Forced Dimerization of gp130 Leads to Constitutive STAT3 Activation, Cytokine-independent Growth, and Blockade of Differentiation of Embryonic Stem Cells

Abstract

The mode of activation of glycoprotein 130 kDa (gp130) and the transmission of the activation status through the plasma membrane are incompletely understood. In particular, the molecular function of the three juxtamembrane fibronectin III-like domains of gp130 in signal transmission remains unclear. To ask whether forced dimerization of gp130 is sufficient for receptor activation, we replaced the entire extracellular portion of gp130 with the c-jun leucine zipper region in the chimeric receptor protein L-gp130. On expression in cells, L-gp130 stimulates ligand-independent signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase 1/2 phosphorylation. gp130 activation could be abrogated by the addition of a competing peptide comprising the leucine zipper region of c-fos. When stably expressed in the interleukin-3-dependent Ba/F3 murine pre-B-cells, these cells showed constitutive STAT3 activation and cytokine-independent growth over several months. Because gp130 stimulation completely suppressed differentiation of murine embryonic stem cells in vitro, we also stably expressed L-gp130 in these cells, which completely blocked their differentiation in the absence of cytokine stimulation and was consistent with high constitutive expression levels of the stem cell factor OCT-4. Thus, L-gp130 can be used in vitro and in vivo to mimic constitutive and ligand-independent activation of gp130 and STAT3, the latter of which is frequently observed in neoplastic diseases. INTRODUCTIONThe interleukin (IL)-6 family of cytokines consists of IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), Oncostatin M (OSM), IL-27, and new neurotrophin-1 (NNT-1). IL-6 and IL-11 induce the formation of a glycoprotein 130 kDa (gp130)-homodimer, whereas signaling by LIF, CNTF, CT-1, and NNT-1 results in the formation of a gp130/leukemia inhibitory factor receptor (LIFR) heterodimer. OSM can induce the formation of a dimer of gp130 with LIFR and the related protein OSMR (Taga and Kishimoto, 1997). IL-27 exclusively signals via a heterodimer composed of gp130 and the WSX-1 receptor (Pflanz et al., 2004).IL-6 has pro-and anti-inflammatory properties and plays a central role in host defense against infection and tissue injuries (Simpson et al., 1997). The activities of IL-6 are highly pleiotropic, stimulating a wide range of biological activities, including B-cell maturation, hepatocyte regeneration, and neuronal growth .IL-6 binds to the specific IL-6 receptor (IL-6R), and this complex associates with two molecules of the ubiquitously expressed gp130 Heinrich et al., 2003). The formation of a hetero-oligomeric receptor complex containing the gp130 signal transducing receptor leads to intracellular activation of Janus tyrosine kinase (JAK)/ Tyk tyrosine kinases as well as the signal transducer and activator of transcription (STAT) family of transcription factors. Furthermore, gp130 activation leads to stimulation of the phosphoinositide 3-kinase and RAS/mitogen-activated protein...

Constitutively Active Mutant gp130 Receptor Protein from Inflammatory Hepatocellular Adenoma Is Inhibited by an Anti-gp130 Antibody That Specifically Neutralizes Interleukin 11 Signaling

“…However, the proliferation of Ba/F3-gp130-gp130⌬YY or Ba/F3-gp130-L-gp130 cells was not affected by B-R3. In L-gp130, the entire extracellular portion of gp130 was replaced with the c-jun leucine zipper region (5). As a control, B-R3 did not inhibit the proliferation of Ba/F3-gp130 cells stimulated with IL-3, indicating that B-R3 specifically blocked the receptor activation of gp130 in Ba/F3-gp130 cells (Fig.…”

“…The marked activation of the gp130 signaling pathway in IHCAs was shown to be directly caused by these gain-of-function somatic mutations within the gp130 receptor chain, resulting in ligandindependent constitutively active mutant gp130 proteins (2). Sustained ligand-independent activation of gp130 homo-and heterotypic signaling pathways was demonstrated recently, showing that long-term activation was not suppressed by negative feedback loops (5,6). Mutant gp130 receptor chains were coexpressed along with wild-type gp130, suggesting a dominant effect of the mutations.…”

GP130 activation induces myeloma and collaborates with MYC

“…Thus, L-GP130 overexpression in BM results in activation of GP130 downstream pre-B cell line BA/F3, which lacks GP130 expression (22), was infected with a retrovirus encoding either WT GP130 or the constitutively active form (L-GP130; ref. 23 and Supplemental Figure 2A). In contrast to WT GP130-expressing BA/F3 cells, cultures of L-GP130-expressing cells grew independently of IL-3 with moderately reduced growth kinetics (Supplemental Figure 2, B and C).…”

Transgenic blockade of interleukin 6 transsignaling abrogates inflammation

“…25 The transfection efficiency as visualized after 24 hours by GFP expression (Axiovert 200 Microscope, Zeiss) was approximately 80%.…”