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Interleukin-4 and Interleukin-13 Signaling Connections Maps

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Science  06 Jun 2003:
Vol. 300, Issue 5625, pp. 1527-1528
DOI: 10.1126/science.1085458

Abstract

Cytokines are inflammatory mediators important in responding to pathogens and other foreign challenges. Interleukin-4 (IL-4) and IL-13 are two cytokines produced by T helper type 2 cells, mast cells, and basophils. In addition to their physiological roles, these cytokines are also implicated in pathological conditions such as asthma and allergy. IL-4 can stimulate two receptors, type I and type II, whereas IL-13 signaling is mediated only by the type II receptor (see the STKE Connections Maps). These cytokines activate the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascades, which may contribute to allergic responses. In addition, stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway through recruitment of members of the insulin receptor substrate family may contribute to survival and proliferation.

The cytokines IL-4 and IL-13 are produced by T helper type 2 (TH2) cells in response to antigen receptor engagement, and by mast cells and basophils upon cross-linkage of the high-affinity receptor for immunoglobulin E (IgE) (1, 2). These cytokines can elicit similar responses, many of which are associated with allergy, asthma, and inhibition of autoimmunity. However, IL-4 is more active in regulating TH2 development, whereas IL-13 is more active in regulating airway hypersensitivity and mucus hypersecretion, and TH2-type inflammation of the bowel (24). The signal transduction pathways activated by these cytokines, and the mechanisms by which they regulate cell growth, survival, and gene expression, were characterized with long-term cell lines (15) (Fig. 1). More recently, studies of signaling in primary cell types are revealing new signaling rules (5). Because therapeutic strategies targeting IL-4 and IL-13 or their specific signaling pathways are currently being tested to treat allergy and asthma (3), understanding these differences is critical. However, the differences in the signaling pathways and target genes of IL-4 and IL-13 are not yet clear.

Fig. 1.

Selected signaling pathways activated by IL-4. The binding of IL-4 to the IL-4Rα induces heterodimerization with the γC (shown) or the IL-13Rα1. This dimerization activates the JAKs that initiate the phosphorylation cascade. Tyrosine residues in the cytoplasmic tail of the IL-4Rα are phosphorylated and act as docking sites for signaling molecules. The first cytoplasmic tyrosine residue (Y1) interacts with PTBs. Members of the IRS family and Shc dock to this site. Tyrosine residues 2 through 4 interact with the SH2 domain of STAT6. The fifth cytoplasmic residue lies in an ITIM consensus motif and interacts with the phosphatase Shp-1. After being recruited to the receptor complex, these signaling molecules are tyrosine phosphorylated. Phosphorylated STAT6 dimerizes, migrates to the nucleus, and binds to promoters of genes such as CD23 and major histocompatibility complex (MHC) class II. STAT6 can be dephosphorylated by the action of Shp-1. Phosphorylated IRS binds to the p85 subunit of PI3K and to Grb2. In cell lines, the IRS pathway has been linked to cellular proliferation in response to IL-4. The activated PI3K regulates the p70S6 kinase and protein kinase B (PKB), both of which have been shown to participate in survival pathways in cell lines. PI3K activity also regulates the IL-4–induced phosphorylation of the DNA binding protein HMG(I)Y. In contrast to cell lines, in primary lymphocytes STAT6 also plays a role in proliferation and protection from apoptosis, although the contribution of IRS2 is unclear.

IL-4 receptors are composed of two transmembrane proteins. The IL-4Rα chain binds IL-4 with high affinity, leading to dimerization with another protein to form either a type I or type II receptor. In cells of hematopoietic lineage, the type I receptor arises by recruitment of a common gamma chain (γC) (also a component of receptors for IL-2, -7, -9, -15, and -21) (13). In nonhematopoietic cells, the type II receptor is formed by interaction of IL-4Rα with IL-13Rα1 instead of γC (24). IL-13 also binds two different receptor complexes. IL-13 binds to IL-13Rα1 with high affinity, inducing heterodimerization with IL-4Rα to form a complex identical to the type II IL-4R [see the IL-4 and IL-13 Pathways in the STKE Connections Maps (6, 7)]. Because IL-4 and IL-13 both use the type II receptor, the molecular basis of the cytokines' differing functional responses is unclear. In contrast to its interaction with the IL-13Rα1, IL-13 binds IL-13Rα2 with greater affinity than IL-13Rα1 but fails to induce a signal, so that IL-13Rα2 acts as a decoy receptor (8).

As with other cytokine receptors, the cytoplasmic tails of IL-4 and IL-13 receptor subunits associate with tyrosine kinases of the Janus family (JAK 1–3, TYK2) (17). IL-4Rα associates with JAK1 and γC with JAK3, whereas IL-13Rα1 interacts with either JAK2 or TYK2, but not JAK3. Dimerization of the receptor subunits enhances JAK activity, leading to phosphorylation of tyrosine residues in the cytoplasmic domain of IL-4Rα. These phosphotyrosines then act as docking sites for signaling molecules containing protein tyrosine binding domains (PTBs) or Src homology 2 (SH2) domains.

