* Department of Immunology, National Jewish Medical and Research Center, University of Colorado Health Sciences Center, Denver; and
Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, China
Correspondence: Dr. Hong-Bing Shu, Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, K516c, Denver, CO 80206. E-mail: shuh{at}njc.org
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B-dependent manner in reporter gene assays. Moreover, our findings indicated BAFF-R, but not TACI, could dramatically up-regulate IL-10 secretion by RPMI-8226 cells. The identification of TALL-1-up-regulated genes will help explain the mechanisms of TALL-1-triggered biological and pathological effects and to identify molecular targets for intervention of lupus-like autoimmune diseases.
Key Words: B lymphocytes • autoimmunity • IL-10 • gene regulation
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TALL-1 signals through three receptors, including BCMA, TACI, and BAFF-R, which are members of the TNF receptor family [5 6 7 8 9 10 11 12 13 14 15 16 ]. All three receptors are mainly expressed by mature B lymphocytes, and TACI is also induced in a subset of T cells following their activation [5 6 7 8 9 10 11 12 13 14 15 16 ]. In vitro, soluble TALL-1 (sTALL-1) can potently stimulate B lymphocyte proliferation alone or in synergy with anti-immunoglobulin (Ig)M [2 , 3 , 10 11 12 13 ]. Administration of recombinant sTALL-1 or overexpression of sTALL-1 in mice leads to increased numbers of mature B lymphocytes, splenomegaly, anti-DNA antibodies, proteinuria, and glomerulonephritis, phenotypes that mimic those of systemic lupus erythemasus (SLE) [3 , 5 , 14 , 17 , 18 ]. Conversely, it has been shown that recombinant sTACI-Ig fusion proteins could significantly inhibit progression of lupus-like autoimmune syndrome and collagen-induced arthritis in animal models [19 ]. Recently, gene knockout studies further confirmed that TALL-1 is required for normal B cell development [20 ]. Surprisingly, gene inactivation studies also indicated that BAFF-R, but not TACI and BCMA, is required for TALL-1-triggered B cell development [15 , 21 22 23 ]. Taken together, these studies suggest that the TALL-1 signaling plays critical roles in regulation of B cell function and autoimmune diseases.
Like many other members of the TNF receptor family, BCMA and TACI can bind to TNF receptor-associated factor proteins and activate the transcription factor nuclear factor (NF)-B and the serine/threonine protein kinase c-jun NH2-terminal kinase [10
11
12
13
, 16
, 24
]. In addition, it has been shown that TACI can activate the transcription factor NF-AT [9
]. These studies suggest that TALL-1 signaling may lead to transcriptional induction of downstream genes.
In this paper, we report the identification of genes transcriptionally induced by TALL-1 stimulation in B cells using a cDNA subtractive hybridization approach.
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B
(SS32/36AA) expression plasmid (Dr. David Goeddel, Tularik, Inc.) were provided by the indicated investigators. The RPMI-8226 B lymphoma cell line was purchased from American Type Culture Collection (Manassas, VA). RPMI-8226 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS). Cells (293) were maintained in high glucose Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum.
Flow cytometry analysis
RPMI-8226 cells (
1x106) were incubated in staining buffer (phosphate-buffered saline/2% FBS) in the absence or presence of Flag-sTALL-1 (100 ng/ml) for 40 min. Cell staining was performed by sequential incubation (each 40 min) with anti-Flag monoclonal antibody (mAb; 1 µg/ml) and RPE-conjugated goat anti-mouse IgG (1:200 dilution) in staining buffer. Cells were washed two times with staining buffer following each incubation. The fluorescence exhibited by the stained cells was measured using a FACScan flow cytometer (Becton Dickson, San Jose, CA).
Electrophoretic mobility shift assays
RPMI-8226 cells (2x106) were treated with His6-sTALL1 (200 ng/ml) and TNF (20 ng/ml) or were left untreated for 30 min, and then nuclear extracts were prepared. Aliquots of the nuclear extracts (20 µg) were incubated with 0.4 ng radiolabeled, double-stranded oligonucleotide containing the following NF-B-binding sequence: 5' GGGGACTTTCCC 3'. Each reaction was supplemented with 0.8 µg poly(dIdC) for blocking nonspecific binding. Nucleoprotein complexes were resolved by electrophoresis on a 6% nondenaturing acrylamide gel in Tris-borate-ethylenediaminetetraacetate buffer.
