanisms underlying constitutive activation of KITG559 or KITV814 may be different, both of the activating mutations have a function to induce a factor-independent and tumorigenic phenotype. Also, the data of this study raise the possibility that the constitutively activating mutations of c-kit may play a causal role in development of hematologic malignancies.     

Heterodimerization of the alpha and beta chains of the interleukin-3 (IL-3) receptor is necessary and sufficient for IL-3-induced mitogenesis.

The high-affinity receptor for interleukin-3 (IL-3) is a complex of the IL-3-binding subunit (alpha(IL-3)) and a larger beta chain-beta(c), or, in the mouse, beta(c) or its close relative beta(IL-3). There is evidence that the critical event that initiates signaling is not the approximation of the cytoplasmic domains of alpha(IL-3) and beta(IL-3), but is, rather, the formation of a beta-beta homodimer. Many of these studies involved the analyses of receptor chimeras where the cytoplasmic domains were derived from alpha(IL-3), beta(c) or beta(IL-3), and the extracellular domains were derived from other cytokine receptors, such as the erythropoietin receptor (EpoR). However, evidence that the EpoR may also associate with other receptors clouds the interpretation of these experiments. Therefore, we reevaluated the structure of the functional IL-3R using chimeric receptors with extracellular domains derived not from members of the cytokine-receptor family, but from CD8 or CD16. We show, by expression of these chimeras in Ba/F3 or CTLL-2 cells, that mitogenic signals were only generated by heterodimerization of the cytoplasmic domains of alpha(IL-3) and beta(IL-3). Homodimers of either alpha(IL-3) or beta(IL-3), alone or in combination, were nonfunctional. Furthermore, the ability of heterodimers to stimulate mitogenesis correlated with their ability to induce tyrosine phosphorylation of JAK-2. These data suggest that the physiological activation of the IL-3R involves the generation of simple heterodimers of alpha(IL-3) and beta(IL-3).     

Growth-promoting effects of insulin-like growth factor-1 (IGF-1) on hematopoietic cells: overexpression of introduced IGF-1 receptor abrogates interleukin-3 dependency of murine factor-dependent cells by a ligand-dependent mechanism.

Although insulin-like growth factor (IGF-1) stimulated 3H-thymidine incorporation upon addition to the interleukin-3 (IL-3)-dependent cell line FDC-P1, IGF-1 did not relieve IL-3 dependency for growth. To further examine the effects of IGF-1 on hematopoietic cells, FDC-P1 cells were infected with a retroviral construct (LISN) containing the human IGF-1 receptor (hIGF-1R) and neo genes. IL-3-independent cells were readily isolated after LISN infection when either IGF-1 or supraphysiologic concentrations of insulin were included in the culture medium. These cells were transformed to IL-3 independence by a ligand-dependent mechanism because their growth was dependent on the presence of either IGF-1 or insulin and growth factors capable of supporting autocrine growth were not detected. Furthermore, a monoclonal antibody (MoAb) directed against the human IGF-1R (alpha IR-3) inhibited IGF-1 but not IL-3-induced proliferation and these cells contained 20- to 200-fold more IGF-1 receptors than uninfected FDC-P1 cells. In contrast, when LISN-infected cells were plated in medium without exogenously supplied IGF-1 or insulin, factor-independent cells were rarely isolated. Growth of these cells was also inhibited by the alpha IR-3 MoAb and they expressed 100- to 400-fold more IGF-1 receptors than uninfected FDC-P1 cells. The endogenous IGF-1 and/or insulin present in the calf serum may have enabled their growth because these cells, unlike the parental cells, would proliferate in serum-free defined media and their growth was again inhibited by the alpha IR-3 MoAb. These results demonstrate that IGF-1 can replace IL-3 for growth when FDC-P1 cells overexpress the IGF-1R. Given the fairly ubiquitous expression of the IGF-1 receptor, these and additional experiments might help to determine whether increased expression of endogenous receptors by cells can lead to leukemogenesis and tumorigenesis. Moreover, hIGF-1R-infected cells will be useful in investigating the mechanisms of IGF1-mediated signal transduction because they are now known to proliferate in response to IGF-1.     

IL-3 receptor signaling is dispensable for BCR-ABL-induced myeloproliferative disease

BCR-ABL expression led to a dramatic up-regulation of the IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptor beta common (IL-3Rbetac) and IL-3 receptor beta (IL-3Rbeta) chains in murine embryonic stem cell-derived hematopoietic cells coincident with an expansion of multipotent progenitors and myeloid elements. This up-regulation required BCR-ABL tyrosine kinase activity and led to IL-3Rbetac/beta chain tyrosine phosphorylation in the absence of detectable IL-3 production. These results suggested that cytokine-independent IL-3 receptor activation could be a dominant signaling component in BCR-ABL-induced leukemogenesis. To unambiguously define the significance of IL-3 receptor-dependent signaling in BCR-ABL-induced leukemogenesis, BCR-ABL-transduced bone marrow cells deficient in either IL-3Rbetac chain or both IL-3Rbetac/beta chain expression were examined for their ability in generating myeloproliferative disease (MPD). These BCR-ABL-expressing knockout cells were capable of generating MPD similar to control cells, demonstrating that IL-3 receptor activation is not essential for BCR-ABL-induced MPD. However, the IL-3Rbetac/beta chain could act as a cofactor in BCR-ABL-induced leukemogenesis by activation of its many known oncogenic signaling pathways.     

