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• W2086626108 abstract "Although it is known that the unfolded protein response (UPR) plays a significant role in the process of plasma cell differentiation, the contribution of the individual sensors of the UPR to this process remains unclear. In this study we examine the death signals and compensatory survival signals activated during B cell activation and the first stages of plasma cell differentiation. During in vitro differentiation of both primary murine B cells and the Bcl1 cell line, we demonstrate that in addition to activation of the physiological UPR, changes in the expression of several Bcl-2 proteins occur, which are consistent with a lowering of the apoptotic threshold of the cell. Specifically, we observed decreased expression of Bcl-2 and Mcl-1 and increased expression of the proapoptotic protein Bim. However, these changes were countered by Bcl-xL induction, which is necessary to protect differentiating cells both from ER stress-induced death by tunicamycin and from the death signals inherent in differentiation. Consistent with differentiating cells becoming dependent on Bcl-xL for survival, the addition of ABT-737 resulted in apoptosis in differentiating cells through the inhibition of sequestration of Bim. Confirming this result, differentiation in the context of RNAi-mediated Bcl-xL knockdown also induced apoptosis. This cell death is C/EBP homologous protein (CHOP)-dependent, connecting these events to the UPR. Thus plasma cell differentiation proceeds through a Bcl-xL-dependent intermediate. Although it is known that the unfolded protein response (UPR) plays a significant role in the process of plasma cell differentiation, the contribution of the individual sensors of the UPR to this process remains unclear. In this study we examine the death signals and compensatory survival signals activated during B cell activation and the first stages of plasma cell differentiation. During in vitro differentiation of both primary murine B cells and the Bcl1 cell line, we demonstrate that in addition to activation of the physiological UPR, changes in the expression of several Bcl-2 proteins occur, which are consistent with a lowering of the apoptotic threshold of the cell. Specifically, we observed decreased expression of Bcl-2 and Mcl-1 and increased expression of the proapoptotic protein Bim. However, these changes were countered by Bcl-xL induction, which is necessary to protect differentiating cells both from ER stress-induced death by tunicamycin and from the death signals inherent in differentiation. Consistent with differentiating cells becoming dependent on Bcl-xL for survival, the addition of ABT-737 resulted in apoptosis in differentiating cells through the inhibition of sequestration of Bim. Confirming this result, differentiation in the context of RNAi-mediated Bcl-xL knockdown also induced apoptosis. This cell death is C/EBP homologous protein (CHOP)-dependent, connecting these events to the UPR. Thus plasma cell differentiation proceeds through a Bcl-xL-dependent intermediate. Plasma cell differentiation involves striking changes in the morphology and physiology of the cell. To become a professional antibody-secreting cell, the B cell must increase the size and capacity of the endoplasmic reticulum (ER) 2The abbreviations used are: ERendoplasmic reticulumUPRunfolded protein responseCHOPC/EBP homologous proteinC/EBPCCAAT-enhancer-binding proteinPERKPKR-like ER kinase. and secretory apparatus (1.Gass J.N. Gunn K.E. Sriburi R. Brewer J.W. Stressed-out B cells? Plasma-cell differentiation and the unfolded protein response.Trends Immunol. 