The Adaptor Protein HSH2 Attenuates Apoptosis in Response to Ligation of the B Cell Antigen Receptor Complex on the B Lymphoma Cell Line, WEHI-231
2004; Elsevier BV; Volume: 280; Issue: 5 Linguagem: Inglês
10.1074/jbc.m407690200
ISSN1083-351X
AutoresBrantley R. Herrin, Alison L. Groeger, Louis B. Justement,
Tópico(s)Immune Response and Inflammation
ResumoSignals transduced by the B cell antigen receptor (BCR) play a central role in regulating the functional response of the cell to antigen. Depending on the nature of the antigenic signal and the developmental or differentiation state of the B cell, antigen receptor signaling can promote either apoptosis or survival and activation. Understanding the molecular mechanisms underlying BCR-mediated apoptosis constitutes an important area of research because aberrations in programmed cell death can result in the development of autoimmunity or cancer. Expression of the adaptor protein hematopoietic Src homology 2 (HSH2) was found to significantly decrease BCR-mediated apoptosis in the murine WEHI-231 cell line. Analysis of signal transduction pathways activated in response to BCR ligation revealed that HSH2 does not significantly alter total protein tyrosine phosphorylation or Ca2+ mobilization. HSH2 does not potentiate the activation-dependent phosphorylation of AKT either. With respect to MAPK activation, HSH2 was not observed to alter the activation of ERK or p38 in response to BCR ligation, but it does significantly potentiate JNK activation. Analysis of processes directly associated with apoptosis revealed that HSH2 inhibits mitochondrial depolarization to a significant degree, whereas it has only a slight effect on caspase activation and poly ADP-ribose polymerase cleavage. BCR-induced apoptosis of WEHI-231 cells is associated with the loss of endogenous HSH2 expression within 12 h, whereas inhibition of apoptosis in response to CD40-mediated signaling leads to stabilization of HSH2 expression. Thus, endogenous HSH2 expression correlates directly with survival of WEHI-231 cells, which supports the hypothesis that HSH2 modulates the apoptotic response through its ability to directly or indirectly promote mitochondrial stability. Signals transduced by the B cell antigen receptor (BCR) play a central role in regulating the functional response of the cell to antigen. Depending on the nature of the antigenic signal and the developmental or differentiation state of the B cell, antigen receptor signaling can promote either apoptosis or survival and activation. Understanding the molecular mechanisms underlying BCR-mediated apoptosis constitutes an important area of research because aberrations in programmed cell death can result in the development of autoimmunity or cancer. Expression of the adaptor protein hematopoietic Src homology 2 (HSH2) was found to significantly decrease BCR-mediated apoptosis in the murine WEHI-231 cell line. Analysis of signal transduction pathways activated in response to BCR ligation revealed that HSH2 does not significantly alter total protein tyrosine phosphorylation or Ca2+ mobilization. HSH2 does not potentiate the activation-dependent phosphorylation of AKT either. With respect to MAPK activation, HSH2 was not observed to alter the activation of ERK or p38 in response to BCR ligation, but it does significantly potentiate JNK activation. Analysis of processes directly associated with apoptosis revealed that HSH2 inhibits mitochondrial depolarization to a significant degree, whereas it has only a slight effect on caspase activation and poly ADP-ribose polymerase cleavage. BCR-induced apoptosis of WEHI-231 cells is associated with the loss of endogenous HSH2 expression within 12 h, whereas inhibition of apoptosis in response to CD40-mediated signaling leads to stabilization of HSH2 expression. Thus, endogenous HSH2 expression correlates directly with survival of WEHI-231 cells, which supports the hypothesis that HSH2 modulates the apoptotic response through its ability to directly or indirectly promote mitochondrial stability. Normal B lymphocyte homeostasis and immune function are critically dependent on regulatory pathways that control programmed cell death (apoptosis) (1Strasser A. Bouillet P. Immunol. Rev. 2003; 193: 82-92Crossref PubMed Scopus (63) Google Scholar, 2Sohn S.J. Rajpal A. Winoto A. Curr. Opin. Immunol. 