B lymphocyte isolation
Dr. Peter Henson (National Jewish Medical and Research Center, Denver, CO) provided lymphocytes from peripheral blood of healthy donors were provided. Primary B lymphocytes were purified using anti-CD19 Dynal beads and DETACHaBead anti-CD19 (Dynal, Great Neck, NY) by following procedures suggested by the manufacturer.
RNA isolation
RPMI-8226 cells (1x108) were treated with His-sTALL-1 (200 ng/ml) or left untreated for 4 h. mRNAs were then isolated with the Message Marker mRNA isolation system (Life Technologies, Gaithersburg, MD) following procedures recommended by the manufacturer.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
RT-PCR was performed as previously described [1
].
cDNA subtractive hybridization screening
cDNA subtractions were performed with the PCR-Select cDNA subtraction kit (Clontech, Palo Alto, CA) following procedures recommended by the manufacturer.
Northern blot hybridization
mRNAs from TALL-1-treated and untreated cells were fractionated in 1.2% agarose gels and transferred to pure nitrocellulose membranes. Hybridization was performed with the Rapid Hybridization Buffer (Clontech) at 65°C for 2 h. Blots were washed under high stringent conditions.
Construction of reporter plasmids
To construct the interleukin (IL)-10-luciferase reporter vector, an 1.5-kb fragment of the human IL-10 promoter was amplified from human leukocyte genomic DNA with the following two primers: 5' primer-5'-AAACCCGGGTCAGTGTTCCTCCCAGTTACAGTCTAAACTG-3' and 3' primer-5'-TCTCGGAGATCTCGAAGCATGTTAGGCAGGTTG-3'. The amplified fragment was digested with XmaI/BglII restriction enzymes and inserted into the XmaI/BglII sites of the pGL3-basic plasmid (Promega, Madison, WI).
To construct the lymphocyte activation gene-1 (LAG-1)-luciferase reporter vector, an 2.6-kb fragment of human LAG-1 promoter was amplified from human leukocyte genomic DNA with the following two primers: 5' primer-5'-AAGGTACCCAATAATGGGATTGCTGGGTTGAATGGTAATTC-3' and 3' primer-5'-AACCCGGGAGTTCCTCTCAGCTTCTCTTCCCCAGGGCG-3'. The amplified fragment was digested with KpnI/XmaI restriction enzymes and inserted into the KpnI/XmaI sites of the pGL3-basic plasmid (Promega).
Reporter gene assays
Cells (293; 2x105) were seeded on six-well (35-mm) dishes and were transfected the following day with 0.5 µg IL-10 or LAG-1 luciferase reporter constructs and the indicated expression plasmids. Within the same experiment, each transfection was performed in triplicate, and where necessary, enough amount of empty control plasmid was added to keep each transfection receiving the same amount of total DNA. To normalize for transfection efficiency and protein amount, 0.5 µg respiratory syncytial virus-ß-gal plasmid was added to all transfections. Fourteen hours after transfection, cells were treated with His-sTALL-1 (200 ng/ml) or were left untreated for 6 h. Luciferase reporter assays were performed using a luciferase assay kit (Pharmingen, San Diego, CA) and following the manufacturer’s protocols. ß-Galactosidase activity was measured using the Galacto-Light chemiluminescent kit (TROPIX Inc., Bedford, MA). Luciferase activities were normalized on the basis of ß-galactosidase expression levels.
Establishing RPMI-8226 stable cell lines
cDNAs encoding for human BCMA, TACI, and BAFF-R were amplified from a B cell cDNA library by PCR and inserted into a C-terminal, HA-tagged pcDNA3 plasmid [10
]. The plasmids were linearized and transfected into RPMI-8226 cells by electroporetation. The transfected cells were selected by G418 (1 mg/ml) for 2 weeks. Cells with high-receptor expression in the stable cell lines were further sorted with Flag-tagged sTALL-1 by flow cytometry. Overexpression of the receptors was confirmed by Western blot with anti-HA antibody.
Enyzme-linked immunosorbent assays (ELISAs)
Human IL-10 ELISAs were performed using the human IL-10 ELISA Ready-Set-Go kit (eBioscience, San Diego, CA) by following procedures recommended by the manufacturer.
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B in these cells, as suggested by gel-shift experiments (Fig. 1C)
. These results suggest that TALL-1 can signal in RPMI-8226 cells.