2004; 25: 17-24Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). To accomplish this end, the cell activates a set of signaling pathways known as the unfolded protein response (UPR) (2.Gass J.N. Gifford N.M. Brewer J.W. Activation of an unfolded protein response during differentiation of antibody-secreting B cells.J. Biol. Chem. 2002; 277: 49047-49054Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). During periods of ER stress when the protein load in the ER outweighs its folding capacity, three sensors of ER stress, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and PKR-like ER kinase (PERK), are activated (3.Schröder M. Kaufman R.J. ER stress and the unfolded protein response.Mutat. Res. 2005; 569: 29-63Crossref PubMed Scopus (1395) Google Scholar). These sensors reside across the ER membrane with their luminal tails bound to the ER chaperone Bip (GRP78) keeping them in an inactive state. When unfolded protein builds in the ER, Bip is titrated off the luminal tails of these signaling molecules. ATF6 translocates to the Golgi where it is cleaved to yield p50ATF6, a transcription factor that up-regulates ER quality control and capacity (4.Yoshida H. Haze K. Yanagi H. Yura T. Mori K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins: involvement of basic leucine zipper transcription factors.J. Biol. Chem. 1998; 273: 33741-33749Abstract Full Text Full Text PDF PubMed Scopus (1015) Google Scholar, 5.Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress.Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1538) Google Scholar, 6.Shen J. Chen X. Hendershot L. Prywes R. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals.Dev Cell. 2002; 3: 99-111Abstract Full Text Full Text PDF PubMed Scopus (1067) Google Scholar, 7.Kokame K. Kato H. Miyata T. Identification of ERSE-II, a new cis-acting element responsible for the ATF6-dependent mammalian unfolded protein response.J. Biol. Chem. 2001; 276: 9199-9205Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8.Yoshida H. Okada T. Haze K. Yanagi H. Yura T. Negishi M. Mori K. Endoplasmic reticulum stress-induced formation of transcription factor complex ERSF including NF-Y (CBF) and activating transcription factors 6α and 6β that activates the mammalian unfolded protein response.Mol. Cell. Biol. 2001; 21: 1239-1248Crossref PubMed Scopus (259) Google Scholar). IRE1 oligomerizes and activates its ribonuclease, which splices x-box-binding protein 1 (XBP1) mRNA (9.Shamu C.E. Walter P. Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus.EMBO J. 1996; 15: 3028-3039Crossref PubMed Scopus (447) Google Scholar, 10.Bertolotti A. Zhang Y. Hendershot L.M. Harding H.P. Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.Nat. Cell Biol. 2000; 2: 326-332Crossref PubMed Scopus (2095) Google Scholar, 11.Liu C.Y. Xu Z. Kaufman R.J. Structure and intermolecular interactions of the luminal dimerization domain of human IRE1α.J. Biol. Chem. 2003; 278: 17680-17687Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12.Sidrauski C. Walter P. The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response.Cell. 1997; 90: 1031-1039Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, 13.Calfon M. Zeng H. Urano F. Till J.H. Hubbard S.R. Harding H.P. Clark S.G. Ron D. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA.Nature. 2002; 415: 92-96Crossref PubMed Scopus (2133) Google Scholar). This spliced XBP1 mRNA yields a highly active transcription factor that up-regulates chaperones and expands ER function and capacity (14.Clauss I.M. Chu M. Zhao J.L. Glimcher L.H. The basic domain/leucine zipper protein hXBP-1 preferentially binds to and transactivates CRE-like sequences containing an ACGT core.Nucleic Acids Res. 1996; 24: 1855-1864Crossref PubMed Scopus (87) Google Scholar, 15.Lee A.H. Iwakoshi N.N. Glimcher L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.Mol. Cell. Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1617) Google Scholar). PERK oligomerizes and phosphorylates eukaryotic translation initiation factor 2 α (eIF2α) (16.Harding H.P. Zhang Y. Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.Nature. 1999; 397: 271-274Crossref PubMed Scopus (2519) Google Scholar). Phosphorylated eIF2α inhibits global protein translation while favoring translation of activating transcription factor 4 (ATF4) mRNA (17.Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells.Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar). ATF4 also increases ER capacity as well as strongly inducing C/EBP homologous protein (CHOP) (17.Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells.Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar, 18.Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response.J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (551) Google Scholar, 19.Jiang H.Y. Wek S.A. McGrath B.C. Lu D. Hai T. Harding H.P. Wang X. Ron D. Cavener D.R. Wek R.C. Activating transcription factor 3 is integral to the eukaryotic initiation factor 2 kinase stress response.Mol. Cell. Biol. 2004; 24: 1365-1377Crossref PubMed Scopus (387) Google Scholar). Activation of these three signaling molecules results in the up-regulation of ER protein folding capacity, quality control, and ER-associated degradation while delaying further translation of mRNA. If ER stress is not resolved through the UPR, CHOP induces apoptosis by inhibition of Bcl-2 and induction of Bim (20.McCullough K.D. Martindale J.L. Klotz L.O. Aw T.Y. Holbrook N.J. Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state.Mol. Cell. Biol. 2001; 21: 1249-1259Crossref PubMed Scopus (1576) Google Scholar, 21.Puthalakath H. O'Reilly L.A. Gunn P. Lee L. Kelly P.N. Huntington N.D. Hughes P.D. Michalak E.M. McKimm-Breschkin J. Motoyama N. Gotoh T. Akira S. Bouillet P. Strasser A. ER stress triggers apoptosis by activating BH3-only protein Bim.Cell. 2007; 129: 1337-1349Abstract Full Text Full Text PDF PubMed Scopus (1121) Google Scholar). endoplasmic reticulum unfolded protein response C/EBP homologous protein CCAAT-enhancer-binding protein PKR-like ER kinase. The expansion of the secretory capacity necessary for a B cell to become a professional antibody-secreting cell makes use of the UPR machinery already present in all cells. It must however overcome the effects of PERK activation including translation inhibition and apoptosis. ATF6 and IRE1 are activated and XBP1 is spliced during plasma cell differentiation, and they are necessary to form plasma cells in vivo as animals deficient in any of these genes lack plasma cells (2.Gass J.N. Gifford N.M. Brewer J.W. Activation of an unfolded protein response during differentiation of antibody-secreting B cells.J. Biol. Chem. 2002; 277: 49047-49054Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 22.Reimold A.M. Iwakoshi N.N. Manis J. Vallabhajosyula P. Szomolanyi-Tsuda E. Gravallese E.M. Friend D. Grusby M.J. Alt F. Glimcher L.H. Plasma cell differentiation requires the transcription factor XBP-1.Nature. 2001; 412: 300-307Crossref PubMed Scopus (1040) Google Scholar, 23.Gunn K.E. Gifford N.M. Mori K. Brewer J.W. A role for the unfolded protein response in optimizing antibody secretion.Mol. Immunol. 2004; 41: 919-927Crossref PubMed Scopus (41) Google Scholar, 24.Lee K. Tirasophon W. Shen X. Michalak M. Prywes R. Okada T. Yoshida H. Mori K. Kaufman R.J. IRE1-mediated unconventional mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate XBP1 in signaling the unfolded protein response.Genes Dev. 2002; 16: 452-466Crossref PubMed Scopus (828) Google Scholar). Conversely, perk−/− animals have plasma cells, and PERK is not significantly activated during plasma cell differentiation (25.Gass J.N. Jiang H.Y. Wek R.C. Brewer J.W. The unfolded protein response of B-lymphocytes: PERK-independent development of antibody-secreting cells.Mol. Immunol. 2008; 45: 1035-1043Crossref PubMed Scopus (104) Google Scholar, 26.Ma Y. Shimizu Y. Mann M.J. Jin Y. Hendershot L.M. Plasma cell differentiation initiates a limited ER stress response by specifically suppressing the PERK-dependent branch of the unfolded protein response.Cell Stress Chaperones. 2010; 15: 281-293Crossref PubMed Scopus (101) Google Scholar). Although it is true that ATF4, p50ATF6, and XBP1s all bind to the promoter of CHOP, only ATF4 has been shown to be necessary for CHOP activation (18.Ma Y. Brewer J.W. Diehl J.A. Hendershot L.M. Two distinct stress signaling pathways converge upon the CHOP promoter during the mammalian unfolded protein response.J. Mol. Biol. 2002; 318: 1351-1365Crossref PubMed Scopus (551) Google Scholar). Therefore it stands to reason that inactivation of PERK would inhibit ATF4 and CHOP, relieving the cell of translation inhibition and the proapoptotic effects of CHOP. However, it has also been shown that some activation of CHOP is necessary for production of a maximally efficient plasma cell as Chop−/− animals have plasma cells that secrete immunoglobulin at a lower rate than wild type animals (27.Masciarelli S. Fra A.M. Pengo N. Bertolotti M. Cenci S. Fagioli C. Ron D. Hendershot L.M. Sitia R. CHOP-independent apoptosis and pathway-selective induction of the UPR in developing plasma cells.Mol. Immunol. 