2003; 15: 209-216Crossref PubMed Scopus (44) Google Scholar, 3Rathmell J.C. Thompson C.B. Cell. 2002; 109: S97-107Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 4Niiro H. Clark E.A. Nat. Rev. 2002; 2: 945-956Google Scholar). Aberrant function of apoptotic pathways can lead to numerous life-threatening problems including the development of immunodeficiency, autoimmunity, or cancer. Therefore, it is essential to develop a complete understanding of the pathways that control apoptosis of B lymphocytes. The B cell antigen receptor complex (BCR) 1The abbreviations used are: BCR, B cell antigen receptor; PLCγ, phospholipase Cγ; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase; HSH2, hematopoietic Src homology 2; PI, propidium iodide; ANV, annexin V; PARP, poly ADP-ribose polymerase; NFAT, nuclear factor of activated T cells; Ab, antibody; mAb, monoclonal antibody; PBS, phosphate-buffered saline; TBST, Tris-buffered saline containing Tween; DiOC6, 3,3′-dihexyloxacarbocynine iodide; HRP, horseradish peroxidase. 1The abbreviations used are: BCR, B cell antigen receptor; PLCγ, phospholipase Cγ; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase; HSH2, hematopoietic Src homology 2; PI, propidium iodide; ANV, annexin V; PARP, poly ADP-ribose polymerase; NFAT, nuclear factor of activated T cells; Ab, antibody; mAb, monoclonal antibody; PBS, phosphate-buffered saline; TBST, Tris-buffered saline containing Tween; DiOC6, 3,3′-dihexyloxacarbocynine iodide; HRP, horseradish peroxidase. regulates the development, homeostasis, and function of B cells through its ability to transduce signals that promote either apoptosis or survival and activation, depending on the developmental stage of the cell and the nature of the antigenic stimulus (4Niiro H. Clark E.A. Nat. Rev. 2002; 2: 945-956Google Scholar). Because BCR-mediated signal transduction plays a central role in regulating B lymphocyte apoptosis, understanding the molecular mechanism by which it does so constitutes an important area of investigation with a high degree of relevance for understanding numerous immunologic disease processes. Nevertheless, significant questions remain concerning the molecular linkage between antigen receptor signaling and apoptosis. Ligation of the BCR leads to the activation of several distinct yet interacting signal transduction pathways that ultimately control the functional response of the cell (4Niiro H. Clark E.A. Nat. Rev. 2002; 2: 945-956Google Scholar, 5Gold M.R. Trends Pharmacol. Sci. 2002; 23: 316-324Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 6Kurosaki T. Curr. Opin. Immunol. 2002; 14: 341-347Crossref PubMed Scopus (96) Google Scholar, 7Justement L.B. Curr. Top. Microbiol. 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Immunol. 2003; 4: 280-286Crossref PubMed Scopus (119) Google Scholar). Finally, signaling through the BCR has been shown to regulate the function of mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 (4Niiro H. Clark E.A. Nat. Rev. 2002; 2: 945-956Google Scholar, 11Healy J.I. Dolmetsch R.E. Timmerman L.A. Cyster J.G. Thomas M.L. Crabtree G.R. Lewis R.S. Goodnow C.C. Immunity. 