One critical signaling pathway activated by the IL-4 receptor leads to tyrosine phosphorylation of STAT6, a latent cytoplasmic transcription factor in the family of signal transducers and activators of transcription (STAT) (9, 10). In cell lines, STAT6 is recruited to IL-4Rα upon phosphorylation of the second, third, or fourth cytoplasmic tyrosine residues (2). STAT6 becomes tyrosine phosphorylated, disengages from the IL-4Rα, dimerizes by reciprocal SH2 domain interaction, migrates to the nucleus, and binds to consensus sequences found within promoters of IL-4– and IL-13–regulated genes. STAT6 is central in gene regulation and the IL-4– and IL-13–regulated allergic responses, including TH2 differentiation, IgE production, and chemokine and mucus production at sites of allergic inflammation (3). STAT6 also regulates gene expression involved in cell survival, including E4 binding protein–4 (E4BP4) and growth factor–induced gene–1 (GFI-1) (2, 11), suggesting that the STAT6 pathway helps regulate lymphocyte growth and survival.

A second mechanism of signal transduction activated by IL-4 and IL-13 leads to the insulin receptor substrate (IRS) family (17), which consists of four proteins (IRS-1 to IRS-4). IRS-1, -2, and -3 can interact with IL-4Rα, but IRS-2 is the main family member expressed in hematopoietic cells. These large cytoplasmic docking proteins contain a PTB domain and many sites for serine, threonine, and tyrosine phosphorylation. IRS proteins are recruited to the IL-4Rα by the first cytoplasmic tyrosine residue (Y1), which lies within the I4R consensus motif (2), also found in receptors for insulin and the insulin-like growth factor type I (IGF-1). Tyrosine-phosphorylated sites within the IRS proteins associate with cytoplasmic signaling molecules containing SH2 domains, including the p85 subunit of phosphatidylinositol 3-kinase (PI3K) (12), which results in activation of the p110 catalytic subunit of PI3K. Pathways downstream of PI3K are important for growth, survival, and regulation of gene expression in response to IL-4 and IL-13. However, results of a recent analysis of cells derived from IRS2-deficient mice question the importance of the IRS2 to the PI3K pathway in regulating lymphocyte proliferation and survival (13, 14). Additional proliferation signals may be mediated by signals emanating from the recruitment of the adaptor protein Shc through a PTB domain, and negative regulation may be mediated by the guanine nucleotide exchange factors (GEFs) p62Dok and FRIP1 to the Y1 position in the IL-4Rα (2).

Shortly after T cell antigen receptor ligation, the IL-4 receptor becomes transiently refractory to activation (15). What mechanisms may be mediating such negative regulation? The fifth conserved cytoplasmic tyrosine residue, near the C-terminus of IL-4Rα, lies in a consensus motif termed an immunoreceptor tyrosine-based inhibitory motif (ITIM). This sequence interacts with the SH2 domains of the tyrosine phosphatase Shp-1, which appears to be involved in the negative regulation of signaling (16). STAT6 phosphorylation appears tightly regulated, because it is rapidly dephosphorylated in the cytoplasm and nucleus in the absence of continued IL-4 stimulation (17, 18). One candidate effector of STAT6 dephosphorylation is the phosphatase Shp-1 (18). Further, STAT6 stimulates the expression of negative regulatory proteins such as those in the suppressor of cytokine signaling (SOCS) family (10). Thus, dephosphorylation of STAT6 by phosphatases and inhibition of JAK activity by SOCS proteins negatively regulate STAT6 function. However, clarifying mechanisms whereby phosphoSTAT6 in the nucleus is eliminated is an important area for further investigation.

The processes involved in STAT6 activation and inhibition are probably more complicated than outlined above. Although their role(s) in the stimulation or inhibition of STAT6 are still being investigated, Src tyrosine kinases, serine kinases, and proteases have all been implicated in STAT6 regulation (10, 1921). More over, emerging evidence indicates that in retrovirally transduced activated T lymphocytes, mutated IL-4Rα proteins lacking all cytoplasmic tyrosines can mediate STAT6 activation in response to IL-4 (5). Because this IL-4Rα tyrosine-independent STAT6 activation appears sufficient to promote TH2 differentiation, the mechanism by which T cell activation leads to this additional IL-4Rα–mediated STAT activation pathway is of considerable importance. Additional differences between resting and activated T cells in the mechanisms by which IL-4Rα transduces signals are also emerging. IL-4 induces the phosphorylation of a related STAT dimer, STAT5, in activated but not resting cells, in a process apparently requiring both the calcineurin phosphatase and activity of the NF-κB transcription factor (5).

Determining the molecular mechanisms by which cellular activation alters activation and specificity in the STAT pathway will be a fruitful ground for future investigation. Furthermore, as suggested by the presence of allergy-associated polymorphisms near docking sites in the IL-4Rα (22), subtle differences in IL-4 and IL-13 signaling in different primary cell types could have profound implications for allergy and asthma.

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