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• W1987441485 abstract "•Chi3l1 binds to IL-13Rα2 and stimulates MAPK, Akt/PKB, and Wnt//β-catenin signaling•Chi3l1 regulates oxidant injury, apoptosis, and pyroptosis via IL-13Rα2•Chi3l1 regulates antibacterial response and inflammasome activation via IL-13Rα2•Chi3l1 regulates melanoma metastasis and TGF-β1 production via IL-13Rα2 Members of the 18 glycosyl hydrolase (GH 18) gene family have been conserved over species and time and are dysregulated in inflammatory, infectious, remodeling, and neoplastic disorders. This is particularly striking for the prototypic chitinase-like protein chitinase 3-like 1 (Chi3l1), which plays a critical role in antipathogen responses where it augments bacterial killing while stimulating disease tolerance by controlling cell death, inflammation, and remodeling. However, receptors that mediate the effects of GH 18 moieties have not been defined. Here, we demonstrate that Chi3l1 binds to interleukin-13 receptor α2 (IL-13Rα2) and that Chi3l1, IL-13Rα2, and IL-13 are in a multimeric complex. We also demonstrate that Chi3l1 activates macrophage mitogen-activated protein kinase, protein kinase B/AKT, and Wnt/β-catenin signaling and regulates oxidant injury, apoptosis, pyroptosis, inflammasome activation, antibacterial responses, melanoma metastasis, and TGF-β1 production via IL-13Rα2-dependent mechanisms. Thus, IL-13Rα2 is a GH 18 receptor that plays a critical role in Chi3l1 effector responses. Members of the 18 glycosyl hydrolase (GH 18) gene family have been conserved over species and time and are dysregulated in inflammatory, infectious, remodeling, and neoplastic disorders. This is particularly striking for the prototypic chitinase-like protein chitinase 3-like 1 (Chi3l1), which plays a critical role in antipathogen responses where it augments bacterial killing while stimulating disease tolerance by controlling cell death, inflammation, and remodeling. However, receptors that mediate the effects of GH 18 moieties have not been defined. Here, we demonstrate that Chi3l1 binds to interleukin-13 receptor α2 (IL-13Rα2) and that Chi3l1, IL-13Rα2, and IL-13 are in a multimeric complex. We also demonstrate that Chi3l1 activates macrophage mitogen-activated protein kinase, protein kinase B/AKT, and Wnt/β-catenin signaling and regulates oxidant injury, apoptosis, pyroptosis, inflammasome activation, antibacterial responses, melanoma metastasis, and TGF-β1 production via IL-13Rα2-dependent mechanisms. Thus, IL-13Rα2 is a GH 18 receptor that plays a critical role in Chi3l1 effector responses. The 18 glycosyl hydrolase (GH 18) gene family contains true chitinases (Cs) that degrade chitin polysaccharides and chitinase-like proteins (CLPs) that bind to but do not degrade chitin (Lee et al., 2011Lee C.G. Da Silva C.A. Dela Cruz C.S. Ahangari F. Ma B. Kang M.J. He C.H. Takyar S. Elias J.A. Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue remodeling, and injury.Annu. Rev. Physiol. 2011; 73: 479-501Crossref PubMed Scopus (603) Google Scholar). They are members of an ancient gene family that exists in species as diverse as plants and humans and has evolved during speciation, with a particularly impressive increase in CLPs coinciding with the appearance of mammals (Aerts et al., 2008Aerts J.M. van Breemen M.J. Bussink A.P. Ghauharali K. Sprenger R. Boot R.G. Groener J.E. Hollak C.E. Maas M. Smit S. et al.Biomarkers for lysosomal storage disorders: identification and application as exemplified by chitotriosidase in Gaucher disease.Acta Paediatr. Suppl. 