1997; 6: 419-428Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). These distinct pathways in turn promote activation of numerous transcriptional regulatory proteins including NFAT, NF-κB, and the AP-1 complex that act in concert to regulate gene transcription and the functional response of the B cell (4Niiro H. Clark E.A. Nat. Rev. 2002; 2: 945-956Google Scholar, 11Healy J.I. Dolmetsch R.E. Timmerman L.A. Cyster J.G. Thomas M.L. Crabtree G.R. Lewis R.S. Goodnow C.C. 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Cancro M.P. J. Immunol. 1998; 160: 107-111PubMed Google Scholar), expression/function of kinases, and phosphatases and the potential for differential expression of adaptor proteins are likely to play a significant role in determining the functional outcome of signaling via the BCR (4Niiro H. Clark E.A. Nat. Rev. 2002; 2: 945-956Google Scholar, 16Leo A. Schaven B. Curr. Opin. Immunol. 2001; 13: 307-316Crossref PubMed Scopus (68) Google Scholar, 17Kelly M.E. Chan A.C. Curr. Opin. Immunol. 2000; 12: 267-275Crossref PubMed Scopus (33) Google Scholar, 18Niiro H. Clark E.A. Immunity. 2003; 19: 637-640Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). A growing body of literature has begun to delineate the key role that adaptor proteins play in modulating signal transduction via lymphocyte antigen receptors. Adaptor proteins have the ability to quantitatively and/or qualitatively change the nature of antigen receptor signaling by generating diverse multimolecular signaling complexes in unique spatial/temporal contexts (16Leo A. Schaven B. Curr. Opin. Immunol. 2001; 13: 307-316Crossref PubMed Scopus (68) Google Scholar, 17Kelly M.E. Chan A.C. Curr. Opin. Immunol. 2000; 12: 267-275Crossref PubMed Scopus (33) Google Scholar, 18Niiro H. Clark E.A. Immunity. 2003; 19: 637-640Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In the B cell, studies have shown that BLNK (SLP-65) is involved in coupling BCR proximal protein tyrosine kinases to PLCγ, thereby promoting its activation, as well as recruiting and promoting activation of Vav and Nck (19Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 20Pappu R. Cheng A.M. Li B. Gong Q. Chiu C. Griffin N. White M. Sleckman B.P. Chan A.C. 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Immunol. 2003; 170: 349-355Crossref PubMed Scopus (10) Google Scholar, 26Koonpaew S. Janssen E. Zhu M. Zhang W. J. Biol. Chem. 2004; 279: 11229-11235Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 27Brdicka T. Imrich M. Angelisova P. Brdickova N. Horvath O. Spicka J. Hilgert I. Luskova P. Draber P. Novak P. Engels N. Wienands J. Simeoni L. Osterreicher J. Aguado E. Malissen M. Schaven B. Horejsi V. J. Exp. Med. 2002; 196: 1617-1626Crossref PubMed Scopus (179) Google Scholar). Importantly, evidence suggests that the expression of specific adaptor proteins may vary with the developmental or differentiation stage of the B cell, thereby increasing their potential for differentially regulating BCR signaling (4Niiro H. Clark E.A. Nat. Rev. 2002; 2: 945-956Google Scholar). In the current study, we have examined the biochemical and functional role of the hematopoietic Src homology 2 (HSH2) adaptor protein using the WEHI-231 B lymphoma cell line. HSH2 is expressed predominantly in cells of the lymphoid lineage but can be detected in cells of the myelomonocytic lineage as well (28Oda T. Muramatsu M. Isogai T. Masuho Y. Asano S. Yamashita T. Biochem. Biophys. Res. Comm. 2001; 288: 1078-1086Crossref PubMed Scopus (23) Google Scholar, 29Greene T.A. Powell P. Nzerem C. Shapiro M.J. Shapiro V.S. J. Biol. Chem. 2003; 278: 45128-45134Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). HSH2 contains a single SH2 domain, three conserved proline-rich regions and two tyrosine residues that are potential sites of phosphorylation. Expression of HSH2 in the WEHI-231 B cell line was found to protect these cells from undergoing apoptosis in response to BCR ligation. Although HSH2 was not observed to cause global changes in BCR-mediated signal transduction, it did