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Figure 1. RPMI-8226 cells are responsive to TALL-1. (A) TALL-1 binds to the plasma membrane of RPMI-8226 cells. RPMI-8226 cells were incubated with control buffer (open histogram) or Flag-sTALL-1 (100 ng/ml; shaded histogram). Cells were washed and analyzed by flow cytometry with an anti-Flag mAb. (B) Expression of TALL-1 receptors in RPMI-8226 cells. Total RNA was isolated from RPMI-8226 cells, and RT-PCR was performed with primers corresponding to the coding sequences of the indicated receptors. *, Expected bands. (C) Activation of NF-B by TALL-1 in RPMI-8226 cells. RPMI-8226 cells were treated with control buffer (lane 1), sTALL-1 (200 ng/ml; lane 2), or TNF (20 ng/ml; lane 3) for 30 min, and NF-B activation was examined by gel-shift assays. The specificity of NF-B bands was confirmed by complete competition with unlabeled B probe (lane 4).
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We sequenced a total of 50 clones derived from the forward subtraction. Blast searches indicate that these clones represent 44 different genes. To confirm whether expression levels of these genes are truly regulated by TALL-1, we performed Northern blot hybridization experiments for all the 44 potential, TALL-1, up-regulated genes. These experiments indicated that the mRNA levels of 10 of the identified genes, including IL-10, LAG-1, granulocyte chemotactic protein-2 (GCP-2), pre-B cell colony-enhancing factor (PBEF), CD27 ligand, PIM-2, ferritin, archain, TRK-fused gene (TFG), and DUSP5 (see below for description of these genes), were significantly up-regulated by TALL-1 stimulation (Fig. 2 ). The mRNA levels of the other clones are not significantly changed by TALL-1 stimulation (data not shown), suggesting these genes are false positives of the cDNA subtraction experiments.
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Figure 2. Northern blot analysis of gene expression up-regulated by TALL-1 in RPMI-8226 B lymphoma cells. RPMI-8226 cells were treated with sTALL-1 (200 ng/ml; +) or were left untreated (-) for 4 h, and total RNAs were isolated for Northern blot analysis with the indicated probes.
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Figure 3. RT-PCR analysis of gene expression up-regulated by TALL-1 in primary B lymphocytes. Primary B lymphocytes were treated with sTALL-1 (200 ng/ml) or sTALL-1 mutant (200 ng/ml), or were left untreated (-) for 4 h, and RT-PCR was performed with primers corresponding to the indicated genes.
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, had a lower mRNA level in TALL-1-treated cells than in untreated cells (Fig. 4
). mRNA levels of the other four clones had no significant difference in TALL-1-treated and untreated cells (data not shown). These clones also represent artificial clones from the subtraction.
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Figure 4. Northern blot analysis of expression level of elongation factor 1 down-regulated by TALL-1. RPMI-8226 cells were treated with sTALL-1 (200 ng/ml; +) or were left untreated (-) for 4 h, and total RNAs were isolated for Northern blot analysis.
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B in 293 cells, and TALL-1 can further potentiate BCMA-mediated NF-B activation [10
]. We examined whether overexpression of BCMA could activate the IL-10 and LAG-1 promoters by reporter gene assays. As shown in Figure 5
, overexpression of BCMA activated IL-10 and LAG-1 promoters. In addition, TALL-1 further potentiated BCMA-mediated activation of the LAG-1 promoter. TALL-1 did not potentiate BCMA-mediated activation of IL-10 promoter, probably because of saturation of IL-10 activation by overexpression of BCMA. Moreover, IB (SS/AA), an IB mutant that has been shown to inhibit NF-B activation, completely inhibited TALL-1/BCMA-triggered activation of the IL-10 and LAG-1 promoters. Taken together, these data suggest that the TALL-1/BCMA ligand/receptor pair activates transcription of IL-10 and LAG-1 in a NF-B-dependent manner.
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Figure 5. Activation of LAG-1 and IL-10 promoters by TALL-1/BCMA. Cells (293; 2x105) were transfected with 0.5 µg LAG-1-luciferase (A) or IL-10-luciferase (B) reporter vectors, together with 1 µg each of the indicated mammalian expression plasmids. Fourteen hours after transfection, cells were treated with sTALL-1 (solid bars) or were left untreated (open bars) for 6 h, and luciferase assays were performed. Data shown are averages and standard deviations of relative luciferase activities from one representative experiment in which each transfection has been performed in triplicate.
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Figure 6. IL-10 protein is up-regulated by BAFF-R. Cell culture supernatants from RPMI-8226 stable cell lines overexpressing TACI, BAFF-R, or a control stable line were examined for IL-10 secretion by ELISA (upper panel). Overexpression of C-terminal, HA-tagged TACI and BAFF-R in the stable cell lines was confirmed by Western blot with anti-HA antibody (lower panel).