2008; 97: 7-14Crossref PubMed Scopus (60) Google Scholar, Funkhouser and Aronson, 2007Funkhouser J.D. Aronson Jr., N.N. Chitinase family GH18: evolutionary insights from the genomic history of a diverse protein family.BMC Evol. Biol. 2007; 7: 96Crossref PubMed Scopus (228) Google Scholar). This retention over species and evolutionary time has led to the belief that these moieties play essential roles in biology. Recent studies have confirmed this speculation (Dela Cruz et al., 2012Dela Cruz C.S. Liu W. He C.H. Jacoby A. Gornitzky A. Ma B. Flavell R. Lee C.G. Elias J.A. Chitinase 3-like-1 promotes Streptococcus pneumoniae killing and augments host tolerance to lung antibacterial responses.Cell Host Microbe. 2012; 12: 34-46Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Lee et al., 2009Lee C.G. Hartl D. Lee G.R. Koller B. Matsuura H. Da Silva C.A. Sohn M.H. Cohn L. Homer R.J. Kozhich A.A. et al.Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13-induced tissue responses and apoptosis.J. Exp. Med. 2009; 206: 1149-1166Crossref PubMed Scopus (333) Google Scholar, Lee et al., 2011Lee C.G. Da Silva C.A. Dela Cruz C.S. Ahangari F. Ma B. Kang M.J. He C.H. Takyar S. Elias J.A. Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue remodeling, and injury.Annu. Rev. Physiol. 2011; 73: 479-501Crossref PubMed Scopus (603) Google Scholar, Lee and Elias, 2010Lee C.G. Elias J.A. Role of breast regression protein-39/YKL-40 in asthma and allergic responses.Allergy Asthma Immunol. Res. 2010; 2: 20-27Crossref PubMed Scopus (64) Google Scholar, Sohn et al., 2010Sohn M.H. Kang M.J. Matsuura H. Bhandari V. Chen N.Y. Lee C.G. Elias J.A. The chitinase-like proteins breast regression protein-39 and YKL-40 regulate hyperoxia-induced acute lung injury.Am. J. Respir. Crit. Care Med. 2010; 182: 918-928Crossref PubMed Scopus (81) Google Scholar). This is particularly true for the prototypic CLP chitinase 3-like-1 (Chi3l1, also called YKL-40 in humans and BRP-39 in mice), which has been shown by our laboratory and others to play major roles in antipathogen, antigen-induced, oxidant-induced, inflammation, repair and remodeling responses by regulating a variety of essential biologic processes including oxidant injury apoptosis, pyroptosis, inflammasome activation, Th1/Th2 inflammatory balance, M2 macrophage differentiation, transforming growth factor β1 (TGF-β1) elaboration, dendritic cell accumulation and activation, and mitogen-activated protein kinase (MAPK) and Akt signaling (Areshkov et al., 2012Areshkov P.O. Avdieiev S.S. Balynska O.V. Leroith D. Kavsan V.M. Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation.Int. J. Biol. Sci. 2012; 8: 39-48Crossref PubMed Scopus (57) Google Scholar, Chen et al., 2011aChen C.-C. Llado V. Eurich K. Tran H.T. Mizoguchi E. Carbohydrate-binding motif in chitinase 3-like 1 (CHI3L1/YKL-40) specifically activates Akt signaling pathway in colonic epithelial cells.Clin. Immunol. 2011; 140: 268-275Crossref PubMed Scopus (75) Google Scholar, Dela Cruz et al., 2012Dela Cruz C.S. Liu W. He C.H. Jacoby A. Gornitzky A. Ma B. Flavell R. Lee C.G. Elias J.A. Chitinase 3-like-1 promotes Streptococcus pneumoniae killing and augments host tolerance to lung antibacterial responses.Cell Host Microbe. 2012; 12: 34-46Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Kim et al., 2012Kim M.N. Lee K.E. Hong J.Y. Heo W.I. Kim K.W. Kim K.E. Sohn M.H. Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death.Biochem. Biophys. Res. Commun. 2012; 421: 790-796Crossref PubMed Scopus (38) Google Scholar, Lee et al., 2009Lee C.G. Hartl D. Lee G.R. Koller B. Matsuura H. Da Silva C.A. Sohn M.H. Cohn L. Homer R.J. Kozhich A.A. et al.Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13-induced tissue responses and apoptosis.J. Exp. Med. 2009; 206: 1149-1166Crossref PubMed Scopus (333) Google Scholar, Sohn et al., 2010Sohn M.H. Kang M.J. Matsuura H. Bhandari V. Chen N.Y. Lee C.G. Elias J.A. The chitinase-like proteins breast regression protein-39 and YKL-40 regulate hyperoxia-induced acute lung injury.Am. J. Respir. Crit. Care Med. 2010; 182: 918-928Crossref PubMed Scopus (81) Google Scholar). The potential importance of YKL-40/Chi3l1/BRP-39-induced responses can also be seen in the large number of diseases in which Chi3l1/YKL-40 excess has been documented and the observation that the degree of Chi3l1/YKL-40 dysregulation often correlates with the severity and natural history of these disorders (reviewed in Coffman, 2008Coffman F.D. Chitinase 3-Like-1 (CHI3L1): a putative disease marker at the interface of proteomics and glycomics.Crit. Rev. Clin. Lab. Sci. 2008; 45: 531-562Crossref PubMed Scopus (98) Google Scholar, Lee et al., 2011Lee C.G. Da Silva C.A. Dela Cruz C.S. Ahangari F. Ma B. Kang M.J. He C.H. Takyar S. Elias J.A. Role of chitin and chitinase/chitinase-like proteins in inflammation, tissue remodeling, and injury.Annu. Rev. Physiol. 2011; 73: 479-501Crossref PubMed Scopus (603) Google Scholar). Surprisingly, the mechanisms via which the GH 18 moieties mediate their biologic effects are poorly understood. Importantly the possibility that GH 18 proteins mediate their biologic effects via a ligand-receptor paradigm has not been addressed, and moieties that bind to and signal in response to any of these regulators have not been defined. To address the possibility that YKL-40/Chi3l1/BRP-39, which does not have known enzymatic activity, mediates its effects via identifiable receptors, we used yeast two-hybrid binding and colocalization assays to define YKL-40/Chi3l1/BRP-39 binding-partner interactions and assessments of signaling, gene expression, and in vivo phenotype generation to evaluate the consequences of these interactions. These studies demonstrate that YKL-40/Chi3l1/BRP-39 binds to interleukin-13 receptor α2 (IL-13Rα2). They also demonstrate that YKL-40/Chi3l1/BRP-39, IL-13Rα2, and IL-13 are in a multimeric complex. Lastly, they demonstrate that YKL-40 activates MAPK, Akt, and Wnt/β-catenin signaling pathways and regulates apoptosis, pyroptosis, inflammasome activation, oxidant injury, antibacterial responses, melanoma metastasis, and TGF-β1 elaboration via IL-13Rα2-dependent mechanisms. To define the binding partners of Chi3l1/YKL-40, yeast two-hybrid analysis was undertaken using Chi3l1/YKL-40 as bait. A number of clones gave positive results in these assays. One of the most intriguing encoded IL-13Rα2 (Figure S1A). Further documentation of the interaction between YKL-40 and IL-13Rα2 was obtained with coimmunoprecipitation (coIP), colocalization, and Biacore assays. In the former, A549 cells were transfected with both of these moieties and subjected to immunoprecipitation (IP) with antibodies to one moiety, and the precipitate was then analyzed via western blotting using antibodies to the other moiety. In these experiments, the two moieties always traveled together with IP using antibodies against YKL-40 always precipitating IL-13Rα2 and vice versa (Figure 1A). Immunohistochemical evaluations of lungs from IL-13 transgenic (Tg) mice (in which Chi3l1/BRP-39 and IL-13Rα2 are both strongly induced) demonstrated that Chi3l1/BRP-39 and IL-13Rα2 frequently colocalize in these tissues (Figure 1B). The major site of this colocalization was in F4/80+ macrophages (Figures 1B and S1B). Interestingly, there were macrophage populations in which colocalization occurred and populations in which Chi3l1 was noted and IL-13Rα2 could not be detected (Figure 1B). Colocalization in some alveolar type II cells was also appreciated (Figures 1B and S1C). To identify the sites in the cell of this colocalization, we employed fluorescence-activated cell sorting (FACS) evaluations of nonpermeabilized cells and immunohistochemistry (IHC) of tissue sections and stained both with anti-Chi3l1 and anti-iL-13Rα2. These studies clearly demonstrate that Chi3l1 and IL-13Rα2 can be seen together on the surface and in the cytoplasm of the cell (Figures 1C and S1D). The Biacore assays also demonstrated that Chi3l1/YKL-40 and IL-13Rα2 bind to one another. At pH 7.4, the binding was quite avid, with a Kd of 23 ± 14 pM (Figure 1D). The koff was approximately 10−5 s−1 and the kon was 3.39 ± 1.54 × 105 M−1 s−1. These studies demonstrate that YKL-40 specifically binds to IL-13Rα2 with high affinity.Figure 1Binding and Localization of Chi3l1/YKL-40 and IL-13Rα2Show full caption(A) A549 cells were transfected with Chi3l1/YKL-40 (Chi3l1) and/or human IL-13Rα2 (IL-13Rα2), lysates were prepared and immunoprecipitated (IP) with either anti-Chi3l1 or anti-iL-13Rα2, and the precipitates were evaluated using immunoblot (IB) analysis as noted.(B) Triple-label IHC to detect the colocalization of IL-13Rα2 and BRP-39 in the macrophages (upper panels) and type 2 alveolar epithelial cells (lower panels) in lungs from IL-13 Tg mice using antibodies to BRP-39, IL-13Rα2, and cell-specific markers of macrophages (anti-F4/80) and type 2 epithelial cells (anti-SP-C). Arrows highlight some of the colocalized cells.(C) Cell surface colocalization of Chi3l1/YKL-40 (Chi3l1) and IL-13Rα2 (IL-13Rα2). THP-1 cells were incubated in the presence or absence of anti-YKL-40-biotin antibody and anti-iL-13Rα2 immunoglobulin G (IgG) antibody without permeabilization. They were then washed and stained with streptavidin (SA)-PE and anti-igG-APC and subjected to flow cytometric analysis.(D) Measurement of the affinity and kinetics of IL-13Rα2 binding to Chi3l1/YKL-40 by surface plasmon resonance (SPR). Chil3l1/YKL-40 was immobilized and IL-13Rα2 was in the mobile phase.(E) Yeast two-hybrid characterization of the structures in hChi31 that bind to IL-13Rα2. The 18 GH catalytic domain (CD), chitin binding motif (CBM), signal peptide (SP), and C-terminal fragment are illustrated.(F) Yeast two-hybrid characterization of the structures in IL-13Rα2 that bind to hChi31. The extracellular domain (ECD), transmembrane domain (TD), intracellular domain (ICD), signal peptide (SP), and sites of N-glycosylation (N) are illustrated.Each panel is representative of a minimum of three evaluations. Scale bars in (B) represent 10 μm. See also Figure S1.View Large Image Figure ViewerDownload (PPT) (A) A549 cells were transfected with Chi3l1/YKL-40 (Chi3l1) and/or human IL-13Rα2 (IL-13Rα2), lysates were prepared and immunoprecipitated (IP) with either anti-Chi3l1 or anti-iL-13Rα2, and the precipitates were evaluated using immunoblot (IB) analysis as noted. (B) Triple-label IHC to detect the colocalization of IL-13Rα2 and BRP-39 in the macrophages (upper panels) and type 2 alveolar epithelial cells (lower panels) in lungs from IL-13 Tg mice using antibodies to BRP-39, IL-13Rα2, and cell-specific markers of macrophages (anti-F4/80) and type 2 epithelial cells (anti-SP-C). Arrows highlight some of the colocalized cells. (C) Cell surface colocalization of Chi3l1/YKL-40 (Chi3l1) and IL-13Rα2 (IL-13Rα2). THP-1 cells were incubated in the presence or absence of anti-YKL-40-biotin antibody and anti-iL-13Rα2 immunoglobulin G (IgG) antibody without permeabilization. They were then washed and stained with streptavidin (SA)-PE and anti-igG-APC and subjected to flow cytometric analysis. (D) Measurement of the affinity and kinetics of IL-13Rα2 binding to Chi3l1/YKL-40 