potentiate JNK activation. Moreover, HSH2 was observed to prevent mitochondrial destabilization, whereas it exerted only a modest effect on caspase activation. A key finding is that the endogenous level of HSH2 was observed to decrease in response to pro-apoptotic BCR signaling, whereas it was maintained by anti-apoptotic CD40-mediated signaling. These findings support the conclusion that HSH2 has the ability to modulate the apoptotic response to signals delivered through the BCR complex. Cells and Cell Culture—WEHI-231 murine B lymphoma cells and Phoenix gp retroviral packaging cells (provided by Gary Nolan, Department of Microbiology and Immunology, Stanford University, Palo Alto, CA) were cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum (HyClone, Logan, UT), 2 μm l-glutamine, 50 μm 2-mercapto-ethanol, 100 μg/ml streptomycin-penicillin, and 50 μg/ml gentamicin (Sigma) at 37 °C under 5% CO2. Antibodies—WEHI-231 cells were stimulated with polyclonal goat anti-mouse IgM (F(ab′)2) Ab purchased from BIOSOURCE International (Camarillo, CA). For Western blot analysis, the following antibodies were purchased: mouse anti-FLAG mAb conjugated to horseradish peroxidase (HRP) (Sigma); anti-phosphotyrosine mAb conjugated to HRP (4G10, Upstate Biotechnology Inc., Waltham, MA); mouse anti-Bcl-xL mAb, and mouse anti-Caspase-7 mAb (eBioscience, San Diego, CA); mouse anti-actin mAb (AC-40; Sigma); rabbit polyclonal anti-phospho-ERK (197G2, Thr-0202/Tyr-204), anti-ERK, anti-phospho-JNK (98F2, Thr-183/Tyr-185), anti-JNK, anti-phospho-p38 (3D7, Thr-180/Tyr-182), anti-p38, anti-phospho-AKT (244F9, Thr-308; 193H12, Ser-473), anti-AKT, anti-caspase-3, anti-caspase-9, and anti-poly ADP-ribose polymerase (PARP) Abs (Cell Signaling Technology, Beverly, MA). The hybridoma producing anti-CD40 mAb (1C10) was obtained from Dr. Frances Lund at the Trudeau Institute (Saranac Lake, NY). Rabbit polyclonal anti-HSH2 Ab was generated by immunizing rabbits with intact recombinant HSH2. Plasmids—The cDNA encoding full-length HSH2 and Bcl-xL were PCR-amplified from total murine splenocyte cDNA using KOD high fidelity polymerase from Novagen (Madison, WI). For HSH2, a BglII site was included in the forward primer (5′-GAGAAGATCTCCGCCATGGCAGAAGCC-3′), and a FLAG tag and HpaI site were included in the reverse primer (5′-GAGAGTTAACTCACTTGTCATCGTC-3′). The HSH2 PCR product was digested with BglII and HpaI restriction enzymes and then ligated into the BglII and HpaI sites of the pMSCV-puro vector from Clontech. For Bcl-xL, an XhoI site was included in the forward primer (5′-GAGACTCGAGCCGCCATGTCTCAGAGCAACCGG-3′), and an EcoRI site was incorporated into the reverse primer (5′-GAGAGAATTCTCACTTCCGACTGAAGAG-3′). The Bcl-xL PCR product was digested with XhoI and EcoRI restriction enzymes and ligated into the XhoI and EcoRI sites of pMSCV-puro. Transfection and Transduction—HSH2:pMSCV-puro, Bcl-xL:pMSCV-puro, or empty pMSCV-puro plasmids were co-transfected with the pCL-ECO plasmid encoding the ecotropic receptor envelope into Phoenix gp retroviral packaging cells using Lipofectamine 2000 from Invitrogen according to the manufacturer's instructions. Virus-containing supernatant was collected from transfectants 36 h after transfection. The viral supernatant was incubated with WEHI-231 cells for 12 h in the presence of polybrene (2.5 μg/ml). After transduction, cells were incubated with (1.5 μg/ml) puromycin (Mediatek, Inc., Herndon, VA) for 48 h to select against nontransduced cells. DNA Content Analysis—WEHI-231 cells (1 × 105 cells/ml) were stimulated with 1 μg/ml polyclonal anti-IgM F(ab′)2 Ab for up to 48 h. After stimulation, cells were washed with PBS and fixed in 70% ethanol. The fixed cells were washed with a solution containing 0.1% sodium citrate and 0.1% Triton X-100 to remove nucleotide fragments. Following the wash step, cells were