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a Department of Cell Biology & Anatomy, Cornell University Medical College, New York, New York 10021, USA
b Chinese University of Hong Kong, Department of Clinical Oncology, Prince of Wales Hospital, Shatin, Hong Kong, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF) receptor. The interaction between the p55 receptor and BRE was verified by an in vitro biochemical assay by using recombinant fusion proteins and by co-immunoprecipitation of transfected mammalian cells. In the yeast two-hybrid assay, BRE specifically interacted with p55 TNF receptor but not with other TNF family members such as the Fas receptor, the p75 TNF receptor, and p75 neurotrophin receptor. Overexpression of BRE inhibited TNF-induced NF
B activation, indicating that the interaction of BRE protein with the cytoplasmic region of p55 TNF receptor may modulate signal transduction by TNF-.—Gu, C., Castellino, A., Chan, J. Y-H., Chao, M. V. BRE: a modulator of TNF- action. FASEB J. 12, 1101–1108 (1998)
Key Words: brain· reproduction·PCR·maltese binding protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous differential screening experiments identified a novel gene, brain and reproductive organs (BRE),2 which is expressed highly in testis, ovary, and brain (1). A unique property of BRE is its sensitivity to ultraviolet irradiation. After treatment of primary fibroblast cells with ultraviolet light, BRE mRNA levels decreased dramatically. A down-regulation of BRE was also observed after treatment of HL-60 cells with retinoic acid and of fibroblasts with nitroquinoline-1-oxide, a potential DNA damaging agent. Cells treated with these different agents—UV, DNA damage and retinoic acid—displayed a 50 to 90% decrease in BRE mRNA levels. In addition, highly malignant tumorgenic cells show a greater decrease in BRE mRNA compared to less aggressive tumorgenic cells (1).
The BRE transcript encodes a message of 1.8 kb. The amino acid sequence of BRE predicts a protein of 383 amino acids, which contains an acidic isoelectric point. No significant motif or homology was found through a Genebank search, and the precise functions of the BRE protein have not been defined.
In defining proteins that interact with the p55 TNF receptor, several BRE cDNA clones were identified in a yeast two-hybrid screen with the tumor necrosis factor (TNF-) receptor. TNF- is produced predominately by activated macrophages in response to inflammatory stimuli (2) and is responsible for a wide variety of responses, including apoptosis, NF-B activation, AP-1 activation, increased antiviral activity, nitric oxide production, phospholipase A2 activity, sphingomyelinase activation, and cell proliferation (3–6). Here we consider the possibility the BRE may be a component of TNF receptor action. In response to TNF- treatment in 293 and MCF7 cells, co-expression of BRE had a modulatory effect on TNF -specific NF-B activation. The interaction of BRE with the p55 TNF receptor may serve to regulate specific responses through NFB-related mechanisms.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was purchased from R & D and TPA was from Sigma (St. Louis, Mo.).
Isolation of mouse BRE cDNA
The yeast plasmid clone 14 (c14) was rescued and transformed into E. coli, and the DNA purified and sequenced (7). To screen for the mouse BRE cDNA, the truncated BRE cDNA (trBRE) clone (c14) was digested with SpeI and Not I and the resulting DNA fragment was labeled by random priming with (a-32P)dCTP and Klenow DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.). The labeled probe was used to screen the lZAP C57 B1/6 mouse brain cDNA library (gift from Cary Lai). Five positive pBluescript phagemids were identified. Upon sequencing, one clone was found to contain the full-length mouse BRE (mBRE).
In vitro binding
GST fusion proteins containing different regions of the cytoplasmic domain of the p55 receptor were incubated with MBP-trBRE, according to published procedures (7). The reaction was briefly centrifuged, washed three times, and subjected to electrophoresis on SDS-polyacrylamide gels and Western blotting.