by surface plasmon resonance (SPR). Chil3l1/YKL-40 was immobilized and IL-13Rα2 was in the mobile phase. (E) Yeast two-hybrid characterization of the structures in hChi31 that bind to IL-13Rα2. The 18 GH catalytic domain (CD), chitin binding motif (CBM), signal peptide (SP), and C-terminal fragment are illustrated. (F) Yeast two-hybrid characterization of the structures in IL-13Rα2 that bind to hChi31. The extracellular domain (ECD), transmembrane domain (TD), intracellular domain (ICD), signal peptide (SP), and sites of N-glycosylation (N) are illustrated. Each panel is representative of a minimum of three evaluations. Scale bars in (B) represent 10 μm. See also Figure S1. Deletion mapping was next employed to define the regions in Chi3l1/YKL-40 and IL-13Rα2 that are required for their interactions in the yeast two-hybrid assay. These studies demonstrated that the region of YKL-40 between amino acids 22 and 357 contained the elements that were required to bind to full-length IL-13Rα2 (Figure 1E). This region is called the catalytic domain (CD) of GH 18 moieties and contains the chitin binding motif (CBM) but does not contain its signal peptide (SP) or the C-terminal peptide (Figure 1E). Interestingly, the CBM was necessary, but not sufficient, for YKL-30/Chi3l1-IL-13Rα2 binding (Figure 1E). These studies also demonstrated that the extracellular domain (ECD) of YKL-40 contained elements that were required for binding to full-length Chi3l1/YKL-40 (Figure 1E). The transmembrane and intracellular motifs of IL-13Rα2 did not play a critical role in this interaction (Figure 1F). The four sites of N-linked glycosylation within the ECD were also able to be mutated without abrogating IL-13Rα2 ECD-Chi3l1/YKL-40 binding (Figure 1F). These studies demonstrate that Chi3l1/YKL-40/IL-13Rα2 binding is dependent on the CD and the CBM of the former and the ECD, but not the sites of N-glycosylation in the latter. An ECD-containing soluble version of this receptor has been described elsewhere (Chen et al., 2008Chen W. Tabata Y. Gibson A.M. Daines M.O. Warrier M.R. Wills-Karp M. Hershey G.K. Matrix metalloproteinase 8 contributes to solubilization of IL-13 receptor alpha2 in vivo.J. Allergy Clin. Immunol. 2008; 122: 625-632Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, Chen et al., 2009Chen W. Sivaprasad U. Tabata Y. Gibson A.M. Stier M.T. Finkelman F.D. Hershey G.K. IL-13R alpha 2 membrane and soluble isoforms differ in humans and mice.J. Immunol. 2009; 183: 7870-7876Crossref PubMed Scopus (52) Google Scholar). We thus speculated that Chi3l1/YKL-40 would also bind to soluble IL-13Rα2 (sIL-13Rα2). To test this hypothesis, we did coIP experiments using bronchoalveolar lavage (BAL) fluids from IL-13 Tg mice. These studies demonstrated that Chi3l1/BRP-39 also binds to sIL-13Rα2 (Figure S1E). To define the role(s) of IL-13Rα2 in Chi3l1/YKL-40/BRP-39-induced intracellular signaling, we compared the effects of Chi3l1/YKL-40/BRP-39 on MAPK, extracellular signal-regulated protein kinase (ERK) 1/2, AKT, and Wnt/β-catenin activation in human THP-1 cells treated with IL-13Rα2 small interfering RNA (siRNA) or scrambled controls and peritoneal macrophages from wild-type (WT) and IL-13Rα2 null mice. As can be seen in Figure 2A, ERK activation, AKT activation, and the induction of nuclear β-catenin and c-fos were seen in THP-1 cells 30 min to 2 hr after the addition of recombinant (r) Chi3l1/YKL-40. These effects were dose dependent, with the activation of ERK and AKT being seen with doses of rYKL-40 as low as 0.1–0.3 μg/ml and the induction of nuclear β-catenin and c-fos being seen with doses as low as 0.3–0.5 μg/ml (Figure 2B). They were also at least partially IL-13Rα2 