2010; 47: 1356-1365Crossref PubMed Scopus (53) Google Scholar). Therefore the proapoptotic aspects of CHOP signaling must be overcome for optimal plasma cell differentiation. During all stages of B cell development, there are prosurvival and proapoptotic proteins responsible for determining which B cells are propagated and which are deleted from the repertoire. The Bcl-2 family of proteins responsible for regulating the intrinsic pathway of apoptosis plays a major role in this survival signaling (28.Strasser A. Bouillet P. The control of apoptosis in lymphocyte selection.Immunol. Rev. 2003; 193: 82-92Crossref PubMed Scopus (64) Google Scholar, 29.Veis D.J. Sorenson C.M. Shutter J.R. Korsmeyer S.J. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair.Cell. 1993; 75: 229-240Abstract Full Text PDF PubMed Scopus (1439) Google Scholar, 30.Vikstrom I. Carotta S. Lüthje K. Peperzak V. Jost P.J. Glaser S. Busslinger M. Bouillet P. Strasser A. Nutt S.L. Tarlinton D.M. Mcl-1 is essential for germinal center formation and B cell memory.Science. 2010; 330: 1095-1099Crossref PubMed Scopus (166) Google Scholar). This is true in mature B cells and in plasma cells, but less is known about this pathway during the transition between these cell types. The multidomain antiapoptotic proteins Bcl-2, Mcl-1, and Bcl-xL are important in B cell survival. They function by binding and sequestering proapoptotic BH3-only proteins, such as Bim (31.Danial N.N. Korsmeyer S.J. Cell death: critical control points.Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4024) Google Scholar, 32.Cory S. Adams J.M. The Bcl2 family: regulators of the cellular life-or-death switch.Nat. Rev. Cancer. 2002; 2: 647-656Crossref PubMed Scopus (3324) Google Scholar). This prevents Bim from activating Bax and Bak, which when active induce mitochondrial outer membrane permeabilization, resulting in cytochrome c release that leads to downstream caspase activation and apoptosis. This function of antiapoptotic proteins can now be targeted by a class of drugs called BH3 mimetics. These are small molecules that bind to and antagonize the antiapoptotic proteins. ABT-737, a mimetic of the BH3-only protein Bad, binds to Bcl-xL, Bcl-2, and Bcl-w, displacing Bim and leading to apoptosis in cells that are dependent on one of these proteins for survival (33.Oltersdorf T. Elmore S.W. Shoemaker A.R. Armstrong R.C. Augeri D.J. Belli B.A. Bruncko M. Deckwerth T.L. Dinges J. Hajduk P.J. Joseph M.K. Kitada S. Korsmeyer S.J. Kunzer A.R. Letai A. Li C. Mitten M.J. Nettesheim D.G. Ng S. Nimmer P.M. O'Connor J.M. Oleksijew A. Petros A.M. Reed J.C. Shen W. Tahir S.K. Thompson C.B. Tomaselli K.J. Wang B. Wendt M.D. Zhang H. Fesik S.W. Rosenberg S.H. An inhibitor of Bcl-2 family proteins induces regression of solid tumours.Nature. 2005; 435: 677-681Crossref PubMed Scopus (2931) Google Scholar). ABT-737 does not bind Mcl-1 and therefore will not cause apoptosis in a cell that is dependent on Mcl-1 for survival. In murine immunization models, it has been shown that germinal center B cells and existing plasma cells are insensitive to ABT-737 (34.Carrington E.M. Vikstrom I.B. Light A. Sutherland R.M. Londrigan S.L. Mason K.D. Huang D.C. Lew A.M. Tarlinton D.M. BH3 mimetics antagonizing restricted prosurvival Bcl-2 proteins represent another class of selective immune modulatory drugs.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 10967-10971Crossref PubMed Scopus (85) Google Scholar). Accordingly, it has also been shown that these cell types are dependent on Mcl-1 for survival (30.Vikstrom I. Carotta S. Lüthje K. Peperzak V. Jost P.J. Glaser S. Busslinger M. Bouillet P. Strasser A. Nutt S.L. Tarlinton D.M. Mcl-1 is essential for germinal center formation and B cell memory.Science. 