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A) Molecular mechanisms of signal transduction through cytokine receptor gp130 in immune response in health and disease: its implication in autoimmune disease

1) gp130 signals in health and disease

2) Project

3) Figures for gp130

5) Publication list

6) OTHER PROJECTS

1) gp130 signals in health and disease

Sixteen years have passed since interleukin 6 (IL-6) was cloned in 1986 ( see a review article by Hirano, Interleukin 6 and its receptor: Ten years later, International Reviews of Immunology, 16:249-284, 1998). During the last decade, many findings were made concerning the structure and function of IL-6 and its receptor, and the role of IL-6 in a variety of diseases.

Many cytokines possess a similar helical structure. The IL-6 receptor and many other cytokine receptors are also structurally similar and constitute the cytokine receptor super family. In addition, cytokine receptor subunits are shared among several cytokine receptors. This sharing of subunits is one of the mechanisms by which the functional redundancy of cytokine activities occurs. A typical example is the gp130 subunit, which is shared among the receptors for IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), IL-11, and cardiotrophin-1 (CT-1). Studies on the signal transduction of IFNs have shown that novel tyrosine kinases, JAKs (Janus kinases), and transcriptional factors, STATs (signal transducer and activator of transcription), play a major role in signal transduction through the receptors for a variety of cytokines and hormones.

The binding of IL-6 to a chain (CD126) resulted in the formation of hexametric complex containing two molecules of each component, IL-6, a chain and gp130, followed by the activation of Janus kinase (JAK) family protein tyrosine kinase(s) and tyrosine phosphorylation of various cellular proteins including gp130 itself. Of the JAK family kinases, Jak1, Jak2 and Tyk2 constitutively associate with gp130 or gp130- related LIF receptor and are activated by the IL-6 family of cytokines. The activated tyrosine kinases, in turn, phosphorylate and activate the signal transducer and activator of transcription (Stat) family proteins, especially STAT3 and Stat1 for IL-6 depending on the phosphorylated YXXQ motif on gp130. Gp130 also has a YXXV motif, which is recognized by SHP-2 (also referred as PTP1D, SHPTP-2, PTP2C, and Syp), a member of the phosphotyrosine phosphatase family containing two SH2 domains, and upon ligand binding SHP-2 has been shown to be tyrosine phosphorylated in a manner that depends on the second tyrosine of gp130. SHP-2 has a higher homology with the Drosophila phosphotyrosine phosphatase, Corkscrew(CSW), which acts downstream of the Drosophila tyrosine kinase, Torso. Like ﾊCSW, SHP-2 has been shown to act downstream of tyrosine kinases such as the epidermal growth factor receptor, the fibroblast growth factor receptor, and the insulin receptor.