dependent, because siRNA that decreased the levels of IL-13Rα2 messenger RNA (mRNA) by greater than 70% (Figures S2A and S2B) significantly decreased each of these activation events (Figure 2C). In accord with these findings, rChi3l1/BRP-39 activated ERK and AKT signaling and induced nuclear β-catenin and c-fos accumulation in peritoneal macrophages from WT mice, and these inductive events were significantly decreased in cells from IL-13Rα2 null animals (Figure 2D). These effects were at least partially Chi3l1/YKL-40/BRP-39 specific because the related GH 18 moiety, acidic mammalian chitinase (AMCase), did not signal in a similar manner via IL-13Rα2 (Figure S2C). Interestingly, Chi3l1/YKL-40/BRP-39 signaling was also significantly decreased in experiments in which soluble IL-13Rα2 was added to the cell culture system (Figures 2E and S2D). These studies demonstrate that Chi3l1/YKL-40/BRP-39 activates ERK and AKT signaling and induces β-catenin nuclear translocation and c-fos accumulation via an IL-13Rα2-dependent mechanism(s) in mice and humans and that this signaling is regulated by sIL-13Rα2.Figure S2Silencing of IL-13Rα2, Signaling of AMCase, Effect of sIL-13Rα2, and Role of Cytoplasmic Tail of IL-13Rα2, Related to Figure 2Show full caption(A and B) In vitro siRNA silencing of IL-13Rα2 in THP-1 cells. The THP-1 cells were treated with control siRNA and IL-13Rα2-specific siRNA for 16 hr and the levels of IL-13Rα2 in the cells were evaluated by real-time RT-PCR (A) and western blot (B) evaluations.(C) Peritoneal macrophages were isolated from WT (+/+) and IL-13Rα2 null (−/−) mice, treated with recombinant(r) AMCase (500 ng/ml) as indicated, then the activation of ERK and AKT was evaluated by western blot analysis.(D) The effects of recombinant soluble IL-13Rα2 (rsIL-13Rα2) on Chi3l1-stimulated signaling was also assessed. In these experiments peritoneal macrophages from WT and IL-13Rα2 null mice were treated with PBS or rsIL-13Rα2 and rChi3l1 as indicated and ERK and AKT activation were evaluated by densitometry analysis after western blot.(E) Peritoneal macrophages from IL-13Rα2 null (−/−) mice was transfected with full length (FL) IL-13Rα2, a truncated construct (TC) that lacked the 17 AA in its cytoplasmic domain or empty vector. After incubation of the cells with rChi3l1 (500 ng/ml) as indicated, cell lysates were prepared and the levels of nuclear β-catenin and c-fos were detected by western blot analysis.View Large Image Figure ViewerDownload (PPT) (A and B) In vitro siRNA silencing of IL-13Rα2 in THP-1 cells. The THP-1 cells were treated with control siRNA and IL-13Rα2-specific siRNA for 16 hr and the levels of IL-13Rα2 in the cells were evaluated by real-time RT-PCR (A) and western blot (B) evaluations. (C) Peritoneal macrophages were isolated from WT (+/+) and IL-13Rα2 null (−/−) mice, treated with recombinant(r) AMCase (500 ng/ml) as indicated, then the activation of ERK and AKT was evaluated by western blot analysis. (D) The effects of recombinant soluble IL-13Rα2 (rsIL-13Rα2) on Chi3l1-stimulated signaling was also assessed. In these experiments peritoneal macrophages from WT and IL-13Rα2 null mice were treated with PBS or rsIL-13Rα2 and rChi3l1 as indicated and ERK and AKT activation were evaluated by densitometry analysis after western blot. (E) Peritoneal macrophages from IL-13Rα2 null (−/−) mice was transfected with full length (FL) IL-13Rα2, a truncated construct (TC) that lacked the 17 AA in its cytoplasmic domain or empty vector. After incubation of the cells with rChi3l1 (500 ng/ml) as indicated, cell lysates were prepared and the levels of nuclear β-catenin and c-fos were detected by western blot analysis. Given that the