Expression vectors and transfection
To construct the HA-trBRE expression vector, the yeast library plasmid containing BRE was digested with EcoRI and XbaI. The fragment was ligated into pCDNA3HA vector (gift from Wen Fan). For the subcloning of the full-length BRE in the pCDNA3 expression vector, both N-terminal fragment (L71) and C-terminal fragment (H1), which are both in the EcoRI site of pGEM3Z, were used as templates for the PCR reactions. To amplify L17 fragment, 5' primer (5'-CCCAAGCTTGGGTACCTGTACCCACGTAGT-3') had a HindIII linker incorporated at the 5'-end and the 3' primer (5'-AGGAATTCAGCATCTTCTCCAAAGATA-3') contained the original EcoRI site. To amplify the H1 fragment, 5' primer (5'-CTGAATTCCTGCCAGACCCCTCAGCTT-3') contained the original EcoRI site and 3' primer (5'-GCTCTAGAGCGAGGGGCATTTAATCTTAGA-3') had a XbaI linker incorporated at the 3'-end. For each PCR reaction, 100 ml of reaction mixture contained the primers at 0.3 mM; the DNA template at 1 ng/ml; 50 mM each dATP, dCTP, dGTP, and dTTP; 1X Pfu buffer; and 2.5 units of Pfu polymerase (Stratagene). The PCR conditions for both reactions were as follows: 5 cycles at 95°C for 1 min, annealing temperature of 48°C for 1.5 min, and extension temperature of 72°C for 2 min; 25 cycles at 95°C for 1 min, 56°C for 1.5 min, and 72°C for 2 min. This was followed by a final cycle of 72°C for 4 min. This condition yield PCR fragments of the expected size. The PCR fragments were phenol/chloroform extracted, ethanol-precipitated, and L17 PCR products were digested with HindIII and EcoRI and ligated into pCDNA3 expression vector. Transformants were screened by restriction digestion, and the plasmid DNA that scored positive was used as the recipient vector for the subsequent ligation of H1 PCR products after digestion with EcoRI and XbaI. Transformants were screened by restriction digestion and verified by DNA sequencing.
To construct the flag-tagged, full-length BRE construct, the pCDNA3-BRE vector was used as the template for the PCR reaction. Both the 5'-primer (5'-CCCAAGCTTATGTCCCCAGAAGTG-3') and the 3'-primer (5'-CCCAAGCTTACTGGTGTTTCCTAG-3') contained a HindIII linker. For the PCR reaction, 100 ml of reaction mixture contained the primers at 0.3 mM; the DNA template at 1ng/ml ; 50 mM each dATP, dCTP, dGTP, and dTTP; 1X Pfu buffer; and 2.5 units of cloned Pfu polymerase (Stratagene, La Jolla, Calif.). The PCR condition was as follows: 5 cycles at 95°C for 1 min, annealing temperature of 42°C for 1.5 min, and extension temperature of 72°C for 2 min ; 25 cycles at 95°C for 1 min, 55°C for 1.5 min, and 72°C for 2 min. This was followed by a final cycle of 72°C for 4 min. This condition yielded PCR fragments of the expected size. The PCR fragment was digested with HindIII and ligated into pFLAG-CMV2 Expression Vector (Eastman Kodak, Rochester, N.Y.). Transformants were screened by restriction digestion and verified by DNA sequencing. To generate a His tagged p55 TNF receptor, pCMVp55 (8) was used as the template for the PCR reaction. 5'-primer (5'-CGGGATCCCATCATCATCATCATCATCTGGTCCCTC-ACCTAGGGGACAGG-3') had a BamH1 linker and 3'-primer (5'-TGCTCTAG-AAGCCTCATCTGAGAAGACTGGGCG-3') had a Xba1 linker. The PCR reaction was carried out as described above. The PCR condition was as the follows: 5 cycles at 95°C for 1 min, annealing temperature of 56°C for 1.5 min, and extension temperature of 72°C for 2.3 min ; 25 cycles at 95°C for 1 min, 67°C for 1.5 min, and 72°C for 2.3 min. This was followed by a final cycle of 72°C for 4 min. This condition yield PCR fragments of the expected size. The PCR fragment was digested with BamH1 and Xba1 and ligated into pCDNA3-Ig
signal peptide expression vector. Transformants were screened by restriction digestion and verified by DNA sequencing. For transfections in immunoprecipitation and co-precipitation experiments, the calcium phosphate method was used according to published procedures (8).
