Cytokine Signaling

Abstract

STAT transcription factors signal from the plasma membrane to the nucleus in response to growth factors and cytokines. We have investigated whether plasma membrane "rafts" are involved in cytokine-activated STAT signaling. Cytokine-free human hepatoma Hep3B cells or cells treated with interleukin-6 (IL-6) or orthovanadate (a general activator of STATs) were fractionated, and plasma membrane raft fractions were obtained by equilibrium sedimentation or flotation through discontinuous sucrose gradients using either non-detergent or detergent-based (saponin or Triton X-100) methods. By Western blotting the plasma membrane raft fractions obtained using either non-detergent or detergent-based methods contained significant amounts of STAT1 and STAT3 (up to ϳ10% of the total cytoplasmic amount) as well as the integral raft proteins caveolin-1 and flotillin-1, the IL-6-receptor signal transducing chain gp130, the interferon-␥ receptor ␣ chain (IFN-␥R␣), and the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57). Upon activation of signaling by IL-6 or orthovanadate the respective Tyr-phosphorylated STAT species were now also observed in the membrane raft fraction but in a form deficient in DNA binding. The data show pre-association of STATs with plasma membrane rafts in flotation fractions, which also contained caveolin-1 and flotillin-1, and suggest that Tyr phosphorylation may not in itself be sufficient to cause the departure of PY-STATs from plasma membrane rafts. Methyl-␤-cyclodextrin, which sequesters cholesterol and disrupts plasma membrane rafts, markedly inhibited IL-6-and IFN-␥-induced STAT signaling. Signaling through specialized raft microdomains may be a general mechanism operating at the level of the plasma membrane through which cytokines and growth factors activate STAT species (the "raft-STAT signaling hypothesis").Signal transduction in mammalian cells is initiated by complex protein-protein interactions between ligands, receptors, and kinases at the level of the plasma membrane. It is now becoming clear that specialized microdomains at the cell surface, known as rafts and/or caveolae, are intimately involved in this process (1, 2). These lipid microdomains contain high concentrations of glycolipids, sphingomyelin, and cholesterol and represent platforms for conducting cellular functions such as vesicular trafficking and signal transduction (1, 2). Raft domains contain several integral raft proteins, which include caveolin and flotillin family members. Signaling processes shown to involve plasma membrane raft domains include immunoglobulin E signaling, T-cell antigen receptor signaling, B-cell receptor signaling, signaling involving epidermal growth factor, platelet-derived growth factor, insulin receptor, Ephrin B1 receptor, neurotrophin, Ha-Ras, nitric-oxide synthase and integrins (reviewed in Refs. 1-4). Caveolin-1 in rafts has been shown to modulate insulin-and Ha-Ras-mediated signaling (the "caveola signaling hypothesis") (3, 4). Despite these major advances in the understanding of rafts as...

IL-6 is required for glioma development in a mouse model

“…Yet, in high-grade gliomas of both human and mouse tumours, we observed, in addition to the expected nuclear localization, a novel distinct plasma membrane-associated immunoreactivity for pY-STAT3. This observation is compatible with recent findings, suggesting that STAT proteins do not float solely as free monomers in the cytosol of mammalian cells but constitute high molecular mass protein complexes in lipid rafts (Sehgal et al, 2002) and/or dimerize prior to its activation (Kretzschmar et al, 2003). Raft proteins reside in specialized microdomains at the cell surface and are thought to play an important role in receptor signalling.…”

“…Raft proteins reside in specialized microdomains at the cell surface and are thought to play an important role in receptor signalling. Intriguingly, treatment of cells with the raftbreaking compound methyl-b-cyclodextrin markedly inhibited IL-6-induced signalling (Sehgal et al, 2002). Recently, STAT3 dimerization and subcellular localization was studied in living cells (Kretzschmar et al, 2003).…”

“…Here, the plasma membrane localization of active STAT3 was most prominent at the tumour margin (Figures 3n and 4d). This distinct plasma membrane localization might represent the preassociation of cytokine-activated pY-STAT3 with raft proteins (Sehgal et al, 2002).…”

Activation of Stat3 by cell confluence reveals negative regulation of Stat3 by cdk2

“…We have shown that the cdk2-inhibitor p27 also may interact with signaling complexes by localizing to lipid rafts (Yaroslavskiy et al, 2001) Such localization could facilitate 'inside-out' signaling of cell cycle regulators to alter signal transduction at the membrane. Indeed, recent reports that Stat3 localizes to rafts (Sehgal et al, 2002) and that stat3 undergoes endocytosis during signaling (Bild et al, 2002) indicate that Stat3 may share topographic proximity with cdk2 and cdk2 inhibitors at the cell membrane. Our finding of Stat3 upregulation by cdk2 inhibitors is consistent with the report that antisense p27Kip1 decreased Stat3 activity in keratinocytes (Hauser et al, 1998).…”

Deregulated TGF-β signaling in leukemogenesis

“…In contrast, the lipid-raft pathway is thought to play a critical role in various physiologic processes, such as protein trafficking, cell polarization and signal transduction (Le Roy and Wrana, 2005). Numerous reports have demonstrated that the lipid-raft pathway is crucial for signal transduction in several pathways such as the T-cell receptordependent signaling cascade (Langlet et al, 2000), B cell receptor signaling (Langlet et al, 2000), H-ras-mediated Raf activation (Parton and Hancock, 2004), tumor necrosis factor (TNF) receptor-mediated nuclear factor (NF)-kB activation (Legler et al, 2003), and interferon-g (IFN-g)-induced signal transducer and activator of transcription (STAT) signaling (Sehgal et al, 2002). Therefore, the lipid rafts represent critical sites for organizing and transmitting the extracellular signal into the cells.…”