intracellular domain of IL-13Rα2 only contains a 17-amino-acid structure that lacks protein binding motifs, studies were undertaken to define the role of this domain in Chi3l1/YKL-30/BRP-39 signaling events. To address this question, we compared the signaling induced by Chi3l1/YKL-40/BRP-39 in macrophages from IL-13Rα2 null mice that were transfected with WT (full-length) IL-13Rα2 constructs or truncated constructs that lacked the intracellular domain. These studies demonstrate that the intracellular domain of IL-13Rα2 is not required for Chi3l1 activation of MAPK or AKT (Figure 2F). Interestingly, this segment was required for Chi3l1 activation of the Wnt/β-catenin pathway (Figure S2E). These studies demonstrate that the intracellular domain of IL-13Rα2 has different roles in Chi3l1-induced MAPK and AKT versus Wnt/β-catenin signaling. Because IL-13 also binds to IL-13Rα2, we used coIP, signaling, and Biacore experiments to define the interactions of IL-13, Chi3l1/YKL-40/BRP-39, and IL-13Rα2. These studies demonstrated that IL-13 and Chi3l1/YKL-40/BRP-39 do not bind to one another (data not shown). They did, however, demonstrate that Chi3l1, IL-13Rα2, and IL-13 colocalize in tissue sections (Figure 3A) and participate in a multimeric complex, because the immunoprecipitation of any one of the three always immunoprecipitated the other two (Figure 3B). In keeping with this finding, studies that demonstrated that IL-13 can activate MAPK (Cho et al., 2006Cho S.J. Kang M.J. Homer R.J. Kang H.R. Zhang X. Lee P.J. Elias J.A. Lee C.G. Role of early growth response-1 (Egr-1) in interleukin-13-induced inflammation and remodeling.J. Biol. Chem. 2006; 281: 8161-8168Crossref PubMed Scopus (88) Google Scholar, Lee et al., 2006Lee P.J. Zhang X. Shan P. Ma B. Lee C.G. Homer R.J. Zhu Z. Rincon M. Mossman B.T. Elias J.A. ERK1/2 mitogen-activated protein kinase selectively mediates IL-13-induced lung inflammation and remodeling in vivo.J. Clin. Invest. 2006; 116: 163-173Crossref PubMed Scopus (108) Google Scholar) and studies that demonstrated that IL-13 stimulates heparin binding-epidermal growth factor (HB-EGF) via an IL-13Rα2-dependent mechanism (Allahverdian et al., 2008Allahverdian S. Harada N. Singhera G.K. Knight D.A. Dorscheid D.R. Secretion of IL-13 by airway epithelial cells enhances epithelial repair via HB-EGF.Am. J. Respir. Cell Mol. Biol. 2008; 38: 153-160Crossref PubMed Scopus (89) Google Scholar) demonstrated that IL-13 also activates macrophage MAPK and AKT via IL-13Rα2- and Chi3l1-dependent pathways (Figure S3A and S3B). IL-13 did not compete with Chi3l1/YKL-40/BRP-39 for IL-13Rα2 binding or signaling (Figure 3C). Similarly, Chi3l1/YKL-40/BRP-39 did not compete with IL-13 for IL-13Rα2 binding or signaling (Figure 3D). IL-13 was also a powerful activator of STAT6, Chi3l1/YKL-40 did not activate STAT6, Chi3l1 did not inhibit IL-13-induced phosphorylation of STAT6, and IL-13 did not inhibit Chi3l1/YKL-40 activation of MAPK, AKT, or Wnt/β-catenin signaling (Figure 3E). Structure-function evaluations also demonstrated that deletion of amino acids corresponding to site II (IL-13Rα2dS2) and site III (IL-13Rα2dS3) of IL-13Rα2, the reported IL-13 binding sites in IL-13Rα2 (Lupardus et al., 2010Lupardus P.J. Birnbaum M.E. Garcia K.C. Molecular basis for shared cytokine recognition revealed in the structure of an unusually high affinity complex between IL-13 and IL-13Ralpha2.Structure. 