c-Met and Its Ligand Hepatocyte Growth Factor/Scatter Factor Regulate Mature B Cell Survival in a Pathway Induced by CD74 This information is current as Maya Gordin, Melania Tesio, Sivan Cohen, Yael Gore, Frida of September 28, 2021. Lantner, Lin Leng, Richard Bucala and Idit Shachar J Immunol published online 16 July 2010 http://www.jimmunol.org/content/early/2010/07/16/jimmun ol.0902566 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/07/16/jimmunol.090256 Material 6.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published July 16, 2010, doi:10.4049/jimmunol.0902566 The Journal of Immunology c-Met and Its Ligand Hepatocyte Growth Factor/Scatter Factor Regulate Mature B Cell Survival in a Pathway Induced by CD74 Maya Gordin,* Melania Tesio,* Sivan Cohen,* Yael Gore,* Frida Lantner,* Lin Leng,† Richard Bucala,† and Idit Shachar* The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Durable B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism involved in mature B cell homeostasis, the hepatocyte growth factor/scatter factor (HGF)/c-Met pathway. We demonstrate that c-Met activation by HGF leads to a survival cascade, whereas its blockade results in induction of mature B cell death. Our results emphasize a unique and critical function for c-Met signaling in the previously Downloaded from described macrophage migration inhibitory factor/CD74-induced survival pathway. Macrophage migration inhibitory factor recruits c-Met to the CD74/CD44 complex and thereby enables the induction of a signaling cascade within the cell. This signal results in HGF secretion, which stimulates the survival of the mature B cell population in an autocrine manner. Thus, the CD74–HGF/c-Met axis defines a novel physiologic survival pathway in mature B cells, resulting in the control of the humoral immune response. The Journal of Immunology, 2010, 185: 000–000. http://www.jimmunol.org/ uring their development, B cells encounter various check- brane region and an ∼150-aa lumenal domain. The CD74 chain points that control cell survival. Under steady-state con- was originally thought to function mainly as an MHC class II chap- D ditions, the number and distribution of B cells are under erone, promoting the exit of MHC class II molecules from the en- homeostatic control maintained by a balance between survival and doplasmic reticulum, directing them to endocytic compartments, apoptosis. preventing peptide binding within the endoplasmic reticulum, and Regulation of mature B cell survival involves multiple mecha- contributing to peptide editing in the MHC class II compartment (8). nisms. The BCR provides survival signals essential for maintaining In addition to its chaperone function, CD74 was shown to have the mature B cell pool. Deletion of the Igh gene (1) or conditional a role as an accessory signaling molecule. A small proportion of deletion of either Igh or the signal-transducing Cd79a (Iga) genes CD74 is modified by the addition of chondroitin sulfate, and this by guest on September 28, 2021 (2) leads to a loss of B cells. In addition, survival of mature naive form of CD74 is expressed on the surface of APCs, including B cells depends on signals delivered by the ligand–receptor pair monocytes and B cells. Ab blocking studies have shown that CD74 B cell-activating factor (BAFF) and BAFFR (3, 4). Mice that lack modified by the addition of chondroitin sulfate interacts with CD44 BAFF expression or that are subjected to treatments designed to (9, 10). In addition, it was shown that macrophage migration in- block the action of BAFF fail to produce or to maintain a mature hibitory factor (MIF) binds to the CD74 extracellular domain on B cell pool (5–7). macrophages, a process that results in initiation of a signaling Recently, we described an additional mechanism that regulates pathway in these cells (11). B cell survival, which depends on CD74 (invariant chain, Ii). CD74 is Our studies have shown that CD74 expressed on B cells is di- a type II integral membrane protein, containing a short N-terminal rectly involved in shaping the peripheral B cell populations by cytoplasmic tail of 28 aa, followed by a single 24-aa transmem- regulating mature B cell survival (12) through a pathway leading to the activation of transcription mediated by the NF-kB p65/RelA *Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; and homodimer and its coactivator TAFII105 (13, 14). CD74 stim- †Department of