incubated with 10 μg/ml RNase A and 50 μg/ml propidium iodide (PI) for 15 min at 37 °C. Samples were then analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences). Events with sub-G0/G1 DNA content were scored as apoptotic. Annexin V Staining of Cell Surface Phosphatidylserine—WEHI-231 cells (1 × 105/ml) were stimulated with 1 μg/ml polyclonal anti-IgM F(ab′)2 Ab for up to 48 h. After stimulation, cells were washed with PBS and resuspended in 100 μl of annexin-binding buffer (10 mm HEPES, 140 mm NaCl, and 2.5 mm CaCl2, pH 7.4). The cells were then incubated with 5 μl of annexin V (ANV) conjugated with Alexa-488 (Molecular Probes, Eugene, OR) and PI (50 μg/ml) (30Wilkins R.C. Kutzner B.C. Truong M. Sanchez-Dardon J. McLean J.R. Cytometry. 2002; 48: 14-19Crossref PubMed Scopus (69) Google Scholar). The samples were incubated protected from light at room temperature for 15 min and then immediately analyzed using a FACScan flow cytometer (BD Biosciences). Two-color ANV/PI staining was analyzed to discriminate between three distinct populations of cells. Cells that were ANV–,PI– were scored as viable cells, ANV+,PIlo cells were scored as early apoptotic cells, and ANV+,PIhi cells were scored as late apoptotic or necrotic as the assay could not differentiate between the two possibilities. Western Blot Analysis—WEHI-231 cells (2 × 107 cells/sample) were stimulated with 1 μg/ml polyclonal anti-IgM F(ab′)2 Ab in the presence or absence of anti-CD40 mAb (1C10, 1.5 μg/ml) for the time points indicated. After stimulation, cells were immediately washed in ice-cold PBS to stop the reaction. Next, cells were washed twice with ice-cold PBS and lysed in 0.5 ml of lysis buffer (25 mm HEPES (pH 7.8), 150 mm NaCl, 10 mm EDTA, 1 mm EGTA, 0.1 mm Na3VO4, 50 mm NaF, and 1% Nonidet P-40). Phenylmethylsulfonyl fluoride and a protease inhibitor mixture (Sigma) were added to the lysis buffer just before use. Cell lysates were incubated for 1 h on ice and then centrifuged at 13,000 × g for 15 min at 4 °C. Detergent-soluble lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were blocked in TBST containing 3% nonfat milk (or TBST with 3% bovine serum albumin for 4G10 blotting) overnight at 4 °C and then washed four times with TBST. Next, the membranes were incubated with primary antibodies for 1 h at room temperature and then washed four times with TBST. For primary antibodies not directly conjugated to HRP, secondary goat anti-mouse Ig or goat antirabbit Ig Abs conjugated to HRP (BIOSOURCE International) were incubated with the membranes for 1 h at room temperature and then washed four times with TBST. Finally, proteins of interest were visualized using ECL West-Pico chemiluminescent substrate (Pierce) and subsequent exposure to autoradiographic film (Eastman Kodak Co.). For Western blot analysis of caspase cleavage, WEHI-231 cells (1 × 105 cells/ml) were stimulated with 1 μg/ml polyclonal anti-IgM F(ab′)2 Ab for up to 48 h in 10 ml of normal culture medium. After stimulation, cells were pelleted and lysed in 0.1 ml of radioimmunoprecipitation buffer (25 mm HEPES (pH 7.8), 150 mm NaCl, 10 mm EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS) containing 1 mm phenylmethylsulfonyl fluoride and protease inhibitor mixture, which were added just before use. The lysates were incubated on ice for 1 h and then centrifuged at 13,000 × g for 15 min at 4 °C. The total protein content of the detergent-soluble lysate was quantitated using the BCA protein assay (Pierce) according to the manufacturer's instructions. Equal quantities of protein for each sample were subjected to SDS-PAGE, and separated proteins were transferred to nitrocellulose membranes. Blotting procedures were carried out as described above. Subcellular Fractionation—WEHI-231 cells (2 × 107/sample) were washed with PBS following stimulation and then