2010; 330: 1095-1099Crossref PubMed Scopus (166) Google Scholar, 35.Peperzak V. Vikström I. Walker J. Glaser S.P. LePage M. Coquery C.M. Erickson L.D. Fairfax K. Mackay F. Strasser A. Nutt S.L. Tarlinton D.M. Mcl-1 is essential for the survival of plasma cells.Nat. Immunol. 2013; 14: 290-297Crossref PubMed Scopus (223) Google Scholar). These studies did show that there was a deficit in newly formed plasma cells in the presence of ABT-737; however, the molecular basis for this deficit was not fully defined. In this study we define the molecular basis of differential Bcl-2 family dependence during plasma cell differentiation. The Bcl1 cell line, clone CW13.20.3B3, was acquired from ATCC (CRL-1699). Primary murine B cells were prepared from splenocytes isolated from C57BL/6 spleens and depleted of non-B cells and activated B cells by magnetic bead column separation (Miltenyi B cell isolation kit 130-090-862, LS columns). All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 μm l-glutamine, 100 IU penicillin/streptomycin, 10 mm HEPES buffer, 1 mm sodium pyruvate, nonessential amino acids, and 50 μm 2-mercaptoethanol. Bcl1 cells were cultured at a concentration of 0.3 × 106 cells/ml in complete growth medium supplemented with 10 ng/ml IL-5 (R&D Systems) and 10 μg/ml lipopolysaccharide (Sigma L-4391) for up to 96 h. UPR was activated with 0.5 μg/ml tunicamycin (Sigma T7765) followed by replacement with complete growth medium. Primary C57BL/6 B cells were cultured at a concentration of 1 × 106 cells/ml in complete growth medium supplemented with 20 ng/ml IL-4 (PeproTech) and 20 μg/ml lipopolysaccharide (Sigma L-6216) for up to 96 h. ABT-737 and ABT-199 were a generous gift of Abbvie (North Chicago, IL). Virus was prepared in 293T cells using the MISSION shRNA TORC1 system (Sigma SHCLNG-NM_004083) or with pLKO.1 vector control. Bcl1 cells were then infected and selected with puromycin. Transductants were kept in selection during passage and verified by Western blot and quantitative RT-PCR. Experiments were carried out in the absence of puromycin. Cells were collected at the various time points and treatments. 0.25–0.5 million cells were washed with PBS and resuspended in 100 μl of FACS buffer (1% BSA in PBS containing 0.01% sodium azide) and the appropriate amount of antibody for 30 min at 4 °C. Cells were then washed in FACS buffer and resuspended in 0.5 ml of FACS buffer with 5 μl of 7-aminoactinomycin D and incubated at room temperature for 5 min. Samples were then assayed on a BD FACSCanto II. CD19-V450 (560375), CD138-PE (553714), and CD44-FITC (553133) antibodies and 7-aminoactinomycin D were purchased from BD Biosciences. IgM-FITC (69819) was purchased from Santa Cruz Biotechnology. Apoptosis was assayed with annexin V-FITC (BioVision 1001-1000) and propidium iodide (2 μg/ml Sigma) staining and measured with a BD FACSCanto II as described previously (36.Morales A.A. Gutman D. Lee K.P. Boise L.H. BH3-only proteins Noxa, Bmf, and Bim are necessary for arsenic trioxide-induced cell death in myeloma.Blood. 2008; 111: 5152-5162Crossref PubMed Scopus (66) Google Scholar). Western blotting was performed on lysates collected at the various time points and treatments as described previously (36.Morales A.A. Gutman D. Lee K.P. Boise L.H. BH3-only proteins Noxa, Bmf, and Bim are necessary for arsenic trioxide-induced cell death in myeloma.Blood. 2008; 111: 5152-5162Crossref PubMed Scopus (66) Google Scholar). Samples were run on 4–15% Bio-Rad TGX mini gels. Gels were transferred to nitrocellulose membrane and blocked as described previously (36.Morales A.A. Gutman D. Lee K.P. Boise L.H. BH3-only proteins Noxa, Bmf, and Bim are necessary for arsenic trioxide-induced cell death in myeloma.Blood. 