2010; 18: 332-342Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), abrogates IL-13 binding without abrogating Ch13l1 binding (Figure 3F). These studies demonstrate that IL-13, Chi3l1/YKL-40, and IL-13Rα2 participate in a multimeric complex. They also demonstrate that Chi3l1/YKL-40 and IL-13 do not compete with one another for IL-13Rα2 binding, do not abrogate each other’s signaling, and do not bind to identical locations on IL-13Rα2.Figure S3IL-13 Activates ERK1/2 and AKT via IL-13Rα2- and Chi3l1-Dependent Pathways, Related to Figure 3Show full captionPeritoneal macrophages were isolated from WT (+/+), IL-13Rα2−/− and Chi3l1 null (−/−) mice and stimulated with rIL-13 (10ng/ml) for the noted periods of time. The activation of ERK and AKT was evaluated using western blot analysis. (A) The effects of IL-13 on macrophages from WT and IL-13Rα2−/− mice are compared. (B) The effects of IL-13 on macrophages from WT (+/+) and Chi3l1−/− mice are compared. Panels (A) and (B) are representative of least 3 separate experiments.View Large Image Figure ViewerDownload (PPT) Peritoneal macrophages were isolated from WT (+/+), IL-13Rα2−/− and Chi3l1 null (−/−) mice and stimulated with rIL-13 (10ng/ml) for the noted periods of time. The activation of ERK and AKT was evaluated using western blot analysis. (A) The effects of IL-13 on macrophages from WT and IL-13Rα2−/− mice are compared. (B) The effects of IL-13 on macrophages from WT (+/+) and Chi3l1−/− mice are compared. Panels (A) and (B) are representative of least 3 separate experiments. Previous studies from our laboratory demonstrated that Chi3l1/YKL-40/BRP-39 inhibits oxidant-induced lung injury and epithelial cell apoptosis (Sohn et al., 2010Sohn M.H. Kang M.J. Matsuura H. Bhandari V. Chen N.Y. Lee C.G. Elias J.A. The chitinase-like proteins breast regression protein-39 and YKL-40 regulate hyperoxia-induced acute lung injury.Am. J. Respir. Crit. Care Med. 2010; 182: 918-928Crossref PubMed Scopus (81) Google Scholar). To determine if IL-13Rα2 plays a role in these responses, we compared the epithelial cell death and pulmonary injury responses in WT, IL-13Rα2 null, YKL-40 Tg, and YKL-40 Tg/IL-13Rα2−/− mice exposed to room air or 100% O2 for 48 hr. Hyperoxia induced epithelial apoptosis/DNA injury, alveolar-capillary protein leak, and caspase-3 and caspase-8 activation in lungs from WT mice (Figures 4A, 4B, and S4A; data not shown). Hyperoxia-induced epithelial TUNEL responses and alveolar protein leak were exaggerated in lungs from Chi3l1/BRP-39 null mice and a phenocopy was seen in IL-13Rα2−/− animals (Figures 4A and 4B). Transgenic YKL-40 ameliorated the hyperoxia-induced responses in WT mice and rescued the exaggerated responses in Chi3l1 null animals (Figures 4A and 4B). Importantly, the protective effects of YKL-40 were significantly decreased in Tg mice that lacked IL-13Rα2 (Figures 4A and 4B). In accord with these in vivo findings, epithelial cells treated with H2O2 in vitro manifest increased levels of apoptosis and necrosis and these responses were exaggerated in cells that lacked Chi3l1/YKL-40 or IL-13Rα2 (Figures 4C and S4B). The addition of rChi3l1/YKL-40 to these cultures markedly decreased the cell death responses in WT cells and rescued the exaggerated responses in Chi3l1/BRP-39 null mice but did not cause comparable cytoprotection in cells that lacked IL-13Rα2 (Figures 4C and S4B). These responses were not H2O2 or lung epithelial cell specific because similar responses were seen with FasL-treated lung epithelial cells and H2O2-treated kidney epithelial cells (Figures S4C and S4D). They were also at least partially mediated by the ability of Chi3l1/YKL-40/BRP-39 to activate AKT, because selective AKT inhibitors abrogated the antiapoptotic" @default.
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• W1987441485 title "Chitinase 3-like 1 Regulates Cellular and Tissue Responses via IL-13 Receptor α2" @default.
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