However, the physiological roles of STAT pathway or SHP-2-mediated pathway through gp130 are largely unknown. To understand the molecular mechanisms which determine the cell fate through gp130, we constructed a chimeric receptor consisting of extracelluar domain of G-CSF receptor or growth hormone receptor and intracellular domain of gp130. We introduced a series of mutated chimeric receptors into M1 leukemic cells in which IL-6 induces growth arrest and macrophage differentiation and pro-B cell line, BAF/B03 cells in which IL-6 induces growth. By using the full-length cytoplasmic domain and mutants with progressive carboxyterminal deletions, we identified the first 133 amino acid residues of gp130 as the minimal region necessary and sufficient for both growth arrest and differentiation signal in M1 cells and growth signal in BAF/B03 cells (Yamanaka et al, 1996: Fukada et al, 1996). Immediate early responses, such as STAT3 activation, junB, egr1 and IRF1 induction, and myb and myc repression, were observed in M1 clones with chimeric receptors containing at least the first 133 amino acids of gp130. The mutants harboring mutations at tyrosine residues that were responsible for STAT3 activation, did not differentiate into macrophage, or show the immediate early responses including down regulation of myb and myc. Our result showed STAT3 activation tightly linked to growth arrest and differentiation, so STAT3 may play an essential role to generate growth arrest and differentiation signal in M1 cell(Yamanaka et al, EMBO J., 1996). Consistent with these observations, we showed that dominant-negative forms of STAT3 inhibited both IL-6-induced growth arrest and macrophage differentiation in the M1 transformants(Nakajima et al, EMBO J., 1996). Blocking of Stat activation resulted in inhibition of IL-6-induced repression of c-myb and c-myc. Furthermore, IL-6 enhanced the growth of M1 cells when STAT3 was suppressed. Thus IL-6 generates both growth-enhancing signals and growth arrest- and differentiation-inducing signals at the same time in M1 cells and STAT3 may be critically involved in the cell decision in response to gp130 signal. We further tried to clarify growth signal in a pro-B cell line, BAF/B03 cells. We showed that at least two distinct signals, cell cycle progression and anti-apoptosis, are required for gp130-induced cell growth(Fukada et al,, Immunity, 1996). The second tyrosine in the YXXV motif (from the membrane) of gp130, which was required for the tyrosine phosphorylation of SHP-2, its association with GRB2, and a MAP kinase activation, was essential for cell cycle progression but not for anti-apoptosis. On the other hand, the tyrosine in the YXXQ motifs essential for STAT3 activation was required for bcl-2 induction and anti-apoptosis. Furthermore, dominant negative STAT3 inhibited anti-apoptosis and bcl-2 induction. These data demonstrate that two distinct signals, cell cycle progression and anti-apoptosis, are required for gp130-induced cell growth and STAT3 is involved in anti-apoptosis. All results suggest that STAT3 play a key role in gp130-mediated regulation of cell growth, differentiation and suvival. We further showed that STAT3 plays a key role in the G1 to S phase cell-cycle transition induced by the cytokine receptor subunit gp130, through the upregulation of cyclins D2, D3 and A, and cdc25A, and the concomitant downregulation of p21 and p27. Furthermore, unexpectedly, we found that gp130 could induce the expression of p21 when STAT3 activation was suppressed. Such contradictory signals regulating cell-cycle progression could be simultaneously delivered from distinct cytoplasmic regions of gp130. We propose an 'orchestrating model' for cytokine and growth factor action in which contradictory signals are orchestrated to produce a specific effect in a target cell.(Fukada et al, EMBO J. ,1998). Thus, the activation of STAT3 by the cytokine receptor gp130 is required for both the G1 to S cell cycle transition and antiapoptosis. To clarify this molecular mechanisms, we tried to identify the target molecules of Stat3. We found that Pim-1 and Pim-2 are targets for the gp130-mediated STAT3 signal. Expression of a kinase-defective Pim-1 mutant attenuated gp130-mediated cell proliferation. Constitutive expression of Pim-1 together with c-myc, another STAT3 target, fully compensated for loss of the STAT3-mediated cell cycle progression, antiapoptosis, and bcl-2 expression. We also identified valosine-containing protein (VCP) as a target gene for the Pim-1-mediated signal. Expression of a mutant VCP led cells to undergo apoptosis. These results indicate that Pim-family proteins play crucial roles in gp130-mediated cell proliferation and explain the synergy between Pim and c-Myc proteins in cell proliferation and lymphomagenesis.(Shirogane et al, Immunity, 1999)

To further elucidate the in vivo roles of gp130-mediated each of signal transduction pathway, we generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. Our results shows that gp130-mediated STAT3 signal plays important roles in B cell differentiation and Ig production. Furthermore, tyrosine 759 of gp130, SHP-2 binding site, is positively involved in gp130-mediated MAPK activation, while it negatively regulates gp130-induced STAT3 activation and Ig production. These results indicate the balance of the contradictory signals generated through gp130 determines the final output of the biological activity of IL-6. (Ohtani et al , Immunity, 2000). Interesting finding is that the knockin mice expressing mutant gp130 with a defective in the SHP2 binding site (F759 mice) spontaneously generate autoimmunity and rheumatoid arthritis (RA)-like disease as aged (Atsumi, T et al., A point mutation of Tyr-759 in interleukin 6 family cytokine receptor subunit gp130 causes autoimmune arthritis. J. Exp Med. 196: 979-990, 2002 (PubMed)).

Pleiotropic Function of Interleukin-6

IL-6 induces the differentiation of B cells to antibody forming cells (Hirano et al, Nature 324, 73, 1986)

IL-6 is a growth factor of myeloma cells (Kawano et al, Nature 332, 83, 1988). Generation of plasmacytosis and plasmacytoma in IL-6 transgenic mice (Suematsu et al, PNAS,86, 7547, 1989; Suematsu et al,PNAS, 89, 232, 1992)

IL-6 induces macrophage differentiation of M1 leukemic cells (Miyaura et al, FEBS Letters, 234, 17, 1988; Yamanaka et al, EMBO J, 15, 1557, 1996; Nakajima et al, EMBO J, 15, 3651, 1996)

IL-6 induces neurite outgrowth in PC12 cells (Satoh et al, Mol. Cell. Biol., 8, 3546, 1988, Ihara et al, EMBO J. 17: 5345-5352, 1997.)

