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Characterization of Bovine FAS-Associated Death Domain Gene1

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Characterization of Bovine FAS-Associated Death Domain Gene1 SHORT COMMUNICATION doi:10.1111/j.1365-2052.2004.01207.x Characterization of bovine FAS-associated death domain gene1 M. E. Szperka*, E. E. Connor*, M. J. Paape*, J. L. Williams† and D. D. Bannerman* *Bovine Functional Genomics Laboratory, USDA Agricultural Research Service, Beltsville, MD 20705, USA. †Roslin Institute, Roslin, Midlothian EH25 9PS, Edinburgh, UK Summary The FAS-associated death domain (FADD) protein is an adapter/signaling molecule that has been shown to function in human cells to promote apoptosis and to inhibit NF-jB activa- tion. Because of the critical role that apoptosis and NF-jB play in a variety of disease states, we mapped the bovine FADD gene, sequenced bovine FADD cDNA, and characterized its expression in endothelial cells (EC). Sequencing of bovine FADD revealed approximately 65 and 58% amino acid sequence identity to its human and murine homologues, respectively. Bovine FADD was mapped to chromosome 29 by radiation hybrid mapping. In addition, the functionality of bovine FADD was studied. Expression of a bovine FADD dominant-negative construct blocked bacterial lipopolysaccharide (LPS)- and TNF-a- induced apoptosis in bovine EC consistent with previous studies of human FADD. In contrast to human FADD, elevated expression of bovine FADD had no effect on LPS- or TNF- a-induced upregulation of NF-jB-dependent gene products as assayed by E-selectin expression. Thus, while the role of FADD in mediating apoptosis is conserved across species, its role in regulating NF-jB-dependent gene expression is not. Keywords apoptosis, caspase, cytokines, endotoxin, inflammation. Inflammation and apoptosis contribute to the pathogenesis enables activation of these pro-enzymes into fully functional and/or deleterious outcomes associated with many bacter- proteases resulting in the onset of apoptosis (Muzio et al. ial-mediated diseases including bovine mastitis (Capuco 1998). et al. 1986; Bayles et al. 1998; Bannerman et al. 2004). In NF-jB is a key pro-inflammatory molecule that promotes humans and mice, the FAS-associated death domain the expression of cytokines and adhesion molecules (Tak & (FADD) protein has been demonstrated to function as an Firestein 2001). In addition to its role in promoting apop- adapter protein that couples receptors, such as those for tosis, human FADD has been established to downregulate bacterial lipopolysaccharide (LPS) and the pro-inflamma- LPS-, but not TNF-a-induced NF-jB activation (Chinnaiyan tory cytokine TNF-a, to a family of proteolytic enzymes et al. 1996; Hsu et al. 1996; Bannerman et al. 2002). referred to as caspases (Chinnaiyan et al. 1996; Choi et al. Because of the important role of FADD in mediating pro- 1998; Yeh et al. 1998). FADD recruitment to death receptor apoptotic and pro-inflammatory signaling, we decided to signaling complexes is mediated through highly conserved investigate whether bovine FADD, similar to its human and death domains (DD) contained within FADD and either the murine homologues, is able to function as a pro-apoptotic receptors, themselves, or DD-containing intermediaries adapter protein and whether bovine FADD could moderate recruited to the receptors (Chinnaiyan et al. 1995; NF-jB dependent gene expression. Hofmann & Tschopp 1995). FADD, in turn, can recruit pro- The complete coding sequence of bovine FADD cDNA caspase-8 via protein–protein interactions of death effector (GenBank accession no. AY725483) was obtained by domains (DED) contained within both proteins (Muzio et al. reverse transcription polymerase chain reaction (RT-PCR) 1996, 1998). FADD recruitment of pro-caspase-8 molecules using total RNA isolated from cultured bovine aortic endothelial cells (EC) (generous gift of Dr L.M. Sordillo, Address for correspondence Michigan State University, East Lansing, MI, USA) as Douglas D. Bannerman, Bovine Functional Genomics Laboratory, USDA template. FADD-specific primers for RT-PCR (5¢-CTGGTA Agricultural Research Service/ANRI, BARC-East, Bldg. 1040, Room#2, GCAAAACGGCATTC-3¢ and 5¢-GAGCTCGTTTAATGGGG Beltsville, MD 20705-2350, USA. ACA-3¢) were designed from bovine expressed sequence tags E-mail: [email protected] (TC290893) available in the Bos taurus Gene Index (http:// 1The nucleotide sequence data reported in this paper were submitted to 2www.tigr.org/tdb/tgi). Reverse transcription was performed GenBank and assigned the accession number AY725483 using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Accepted for publication 18 October 2004 Hercules, CA, USA) and subsequent PCR was done using Ó 2004 International Society for Animal Genetics, Animal Genetics, No claim to original US government works, 36, 63–66 63 64 Szperka et al. 2 ll of first-strand cDNA and iQ Supermix (Bio-Rad Labor- subcloned into the BamHI and NotI restriction sites of the atories) according