2008; 111: 5152-5162Crossref PubMed Scopus (66) Google Scholar). ATF4 rabbit polyclonal antibody was purchased from Abcam. Bcl-2 hamster (3F11) and Syrian and Armenian hamster-HRP mouse antibodies were purchased from BD Biosciences. Bcl-2 rabbit (50E3), Bip rabbit (C50B12), CHOP rabbit (D46F1), phospho-eIF2α (Ser-51) rabbit (D9G8), phospho-eIF2α (Ser-51) rabbit (119A11), eIF2α mouse (L57A5), GRP94 rabbit polyclonal antibodies and p58IPK rabbit (C56E7) and Mcl-1 rabbit (D35A5) antibodies were purchased from Cell Signaling Technology. Rabbit IgG-HRP donkey and mouse-IgG-HRP sheep antibodies were purchased from GE Healthcare. Bim rabbit polyclonal antibody was purchased from Millipore. β-Actin mouse (AC-15) and β-actin rabbit polyclonal antibodies were purchased from Sigma Aldrich. Bcl-xL mouse (2A1) and Bcl-xL rabbit polyclonal antibodies (13.6) have been previously described (37.Boise L.H. Minn A.J. Noel P.J. June C.H. Accavitti M.A. Lindsten T. Thompson C.B. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL.Immunity. 1995; 3: 87-98Abstract Full Text PDF PubMed Scopus (1077) Google Scholar). Co-immunoprecipitation was performed with the ImmunoCruz Optima C kit (Santa Cruz Biotechnology) as described previously (36.Morales A.A. Gutman D. Lee K.P. Boise L.H. BH3-only proteins Noxa, Bmf, and Bim are necessary for arsenic trioxide-induced cell death in myeloma.Blood. 2008; 111: 5152-5162Crossref PubMed Scopus (66) Google Scholar). Lysates were prepared in 2% CHAPS buffer. 100 μg of protein was used for each antibody for each sample. Samples were precleared with protein G and preclearing matrix for 2 h. Immunoprecipitation matrices were prepared using 3 μg of the appropriate antibody. Precleared lysates were rocked in immunoprecipitation matrices overnight. Samples were eluted in 2:1 radioimmunoprecipitation assay buffer: 6× loading dye under nonreducing conditions after a 5-min incubation at 95 °C. Mcl-1 mouse (B6) antibody was purchased from Santa Cruz Biotechnology. Bcl-2 mouse (Bcl/10C4) antibody was purchased from Novus Biologicals. Bcl-xL mouse (7B2.5) was previously described (37.Boise L.H. Minn A.J. Noel P.J. June C.H. Accavitti M.A. Lindsten T. Thompson C.B. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL.Immunity. 1995; 3: 87-98Abstract Full Text PDF PubMed Scopus (1077) Google Scholar). cDNA was prepared from RNA harvested at specified time points using the ABI high capacity cDNA kit (Applied Biosystems). Real-time PCR was performed using TaqMan gene expression master mix (ABI 4368814) with an ABI 9600 Fast thermocycler as described previously (38.Morales A.A. Kurtoglu M. Matulis S.M. Liu J. Siefker D. Gutman D.M. Kaufman J.L. Lee K.P. Lonial S. Boise L.H. Distribution of Bim determines Mcl-1 dependence or codependence with Bcl-xL/Bcl-2 in Mcl-1-expressing myeloma cells.Blood. 2011; 118: 1329-1339Crossref PubMed Scopus (99) Google Scholar). The following probes were used: Mcl-1 (mcl1) Mm00725832_s1, Bcl-2 (bcl2) Mm00477631_m1, Bcl-xL (bcl2l1) Mm00437783_m1, Bim (bcl2l11) Mm00437796_m1, Blimp-1 (prdm1) Mm004761289_m1, Bcl-6 (bcl6) Mm00477633_m1, and GAPDH 4352932-0912031 were purchased from Applied Biosystems. ELISA was performed according to protocol using the mouse IgM ELISA kit (Bethyl Laboratories). Supernatants were diluted 1:4 and loaded at 25 μl/well and then calibrated to live cell number counted using trypan blue exclusion. Lysates were loaded at 2.5 μg of total protein/well. 3,3′, 5,5;-Tetramethylbenzidine (TMB) substrate (Invitrogen) and color change were used to measure concentration. Color change was measured as A450–A550. IgM concentrations were reported as IgM per cells in culture or IgM per cells contributing to the lysate loaded and were calculated against the four parameter logistic curve fit of dilutions of standard mouse serum with known IgM concentration. cDNA was prepared from RNA collected from samples at specified time points with the various treatments. The region of the splice site was amplified using primers flanking the splice site. Products were amplified using the DreamTaq kit (Fermentas) and a custom PCR routine: 94 °C/4 min, 40 cycles of 94 °C/30 s, 65 °C/30 s, 72 °C/30 s, and then 72 °C/10 min. PCR products were run on 3% high resolution agarose gels (Invitrogen). 