Immunoprecipitation
Transfected cells growing in 10 cm plates were washed two times with phosphate-buffered saline, scraped, and centrifuged. The cell pellets were lysed in 1 ml of NP-40 lysis buffer (150 mM NaCl, 50 mM Tris pH 8, 1% NP-40) supplemented with protease inhibitors. Lysates were incubated on ice for 15 min; the cell debris was removed by centrifugation; and an aliquot of the lysate was used to determine protein concentration by using the Bio-Rad reagent. Protein A beads (20 ml) were added to the lysates and incubated for 1 h on a rotator at 4°C; the precleared lysates were obtained by centrifugation. Mouse monoclonal antibody against HA (12CA5 from Boehringer Mannheim) was added to the precleared lysates, and the mixture was incubated at 4°C for 2 h, followed by incubating with protein A beads for another 1 h. The immunoprecipitates were washed five times in the NP-40 lysis buffer and boiled for 5 min in SDS-PAGE buffer. The sample was subjected to electrophoresis on a 12 or 10% SDS-polyacrylmide gel.
Co-precipitation
Cells lysates were prepared as described above in NP-40 lysis buffer with protease inhibitors. 50 ul of preequalibrated Ni2+ -NTA agarose (Qiagen, Santa Clarita, Calif.) were incubated with the lysates for 3 h at 4°C and followed by washing three times with the lysis buffer and three times with lysis buffer containing 50 mM imidazole pH 7.3. The sample was boiled in SDS-PAGE buffer containing 10 mM EDTA for 5 min and run on 12% SDS-polyacrylmide gel.
Western blotting
Proteins were resolved by SDS-PAGE gel and transferred onto nitrocellulose. After blocking with 5% milk in TBST (0.1 M NaCl, 0.01 M Tris pH 8.0, 1 mM EDTA, 0.1% Tween-20) for 1 h, the membrane was incutated with the primary antibodies 12CA5, anti-FLAG M2 antibody (Eastman Kodak), or 1:1000 dilution of anti-MBP antibody (7) for another 1h. After incutation with the 1:5000 dilution of the secondary antibodies (peroxidase-coujugated anti-rabbit or anti-mouse IgG), the membrane was visualized with enhanced chemiluminescence (Amersham, Arlington Heights, Ill.).
Gel-shift assay
For the NF-B activation experiments, cells in six-well plates were transfected with 1.5 ug of total DNA per well by using lipofectamine (GIBCO BRL, Grand Island, N.Y.) according to the manufacturer's protocol. Cells from 1 day posttransfection were treated with or without 15 ng/ml of TNF- for 30 min. The cells were then gently rinsed with ice cold PBS and harvested by scraping with ice cold PBS. Cells were pelleted at 4°C (730xg) and lysed in 50 ul of a lysis buffer (20 mM Hepes, pH 7.9, 0.35 M NaCl, 20% glycerol, 1% NP40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF) on ice, and after 15 min the cell debris was removed by centrifugation at 16,000 x g for 10 min. The protein concentration of the whole-cell lysate was measured and then immediatedly frozen and stored at -80°C. Equal amounts of the whole-cell lysates were used in the electrophoretic mobility shift assay (EMSA). A final volume of 20 ml of NF-B-EMSA mix containing 20,000–50,000 cpm of 32P-labeled oligonucleotide corresponding to the light chain enhancer and the binding buffer (25 mM Hepes, PH 7.9, 5% glycerol, 70 mM KCl, 0.2 mM EDTA, 0.27% NP-40, 4% Ficoll 400, 1 mg/ml BSA, 0.1 mg/mg poly [dIdC], 2 mM DTT, 0.2mM PMSF) was incubated on ice for 30 min. The reactions were separated on a nondenaturing 4% acrylamide gel, dried, and visulized with a phosphoimager (Molecular Dynamics, Sunnyvale, Calif.).