Signal transduction through gp130

　

2) Project

1. As described above, the gp130 point mutant knockin mice with a defective in the gp130-mediated SHP2/Gab/MAPK signal (F759 mice) spontaneously generate RA-like disease with a frequency of 100 % at the age of 18 months. We wish to elucidate what cells and what genes are required for the initiation of the disease and the effecter phase of RA-like disease in F759 mice. To identify the genetic background affecting disease development, we will backcross the original F759 mice (129/Bl6 hybrid) to different strains, such as C57BL/6, Balb/c, DBA/1, 129, C3H/J. To examine whether hematopoietic cells, non-hematopoietic microenvironment, or both are necessary for the initiation of RA-like disease in F759 mice, bone marrow transfer experiment in several combinations will be made. We have already known that RA-like disease can be transferred to RAG2-/- mice by spleen cells obtained from the diseased mice. We will examine what population of spleen cells of the diseased mice can transfer the disease to RAG2-/- mice. Another important question is what cells and molecules are required for the initiation of the disease. To answer this issue we will cross the F759 mice with several kinds of knockout mice, such as CD4-/-, CD8-/-, mMT-/-, RAG2-/-, IL-6-/-, stat3-promoter mutant mice so on. We have already made knockin mice expressing low level of STAT3 due to the point mutation of the stat3 promoter region. We will also try to identify the genes abnormally expressed in the F759 mice.

2. Elucidation of the effect of environmental factors on autoimmune diseases in F759 mice. We wish to search the environmental factors, which affect the onset of RA-like disease in F759 mice. We will examine the effect of LPS, CFA, bacterial and viral infection and over expression of IL-6 on the generation of RA in F759 mice. We have already found that HTLV1-tax expression accelerates the onset of RA in F759 mice by crossing F759 mice and Tax-transgenic mice in C57BL/6 genetic background. Under these conditions, RA-like disease developed as early as 3 months of age. We wish to examine if F759 mutation in hematopoietic lineage cells is enough for the generation of RA-like disease. If this is the case, we will examine whether the retrovirus-mediated expression of dominant negative STAT3 in hematopoietic lineage cells can prevent the disease.　We also take advantage of Tax-induced acceleration of disease onset to investigate what populations of hematopoietic cell lineages are required for the disease initiation and effecter phase for the disease.

3. Elucidation of the immunological state observed in the F759 mice.

To investigate the effect of F759 mutation on thymic negative selection, we will cross the F759 mice with anti-HY TCR transgenic mice, and anti-OVA TCR transgenic mice. We also examine if F759 mutaion has any effect on anti-CD3- and superantigen-induced clonal deletion and activation induced cell death. We have already observed that activated memory CD4 cells are increased in the diseased mice. We wish to identify the molecular and immunological mechanisms by which memory T cells are increased in the F759 mice.

The final goal of our project is the clarification of the regulatory mechanisms of the immune response from the view point of signal trasnduction, in particular, cytokine mediated signaling pathways and establishment of the methods by which we can modurate and control immune response to prevent or cure autoimmune diseases. We will continue and extend the experiments planned above. Furthermore, based on the results obtained from the experiments performed, we will make several knockout mice to examine if the genes identified are actually involved in the regulation of the immune response and autoimmune diseases. We also try to examine the effect of retrovirus-mediated gene transfer of the dominant negative form of newly identified genes to bone marrow cells on the generation of the disease. Moreover, based on the information on the genetic background affecting the generation of autoimmune disease in F759 mice, we will try to identify the other genes affecting disease development in F759 mice by simple sequence length polymorphisms (SSLP). Finally, we will try to search inhibitors for the molecules critically involved in the immune response, allergic response and autoimmune diseases based on the results obtained from our research plans.

This project would shed light on the molecular mechanisms of immune responses, homeostasis of immune system and autoimmune diseases. Furthermore, F759 mouse is a very useful model mouse to investigate the molecular mechanisms by which autoimmune disease is developed and search new drugs to prevent or cure the autoimmune diseases, such as RA. In summary, this project would greatly contribute to clarification of the molecular mechanisms of immune responses and development of new drugs to regulate immune response, allergic response, and autoimmune diseases.

　

Figures for gp130

　

　

　

Publications:

Reviews