Medicine, Yale University School of Medicine, New Haven, CT ulation by MIF activates the Syk and PI3K/Akt pathways, leading 06520 to NF-kB activation, enabling entry of the stimulated B cells Received for publication August 6, 2009. Accepted for publication June 3, 2010. into the S phase, an increase in DNA synthesis, cell division, This work was supported by the Israel Science Foundation (Morasha), the Israel Sci- and augmented expression of antiapoptotic proteins in a CD44- ence Foundation, the Israel Cancer Association, and the Minerva Foundation. I.S. is dependent manner. These findings confirmed that surface CD74 the incumbent of the Dr. Morton and Ann Kleiman Professorial Chair. R.B. and L.L. are supported by National Institutes of Health Grants AR049610, AR050498, and functions as a survival receptor (15–17). AI042310 and the Alliance for Lupus Research. Interestingly, the cell surface receptor, CD44, has also been Address correspondence and reprint requests to Dr. Idit Shachar, Department of implicated in the regulation of the signaling of the tyrosine kinase Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address: receptor c-Met (18–20), although the precise mechanism of this [email protected] interaction is unknown. The online version of this article contains supplemental material. c-Met is a unique disulfide-linked a–b heterodimeric receptor Abbreviations used in this paper: BAFF, B cell-activating factor; FLICA, fluorescent- labeled inhibitor of caspases; HGF, hepatocyte growth factor/scatter factor; HPRT, tyrosine kinase with a versatile role in regulating numerous biolog- hypoxanthine ribosyltransferase; MIF, macrophage migration inhibitory factor; MM, ical functions in response to its natural ligand hepatocyte growth multiple myeloma; MZ, marginal zone; PI, propidium iodide; T1, transitional 1; T2, factor/scatter factor (HGF). HGF is a multifunctional cytokine with transitional 2. a domain structure and proteolytic mechanism of activation similar Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 to that of the serine protease plasminogen. Activation of the HGF/ www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902566 2 CD74–HGF/c-Met AXIS DEFINES A SURVIVAL PATHWAY IN B CELLS c-Met signaling pathway, which requires phosphorylation of var- 37˚C. For c-Met blocking, 1 3 107 B cells were incubated in 1 ml RPMI ious specific tyrosine residues on c-Met itself, leads to cellular 1640 medium containing 0.1% (v/v) FCS in the presence of 0.3 ng/ml responses, including increased motility, proliferation, morphogen- PHA-665752, a c-Met inhibitor (a gift from Pfizer, New York, NY), at 37˚C. esis, and cell survival (21–27). RNA isolation and reverse transcription Little is known about the role of HGF/c-Met in the regulation of lymphopoiesis in general and specifically that of B cells. HGF has Total RNA was isolated from cells using the TRIzol Reagent kit (MRC, Cincinnati, OH). Reverse transcription was carried out using Superscript II been reported to regulate hematopoiesis in mouse fetal liver and in reverse transcriptase (Life Technologies, Carlsbad, CA). Primers that were adult bone marrow (28, 29) and was found to have a stimulatory used included: c-Met, 59-GACTTCAGCCATCCCAATGT-39,39-GGTGAA- effect on Ig secretion in cultures of mouse splenocytes (30). CTTCTGCGTTTGC-59; HGF, 59-AGCGCTCTCCCTTCTTCTTT-39,39-T- 9 9 9 On the basis of the known interactions between c-Met and CD44, CACACAGAATCAGGCAAGA-5 ;Bcl-2,5-CAGGGCGATGTTGTCC-3 , 39-CTGGCATCTTCTCCTTCC-59; cyclin E, 59-GAAAATCAGACCAC- we wished to determine whether c-Met is expressed in naive B cells CCAGAGCC-39,39-GAAATGATACAAAGCAGAAGCAGCG-59; hypo- and whether it is involved in the regulation of their survival. In this xanthine ribosyltransferase (HPRT), 59-GAGGGTAGGCTGGCCTATGG- study, we demonstrate that naive murine B cells express both c-Met CT-39,39-GTTGGATACAGGCCAGACTTTGTTG-59. and its ligand HGF, which regulate peripheral B cell survival. In Real-time RT-PCR analysis addition, our study shows that c-Met participates in controlling MIF-induced signaling by forming a survival complex together Levels of mRNA of actin, c-Met, and Bcl-2 were analyzed by quantitative with CD74 and CD44 in B cells. These findings establish a key real-time RT-PCR using a LightCycler instrument (Roche Diagnostic Sys- and novel role for HGF/c-Met pair in the regulation of B cell sur- tems, Burgess Hill, West Sussex, U.K.).