Luciferase assay
A luciferase construct containing two B sites (pBIIX-luc) was utilized in the transfection experiments (9). Cells were washed three times with phosphate-buffered saline and then lysed with 300 ml of reporter lysis buffer (Promega). Aliquots of cell lysates (2 ml) were mixed with 100 ml of luciferase assay reagent (Promega), and the luciferase activity was measured using a ß-counter. Aliquots from the same cell lysates (5 ml) were mixed with 600 ul of ß-Gal reaction buffer (100 mM sodium phosphate buffer pH 7.0, 10 mM KCl, 1 mM MgSO4, 83 mM ß-mercaptoethanol, 1.5% o-nitrophenyl-ß-D-galactopyranoside (ONPG). Samples were incubated at 37°C until color development. The absorbance was measured at 420 nm. These values were then used to normalize the luciferase activities.
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RESULTS |
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Fig. 1)
and is referred to as truncated BRE (trBRE).
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The TNF receptors belong to a receptor superfamily that also includes the Fas antigen, CD40, the p75 neurotrophin receptor, and DR3 (10, 11). The major characteristic of these receptors is the presence of canonical cysteine-rich repeats in the extracellular domain. A subset of the members, such as the Fas receptor, DR3, DR4, and p75 neurotrophin receptor also share a ‘death domain’ sequence in their cytoplasmic region. The binding to these receptors of their respective ligands induces receptor oligomerization that initiates downsteam signaling.
Interaction of BRE with the TNF receptor
To verify the authenticity of the BRE interaction with the TNF receptor, several specificity tests were carried out. Yeast S260 cells were cotransformed with the library vector containing BRE and a bait vector containing different regions representing the p55 TNF receptor, as well as cytoplasmic regions from other members of the TNF receptor superfamily including Fas, the p75 TNF receptor, and p75 neurotrophin receptor. Filter assays for ß-galactosidase were performed after overnight galactose induction. Several p55 TNF receptor constructs containing different juxtamembrane sequences (Y1, Y4, Y5) and produced ß-galactosidase activity ( Fig. 2).
Other receptor constructs containing only the death domain (Y3), or lacking the juxtamembrane region (Y4) as well as other TNFR superfamily members tested here (Fas, p75 TNF receptor, and p75 NGF receptor) did not yield any ß-galactosidase activity. Therefore, the yeast two-hybrid results indicate a specific interaction between BRE and the juxtamembrane region of the p55 TNF receptor.
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To confirm the interaction of BRE with p55 TNF receptor in vitro, the 1.2 kb BRE cDNA was cloned in frame with the coding sequences for the maltose binding protein (MBP). After induction of the MBP-BRE protein in transformed E.coli, the cell extract was passed over an amylose column and eluted with maltose. The eluted fractions were pooled and the proteins were verified by SDS-gel electrophoresis. A prominent protein representing the MBP-BRE fusion protein was observed at 65kDa.
The purified 65 kDa MBP-BRE fusion protein was then incubated with glutathione-sepharose beads containing GST fusion proteins with different cytoplasmic regions of p55 TNF receptor as described (7). The mixture was washed extensively and then subjected to electrophoresis on an SDS-acrylamide gel. An anti-MBP antibody was used to detect MBP-BRE by western blot analysis. A specific interaction was detected between BRE and the juxtamembrane region of the p55 TNF receptor, but not with the death domain of p55 or with the GST protein ( Fig. 3).
The antibodies against MBP were not cross-reactive with either GST or p55 TNF receptor proteins (7). Therefore, the in vitro binding experiment indicated that BRE binds selectively to the juxtamembrane region of the p55 TNF receptor. The yeast two hybrid assay and the in vitro binding experiments both demonstrated that BRE protein specifically interacted with p55 TNF receptor.