10-cm gels were run at 50 V for 6 h. Primers were: forward, 5′-GAACACGCTTGGGAATGGACAC-3′, and reverse, 5′-AGAAAGGGAGGCTGGTAAGGAAC-3′. The murine B cell leukemia cell line Bcl1 can be stimulated to differentiate to an antibody-secreting cell using lipopolysaccharide (LPS) and cytokines (39.Blackman M.A. Tigges M.A. Minie M.E. Koshland M.E. A model system for peptide hormone action in differentiation: interleukin 2 induces a B lymphoma to transcribe the J chain gene.Cell. 1986; 47: 609-617Abstract Full Text PDF PubMed Scopus (72) Google Scholar, 40.Mita S. Harada N. Naomi S. Hitoshi Y. Sakamoto K. Akagi M. Tominaga A. Takatsu K. Receptors for T cell-replacing factor/interleukin 5: specificity, quantitation, and its implication.J. Exp. Med. 1988; 168: 863-878Crossref PubMed Scopus (68) Google Scholar). We tested combinations of previously described differentiation stimuli including IL-2, IL-5, and LPS and found that the combination of IL-5 and LPS was as efficient a differentiation stimulus as the combination of all three (not shown). Consistent with published data (40.Mita S. Harada N. Naomi S. Hitoshi Y. Sakamoto K. Akagi M. Tominaga A. Takatsu K. Receptors for T cell-replacing factor/interleukin 5: specificity, quantitation, and its implication.J. Exp. Med. 1988; 168: 863-878Crossref PubMed Scopus (68) Google Scholar), this cell line has high basal expression of the IL-5 receptor (not shown). Differentiating Bcl1 cells display phenotypic changes associated with the early stages of plasma cell differentiation including up-regulation of CD138, CD19, and CD44 along with down-regulation of surface IgM (Fig. 1A). To examine the effects of UPR activation on plasma cell differentiation, we induced ER stress with an inhibitor of N-linked glycosylation, tunicamycin. A 5-h pulse of tunicamycin activated the UPR as demonstrated by increased expression of CHOP and XBP1 (not shown). These cells rapidly underwent apoptosis unless LPS was included in the differentiation stimulus following the tunicamycin pulse (Fig. 1B, and not shown). Although IL-5 was not sufficient on its own to protect cells, only those cells that received both LPS and IL-5 produced and secreted antibody at a rate similar or greater than cells that did not receive tunicamycin pretreatment (Fig. 1, C and D, and not shown). These data demonstrate that Bcl1 cells are sensitive to ER stress-induced cell death yet are protected by LPS signaling during differentiation. Because plasma cell differentiation induces parts of the unfolded protein response, we chose to further examine the activation of this pathway during ER stress and differentiation. All cells treated with the combination of LPS and IL-5 displayed a large increase in the ER chaperones GRP94 and GRP78 (Bip) regardless of tunicamycin treatment (Fig. 2A). Consistent with this observation, activation of IRE1, as measured by XBP1 splicing, was highest in cells treated with both LPS and IL-5 throughout the time course (Fig. 2B). Together these data suggest that differentiation of Bcl1 cells results in the activation of a physiologic UPR and that tunicamycin does not influence this response. In cells pulsed with tunicamycin for 5 h, we observed phosphorylation of eIF2α at higher levels than untreated cells at 24- and 48-h time points by Western blot (Fig. 2A). Phosphorylated eIF2α was not observed when cells were differentiated in the absence of tunicamycin, a finding consistent with PERK activation not being part of plasma cell differentiation. Protein levels of ATF4 and CHOP, both downstream effectors of the PERK arm of the UPR, were also increased by tunicamycin addition. Cells that received tunicamycin without differentiation stimulus do not show as robust activation of this arm downstream of eIF2α phosphorylation, but it is important to note that the viability of these cells was very low at these time points (Fig. 1B). Surprisingly, all cells treated with LPS and IL-5 in the absence of tunicamycin also demonstrated robust and prolonged expression of b" @default.
• W2086626108 created "2016-06-24" @default.
• W2086626108 creator A5016626239 @default.
• W2086626108 creator A5036097834 @default.
• W2086626108 creator A5090680688 @default.
• W2086626108 date "2014-08-01" @default.
• W2086626108 modified "2023-09-27" @default.
• W2086626108 title "Bcl-xL Protein Protects from C/EBP Homologous Protein (CHOP)-dependent Apoptosis during Plasma Cell Differentiation" @default.
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