to the manufacturer’s instructions. Cyc- retroviral expression vector pBMN-IRES-PURO (kindly pro- ling conditions were 95 °C for 3 min, followed by 30 cycles vided by Dr Gary Nolan, Stanford University, Stanford, CA of: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The and subsequently modified by Dr Kyle Garton, University of resulting 887-bp product was cloned into pCR2.1 vector Washington, Seattle, WA, USA). (Invitrogen, Carlsbad, CA, USA) and sequenced using M13 High-titre retrovirus was used to infect EC with viral primers. All sequencing was performed on a CEQ8000 particles containing the expression vectors as previously automated DNA sequencer (Beckman Coulter, Fullerton, described (Bannerman et al. 2002). Twenty-four hours post- CA, USA). The bovine FADD cDNA encodes a predicted 209 infection, growth media was supplemented with 2 lg/ml amino acid protein sharing 65 and 58% amino acid puromycin (Sigma Chemical Co., St Louis, MO, USA) to sequence identity with human and murine sequences, select for cells stably expressing the construct of interest. respectively. Flow cytometry demonstrated that 99% of the cells infec- For physical mapping of the bovine FADD gene, targeted ted with the control vector encoding enhanced green fluor- PCR amplification of a 490-bp region of putative exon 2 escent protein (EGFP) were positive for EGFP expression (based on homology with human genomic sequence) was following puromycin selection (data not shown). Western carried out using primers 5¢-CTGTGATAACGTGGGGAA blotting performed as previously described (Bannerman et al. GG-3¢ (sense) and 5¢-TGGAGGAGCTCGTTTAATGG-3¢ 2002) with 1:2000 diluted anti-FLAG antibody conjugated (antisense) to amplify DNA from 94 clone lines of a 3000- to horseradish peroxidase (Sigma Chemical Co., St Louis, rad bovine radiation hybrid (RH) panel (ResGen; Invitro- MO, USA) confirmed expression of both the FADD D/N gen). Amplification conditions for PCR were as follows: 10 (Fig. 1a) and FADD full-length constructs (Fig. 2a). cycles of 94 °C for 30 s, 65 °C for 30 s (-1 °C/cycle), 68 °C To determine whether bovine FADD participates in pro- for 2 min; then 30 cycles of 94 °C for 30 s, 55 °C for 30 s, apoptotic signaling, a truncated construct of bovine FADD, 68 °C for 2 min; with a final extension of 68 °C for 10 min. encoding solely the DD and that corresponds to a human The reaction products were scored by agarose gel electro- construct previously demonstrated to function in a D/N phoresis and concordancy data for each cell line were manner (Choi et al. 1998), was expressed in bovine EC. Cells submitted to the Roslin RH database (http://databases. expressing either the EGFP control vector or the FADD D/N 3roslin.ac.uk.radhyb) for two-point analysis using a modifi- were treated with 100 ng/ml of TNF-a or LPS for 8 or 12 h, cation of the RHmap Program (Lange et al. 1995). Using respectively, and assayed for caspase activity as previously this approach, FADD was mapped to BTA29 nearest marker described (Erwert et al. 2002). In those experiments where BMS1948 at approximately 61.6 cM (LODS ¼ 13.87, dis- TNF-a-induced apoptosis was assayed, cells were sensitized tance ¼ 0.16 cR3000). by co-incubation with 40 lg/ml of cycloheximide. All To overexpress a full-length or dominant-negative (D/N) experiments were performed in triplicate. Control EC construct of bovine FADD, PCR was used to generate expressing EGFP were sensitive to LPS- or TNF-a-induced products with compatable ends for insertion into the apoptosis as evidenced by increased caspase activation fol- pCR3.V64 Met FLAG vector (generous gift of Dr Jurg lowing treatment (Fig. 1b and c). In contrast, EC expressing Tschopp, Institute of Biochemistry of the University of the bovine FADD D/N construct were almost completely Lausanne, Switzerland) to yield coding regions with an resistant to LPS and TNF-a-induced apoptosis. These data initiator methionine, FLAG tag (DYKDDDDK), and a spacer demonstrate a role for bovine FADD in mediating LPS and of two amino acids (EF) before the start of the desired coding TNF-a pro-apoptotic signaling. region. Template for PCR was the cloned coding region of In addition to its role in promoting apoptosis, human bovine FADD. The primers used to generate full-length FADD has been reported to downregulate NF-jB, the latter FADD (AA 1–209) were 5¢ CCG GAA TTC ATG GAC CCG of which regulates the expression of several pro-inflamma- TTC CTG GTG CTG 3¢ and 5¢ G CTC TAG AGC GGC CGC CGC tory gene products (Bannerman et al. 2002). Specifically, TGG GTC GTC AGG AGG CT 3¢. The FADD D/N construct increased expression of full-length human FADD has been encoding just the death domain of bovine FADD (AA 91– shown to inhibit NF-jB-dependent gene expression. To 209) was amplified using 5¢ CCG GAA TTC GCG CCG GAG determine whether bovine FADD could similarly influence GAC CGA GAC CTG 3¢ and 5¢ G CTC TAG AGC GGC CGC NF-jB, EC overexpressing full-length bovine FADD were CGC TGG GTC GTC AGG AGG CT 3¢.
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