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Co-precipitation experiments
To determine whether the interaction of BRE occurs in mammalian cells, a co-precipitation assay was performed by using transfection of human embryonic kidney 293 cells. The N-terminus of BRE protein was also tagged with an HA-epitope and the full-length cDNA was subcloned into the pCDNA3 expression vector. The HA-tagged BRE expression vector was transiently transfected into 293 cells. Twenty-four hours after transfection, cell lysates were prepared and immunoprecipitated with monoclonal antibody to HA. The HA-BRE protein was detected in 293 cells by Western blotting with the anti-HA antibody. The HA-BRE protein was detected in transfected, but not in the mock (vector alone) transfected cells ( Fig. 4A).
Therefore, the epitope tagged HA-BRE was expressed and could be immunoprecipitated by anti-HA antibody.
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To detect the BRE and p55 TNF receptor interaction in mammalian cells, an expression vector coding for the full-length p55 receptor containing an N-terminal His epitope was transiently co-transfected with HA-BRE into 293 cells. Extracts were prepared and incubated with Ni2+ beads. The extract was extensively washed, separated by SDS-gel electrophoresis, and then analyzed by Western blotting by using the anti-HA antibody. A 34-kDa protein representing the HA-BRE protein was detected in cells coexpressing His-p55 (
Fig. 4B). A parallel experiment with HA-BRE and vector or with the His-tagged p55 receptor alone did not produce any BRE protein; therefore, the p55 receptor directly associates with a truncated form of BRE in mammalian cells.
NF-B activity
The functional significance of TNF receptor signaling and the association of BRE was addressed by testing the hypothesis that a juxtamembrane-interacting protein such as BRE might modulate a known function of p55 TNF receptor. The major known functions of p55 TNF receptors are initiated by binding of proteins to the C-terminal death domain.
A prominent action of TNF is the ability to stimulate NF-B activity, which is dependent on signal transduction through the RING-finger protein TRAF2 and the adapter protein TRADD (12). MCF7 cells, a human breast carcinoma cell line, were used because of their relatively higher TNF receptor levels and responsiveness to TNF-.
MCF7 cells were therefore transfected with an expression construct containing flag-tagged, full-length BRE, HA-truncated BRE ( Fig. 1), or pCDNA3 vector alone. MCF7 cells were treated with 15 ng/ml of TNF- for 30 min after they had been transfected for 24 h. An electrophoretic mobility shift assay was then performed. TNF treatment of MCF7 cells, which were mock transfected, lead to a strong activation of NFB, as evidenced by the gel mobility shift ( Fig. 5A).
This response could be abolished with the incorporation of excess unlabeled oligonucleotide. In contrast, cells transfected with the full-length BRE protein displayed a significant decrease in the levels of NF-B. The truncated form of BRE did not produce any effect on the induction of NFB. These results were confirmed in similar experiments using 3T3 cells ( Fig. 5B). In the absence of TNF, vector, trBRE or BRE, transfected cells gave the same basal level of NF-B.
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To verify this response in an independent manner, a luciferase reporter gene assay was utilized in transfected 293 cells. A luciferase reporter construct containing two B sites in tandem was cotransfected with LacZ plasmid, together with either truncated BRE (trBRE) or full-length BRE, or with the expression vector alone. After 20 h, the cells were treated with 15ng/ml of TNF- for 16 h. The luciferase activity was measured and normalized to the level of ß-galactosidase activity. As shown in Fig. 6B,
transfection of the empty vector resulted in a substantial NFB activation (nearly 80-fold) upon TNF- treatment, while full-length BRE expression resulted in almost complete inhibition of TNF-induced NFB activation. It is interesting that transfection of the truncated BRE expression construct resulted in a partial inhibition of TNF-induced NFB activation (approximately 55-fold activation). The inhibition of BRE expression was specific to TNF since NFB activation induced by phorbol 12-myristate 13-acetate (PMA) was not inhibited by the expression of BRE ( Fig. 6B). Expression of BRE can therefore affect TNF-induced NFB activity, but not all pathways leading to NFB activation. These findings indicate that BRE might regulate TNF signaling through its interactions with the p55 receptor.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (15), and activation of caspases (16). These signaling pathways can act to mediate a wide range of biological activities including inflammation and cellular immune responses, cell proliferation, differentiation, and necrosis and apoptosis (5).