resuspended in 1 ml of homogenization buffer (20 mm Hepes, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 250 mm sucrose, 1 mm dithiothreitol, and protease inhibitor mixture). Cells were incubated in homogenization buffer on ice for 15 min to allow cells to swell and then lysed with 20 passages through a 28-gauge syringe. Lysates were centrifuged at 700 × g for 10 min at 4 °C to remove nuclei and cell debris. The supernatant was collected and centrifuged at 10,000 × g for 15 min at 4 °C. The resulting supernatant was removed and used as the cytoplasmic fraction. The heavy membrane pellet was resuspended in radioimmunoprecipitation buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) and is designated the mitochondrial fraction. The BCA assay (Pierce) was used to quantitate total protein content of the fractions for equal loading on SDS-PAGE gels. Rabbit polyclonal anti-Apaf-1 (eBioscience, San Diego, CA) and mouse monoclonal anti-Hsp60 (Pharmingen) Abs were used to validate the purity of the cytoplasmic and mitochondrial fractions, respectively. Analysis of Mitochondrial Membrane Depolarization (ΔΨm)—WEHI-231 cells (1 × 105/ml) were stimulated with 1 μg/ml polyclonal anti-IgM F(ab′)2 Ab for up to 48 h. At the end of the stimulation time course, 3,3′-dihexyloxacarbocynine iodide (DiOC6) (Molecular Probes) was added directly to the cell culture to a final concentration of 40 nm, and the cells were incubated at 37 °C for 30 min. After incubation, the cells were pelleted by centrifugation and resuspended in 0.2 ml of RPMI 1640 with 5% fetal bovine serum and 40 nm DiOC6 and kept on ice until analyzed by flow cytometry. HSH2 Protects WEHI-231 Cells from Undergoing BCR-induced Apoptosis—Oda et al. (28Oda T. Muramatsu M. Isogai T. Masuho Y. Asano S. Yamashita T. Biochem. Biophys. Res. Comm. 2001; 288: 1078-1086Crossref PubMed Scopus (23) Google Scholar) first identified human HSH2 after searching existing databases for genes that encode proteins with regions homologous to SH2 domains. The hsh2 gene encodes a 352-amino-acid protein with expression of transcripts restricted to cells of the hematopoietic lineage, including B and T lymphocytes. Based on a search of expressed sequence tag databases using the BLAST algorithm, we identified the mouse and rat homologues of human HSH2 consisting of 334 and 335 amino acids, respectively. Recently, another group has cloned the mouse homologue of HSH2 referred to as ALX (29Greene T.A. Powell P. Nzerem C. Shapiro M.J. Shapiro V.S. J. Biol. Chem. 2003; 278: 45128-45134Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Primary amino acid sequence analysis of mouse HSH2 revealed an N-terminal SH2 domain and three conserved PXXP motifs that are likely to facilitate protein-protein interactions; however, no domains associated with catalytic activity were detected. Based on this sequence analysis, it was logical to hypothesize that HSH2 functions as an adaptor protein. Moreover, the expression of HSH2 in lymphocytes raised the possibility that it might function as a regulator of antigen receptor-mediated signal transduction. To determine whether HSH2 is capable of regulating antigen receptor signal transduction, the WEHI-231 B lymphoma cell line was chosen. Cross-linking of membrane IgM on WEHI-231 cells results in growth arrest and the induction of apoptosis (31Wu M. Yang W. Bellas R.E. Schauer S.L. FitzGerald M.J. Lee H. Sonenshein G.E. Curr. Top. Microbiol. Immunol. 1997; 224: 91-101PubMed Google Scholar, 32Benhamou L.E. Cazenave P.A. Sarthou P. Eur. J. Immunol. 