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PBEF is a 52-kDa-secreted protein that is dramatically induced by pokeweed mitogen in peripheral blood leukocytes [29 ]. It has been shown that PBEF could enhance the pre-B cell colony-formation activity of stem-cell factor and IL-7 [29 ].
TALL-1 stimulation of B lymphocytes up-regulates expression levels of two chemokine genes, GCP-2 and LAG-1. GCP-2 [30 ], also called small, inducible cytokine subfamily B, member 6 (SCYB6) [31 ], is a member of the CXC chemokine family [32 33 34 35 ]. LAG-1 [36 ], also called small inducible chemokine A4 (SCYA4) [37 ] and Act-2 [38 ], is a member of the CC chemokine family and is the human counterpart of mouse macrophage inflammatory protein-1ß (MIP-1ß) [32 33 34 35 36 37 38 ]. Interestingly, the receptors for GCP-2 (CCR1 and CCR2) and the receptor for MIP-1ß (CCR5) are mostly expressed by Th1 cells and macrophages [32 33 34 35 ]. Gene knockout experiments have demonstrated that CCR2 is critically involved in experimental autoimmune encephalomyelitis [35 ]. As the macrophage-produced TALL-1 can activate B lymphocytes to produce GCP-2 and LAG-1, whose receptors are expressed by macrophages, it is possible that TALL-1-regulated expression of GCP-2 and LAG-1 is involved in functional interaction between macrophages and B lymphocytes.
PIM-2 is a protooncogene belonging to the PIM kinase family. PIM-2 is highly expressed in mitogen-stimulated hematopoietic cells and is induced by a variety of cytokines that also induce NF-B [39
]. PIM-2 collaborates with c-myc to promote progression of cell cycle [40
] and to induce neonatal pre-B cell leukemia in transgenic mice [39
]. Recently, it has been shown that PIM-2 is a NF-B target gene at the pre-B to immature B cell transition [41
]. As TALL-1 can activate NF-B, it is not surprising that PIM-2 is identified as a downstream gene of TALL-1 in this study. Based on the known function of PIM-2, we speculate that PIM-2 is one of the downstream genes that is involved in TALL-1-induced B cell proliferation.
CD27L (CD70) is a member of the TNF family of ligands that binds to CD27. CD27L and CD27 are mostly expressed by activated lymphocytes, and their interaction plays a key role in T-dependent B cell responses [42 ].
Ferritin is an iron storage protein and is critically involved in iron homeostasis. Although it is not known how ferritin may be involved in TALL-1-triggered effects, it has been shown that serum ferritin is dramatically increased with the progression of SLE [43 , 44 ]. This is in good correlation with TALL-1, which is also increased with the progression of SLE [5 ].
Archain, also called coatomer delta subunit, is one component of the cytosolic protein complex called coatomer. Coatomer associates with Golgi nonclathrin-coated vesicles and is involved in vesicle transport and protein secretion [45 ]. It is possible that up-regulation of coatomer components such as archain may be necessary for TALL-1-triggered secretion of Igs and other related activities.
TFG was originally identified in human as the N-terminus of an oncogenic fusion protein TRK-T3, associated with papillary thyroid carcinoma [46 ]. An amino-terminal, coiled-coil domain of TFG is responsible for mediating oligomerization of the TRK-T3 oncoprotein, which results in constitutive activation of the TRK protein tyrosine kinase and oncogenesis [46 ]. DUSP5 is a dual-specific phosphatase, and the physiological functions of DUSP5 are not known. The involvement of TFG and DUSP5 in TALL-1 signaling needs to be further explored.
Our cDNA subtraction hybridization experiments also suggest that elongation factor 1 is down-regulated by TALL-1. The functional significance of this observation is still unclear.
In conclusion, we have identified 10 genes up-regulated by TALL-1. Our findings provide important groundwork to understand the mechanisms of TALL-1-triggered, biological effects and to identify potential molecular targets for intervention of TALL-1-triggered diseases such as SLE.
Received February 1, 2002; revised March 12, 2002; accepted March 27, 2002.
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B, and c-Jun NH2-terminal kinase J. Biol. Chem. 274,15978-15981B and ELF-1 Cytokine 13,25-31[Medline]B, Elk-1, c-jun N-terminal kinase, and p38 mitogen-activated protein kinase J. Immunol. 165,1322-1330B kinase and NF-B target genes at the pre-B to immature B cell transition J. Biol. Chem. 276,18579-18590This article has been cited by other articles:
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