Here we have identified a new protein interaction between the juxtamembrane region of p55 TNFR and a DNA damage responsive gene, BRE. This specific interaction was confirmed in both the yeast two-hybrid system and an in vitro biochemical system, as well as an in vivo mammalian cell assay. BRE specifically interacts with p55 TNF receptor, but not with other TNF receptor family members. BRE interacts with the juxtamembrane region of p55 TNF receptor, but not with the death domain of this receptor. Overexpression of BRE in 293 cells inhibited TNF-induced NF-B activities.
The transcription factor NF-B is a critical regulator of cytokine-inducible gene expression (17). Genes regulated by nuclear NF-B include those involved in inflammatory responses such as other cytokines, immunoregulatory genes, and metalloproteinases. Recently, NF-B activities have been demonstrated in several cell systems to prevent TNF--induced apoptosis (18–21). The activation of NF-B by TNF requires the phosphorylation and degradation of IB, which leads to the nuclear translocation of NF-B. A novel set of IB protein kinases have been identified that lead to the phosphorylation of IB (22, 23). Overexpression of BRE appears to induce cell death in HeLa cells, L929 and MCF7 cells (data not shown), suggesting that upregulation of BRE may decrease NF-B activities and thereby promote TNF-mediated cytotoxicity.
How might TNF-induced NF-B activities be regulated by BRE? Overexpression of TNF receptor interacting molecules TRAF2 and RIP induce NFB activation. In addition, the association of TRAF2 with a newly identified MAP kinase family member NIK (NF-B-inducing kinase) with TRAF2 is upstream of cytokine-induced NFB activation events (24). BRE's association with the TNF receptor may inhibit or disrupt interactions between TRAF2 and NIK, or repress IB phosphorylation activities. In this regard, the isolation of a large 700–900,000 dalton complex containing the IB kinase activities (23, 25) implies many proteins participate in the regulation of NFB activation. It is conceivable that BRE may associate with or be a component of this large complex of proteins. Overexpression of BRE inhibits TNF-induced NF-B may because of its disruption of any of these pathways or by direct interference with TRAF2, TRADD, or RIP. It is also possible that BRE interacts with an unidentified molecule that affects TNF-induced NFB activation.
A Genebank homology search for BRE reveals no significant motifs or sequence similarity except a weak homology to a Saccharomyces cerevisiae protein, NSP1/TFS1. NSP1/TFS1 displays 47–53% identity in only two stretches of 15–17 amino acids in BRE (see Fig. 1). NSP1/TFS1 was identified originally as a suppressor of cdc25 mutations in Saccharomyces cerevisiae (26, 27). Genetic studies in the yeast suggest that NSP1/TFS1 acts upstream of ras and adenylyl cyclase and it could act in conjunction with cdc25 to effect guanine nucleotide exchange on ras or have a role in localizing adenylyl cyclase to the plasma membrane. The weak homology between BRE and NSP1/TFS1 provides another suggestion that BRE may be involved in regulating phosphorylation events. Further biochemical experiments are necessary to test this hypothesis and the relationship of BRE to the emerging pathway leading from the TNF receptor to the activation of NFB.
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ACKNOWLEDGMENTS |
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B luciferase promoter construct and for continual advise. This project was supported by the NCI (CA45670) and the Dorothy Rodbell Cohen Foundation. |
FOOTNOTES |
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2 Abbreviations: trBRE, truncated brain and reproductive organs; mBRE, mouse BRE; TNF-, tumor necrosis factor; EMSA, electrophoretic mobility shift assay; MBP, maltose binding protein; PCR, polymerase chain reaction.
Received for publication January 8, 1998. Accepted for publication April 24, 1998.
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B in the immune system. Ann. Rev. Immunol. 12, 141–179[Medline]
B in preventing TNF--induced cell death. Science 274, 782–784B. Science 274, 784–787B kinase that activates the transcription factor NF-B. Nature 388, 548–554[Medline]
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