1990; 20: 1405-1407Crossref PubMed Scopus (219) Google Scholar, 33Page D.M. DeFranco A.L. Mol. Cell. Biol. 1990; 10: 3003-3012Crossref PubMed Scopus (46) Google Scholar). Because of this functional response, WEHI-231 cells have been used extensively as a model for immature B cell negative selection. Additionally, stimulation of WEHI-231 cells through the BCR initiates a tyrosine phosphorylation-based signaling cascade, which makes them a useful model for studying BCR-mediated signal transduction. Finally, WEHI-231 cells can be efficiently transduced by retroviruses, thus facilitating their genetic manipulation (34Krebs D.L. Yang Y. Dang M. Haussmann J. Gold M.R. Methods Cell. Sci. 1999; 21: 57-68Crossref PubMed Scopus (35) Google Scholar). We utilized these properties of WEHI-231 cells to examine the functional and biochemical consequences of HSH2 expression on antigen receptor signaling following surface immunoglobulin M cross-linking. Retrovirus encoding full-length FLAG-tagged HSH2, Bcl-xL, and the empty pMSCV-puro vector were used to transduce WEHI-231 cells to express these proteins. Bcl-xL overexpression was used as a control for the apoptosis assays that follow because it has been demonstrated to function as a potent inhibitor of antigen receptor-induced apoptosis in WEHI-231 cells (35Merino R. Grillot D.A. Simonian P.L. Muthukkumar S. Fanslow W.C. Bondada S. Nunez G. J. Immunol. 1995; 155: 3830-3838PubMed Google Scholar, 36Wiesner D.A. Kilkus J.P. Gottschalk A.R. Quintans J. Dawson G. J. Biol. Chem. 1997; 272: 9868-9876Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Cells were transduced with retrovirus encoding empty pMSCV-puro to control for any effects that retroviral transduction might have on WEHI-231 function. After transduction, nontransduced cells were eliminated using puromycin drug selection. Even before drug selection, transduction efficiencies of 70–80% were achieved as determined by the percentage of enhanced green fluorescent protein-positive WEHI-231 cells after transduction with an enhanced green fluorescent protein-expressing retrovirus (data not shown). As expected, HSH2 was detected by Western blotting with anti-FLAG mAb only in HSH2-transduced WEHI-231 cells. Similarly, Bcl-xL expression could only be detected by Western blotting with anti-Bcl-xL mAb in WEHI-231 cells transduced with retrovirus encoding Bcl-xL (data not shown). The extent of apoptosis initiated by antigen receptor cross-linking can be quantitated based on the analysis of DNA content. The induction of apoptosis results in activation of nucleases that cleave the genome of apoptotic cells into nucleotide fragments. Cells undergoing apoptosis will therefore have less total DNA than nonapoptotic cells. Using a dye that quantitatively binds DNA, such as PI, these populations can be discriminated. To determine whether HSH2 expression alters the apoptotic response of WEHI-231 cells to stimulation through the BCR, cells expressing empty vector, HSH2, or Bcl-xL were stimulated with polyclonal anti-IgM F(ab′)2 Ab. Cells were then assayed for the induction of apoptosis based on PI staining (Fig. 1A). As expected, ∼70% of the WEHI-231 cells transduced with empty vector had undergone apoptosis by the 48-h time point. Also, as expected, WEHI-231 cells expressing Bcl-xL were almost completely resistant to BCR-induced apoptosis with only 10% of the population containing sub-G0/G1 levels of DNA. In contrast to control WEHI-231 cells, only 37% of HSH2-expressing WEHI-231 cells contained sub-G0/G1 levels of DNA at the 48-h time point. Indeed, at both the 24- and 48-h time points, approximately half of the number of HSH2-expressing cells had undergone apoptosis when compared with control cells transduced with empty vector. The results of these initial experiments suggest that HSH2 expression is capable of inhibiting apoptosis initiated in response to stimulation of cells through the BCR. To confirm that HSH2 is capable of inhibiting apoptosis initiated by antigen receptor cross-linking, WEHI-231 cells transduced with empty vector, HSH2, or Bcl-xL were stimulated
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