Interleukin-2 (IL-2) upregulates BAG-1 gene expression through serine- rich region within IL-2 receptor beta c chain

BAG-1 is a Bci-2-binding protein which functions in protection from apoptotic cell death. Here we provide evidence for interleukin-2 (IL-2)- mediated upregulation of BAG-1 expression. In hematopoietic cell line BAF-B03 F7 cells, gene transfer mediated expression of the IL-2R beta c chain is sufficient to confer proliferation and cell survival responses to IL-2. In these IL-2R beta c-expressing cells, BAG-1 mRNA was dramatically induced by IL-2. The IL-2-mediated induction of BAG-1 expression required the activation of tyrosine kinase(s) and was sensitive to rapamycin as the induction of bcl-2 expression was. Analysis of the transfectants which express mutant IL-2R beta c chains or mutant Janus family protein tyrosine kinase Jak3 lacking the kinase domain showed that the IL-2-mediated BAG-1 gene expression required the serinerich region within the IL-2R beta c chain, but Jak3 activation was dispensable. The signaling pathway for BAG-1 gene expression thus highly resembles that for bcl-2 gene expression, strongly suggesting that their induction shares the same signaling pathway. In addition, deletion of the serine-rich region led to loss of IL-2-mediated protection from apoptotic cell death. Taken together, these studies demonstrate that the serine-rich region of the IL-2R beta c chain mediates the coordinated expression of bcl-2 and BAG-1 genes, thereby contributing to suppression of apoptosis.     

Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain

We have identified a type I cytokine receptor, which we have termed novel interleukin receptor (NILR), that is most related to the IL-2 receptor beta chain (IL-2Rbeta) and physically adjacent to the IL-4 receptor alpha chain gene on chromosome 16. NILR mRNA is most highly expressed in thymus and spleen, and is induced by phytohemagglutinin in human peripheral blood mononuclear cells. NILR protein was detected on human T cell lymphotropic virus type I-transformed T cell lines, Raji B cells, and YT natural killer-like cells. Artificial homodimerization of the NILR cytoplasmic domain confers proliferation to Ba/F3 murine pro-B cells but not to 32D myeloid progenitor cells or CTLL-2 murine helper T cells. In these latter cells, heterodimerization of IL-2Rbeta and the common cytokine receptor gamma chain (gamma(c)) cytoplasmic domains allows potent proliferation, whereas such heterodimerization of NILR with gamma(c) does not. This finding suggests that NILR has signaling potential but that a full understanding of its signaling partner(s) is not yet clear. Like IL-2Rbeta, NILR associates with Jak1 and mediates Stat5 activation.     

Soluble type II interleukin 1 (IL-1) receptor binds and blocks processing of IL-1 beta precursor and loses affinity for IL-1 receptor antagonist.

Two IL-1 receptors have been identified, termed type I and type II. The extracellular domain of the type II IL-1 receptor is released from certain cells and can function as a specific inhibitor of IL-1 beta activity. We assessed the ligand-binding properties of the type II membrane-bound and soluble IL-1 receptor (sIL-1R) from the human B cell line Raji by competition. Upon release, the affinity of sIL-1R for IL-1 alpha and IL-1 beta remained constant, and both soluble and cell surface IL-1 receptors bound to the same regions on the IL-1 beta molecule as defined by binding of a series of IL-1 beta mutant molecules. However, the affinity of sIL-1R for the IL-1 receptor antagonist (IL-1ra) decreased by a factor of 2000 when compared with the cell surface receptor. Type II sIL-1R and IL-1ra had an additive effect in inhibiting the binding of IL-1 beta to cell surface IL-1 receptors. In contrast, the combination of recombinant type 1 sIL-1R with IL-1ra abrogated the inhibition seen with each of the individual agents alone. The type II cell surface IL-1 receptor failed to bind the biologically inactive IL-1 beta precursor molecule, but binding to the IL-1 beta precursor was observed on cellular release of the receptor; this was confirmed with 35S-labeled IL-1 beta. Binding of IL-1 beta precursor by sIL-1R inhibited the precursor's ability to be processed to the mature, biologically active 17-kDa species. These observations suggest that the type II sIL-1R inhibits IL-1 beta at two steps, by preventing processing of propeptide and by blocking the interaction of mature IL-1 beta with type I IL-1 receptor. In addition, type II sIL-1R does not interfere with inhibition mediated by IL-1ra.