The authors have declared that no competing interests exist.
Performed the experiments: PM IS AF CR M. Pizzulo LED CDM GT TZ. Analyzed the data: PV M. Paolucci RS EV. Wrote the paper: PV RS.
The complexes formed by BCL10, MALT1 and specific members of the family of CARMA proteins (CBM complex), have recently focused much attention because they represent a central hub regulating activation of the transcription factor NF-κB following various cellular stimulations. In this manuscript, we report the functional characterization of a Danio rerio 241 amino acids polypeptide ortholog of the Caspase recruiting domain (CARD)-containing protein BCL10. Biochemical studies show that zebrafish Bcl10 (zBcl10) dimerizes and binds to components of the CBM complex. Fluorescence microscopy observations demonstrate that zBcl10 forms cytoplasmic filaments similar to that formed by human BCL10 (hBCL10). Functionally, in human cells zBcl10 is more effective in activating NF-κB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that zBcl10 can functionally replace the human protein. Finally, we show that the zebrafish cell line PAC2 is suitable to carry out reporter assays for monitoring the activation state of NF- kB transcription factor. In conclusion, this work shows that zebrafish may excellently serve as a model organism to study complex and intricate signal transduction pathways, such as those that control NF-κB activation.
NF-κB is an inducible and ubiquitously expressed transcription factor for genes involved in immune and inflammatory responses, cell survival, cell adhesion, differentiation, and growth [
Genetic disruption of the BCL10 locus in murine strains results in immunodeficiency, having these genetically modified mice profound defects in humoral and cellular immune responses [
The biological function of BCL10 is explicated through formation of the CBM complex, a molecular complex that includes one of three members of the family of CARMA proteins and the protein MALT1 [
Recently, extensive analysis of the zebrafish (
All the procedures involving animals were conducted as indicated in the Italian National Guidelines (D.L. No. 116 G.U., suppl. 40, 18.2.1992, circolare No. 8, G.U. July 1994) and in the appropriate European Directives (EEC Council Directive 86/609, 1.12.1987), adhering to the Guide for the Care and Use of Laboratory Animals (United States National Research Council, 1996). All the in vivo experimental activities were approved by the Animal Ethics Committee (CESA) of Biogem (Italy).
Total RNA samples were extracted from whole 6-day larvae using Trizol RNA isolation reagent (GIBCO-BRL) according to the manufacturer’s instructions. 1 μg of total RNA was primed with oligo(dT) and reverse-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions to generate a first-strand cDNA. Primers used to amplify zBcl10 were the following: forward 5’-ATGGATGTTACTCACCTG-3’ and reverse 5’-GACGTTTACGGAGACAAA-3’. PCR conditions were as follows: 98°C for 30 s, 30 cycles (98°C/5 s; 63°C/22 s; 72°C/30 s), and then 72°C for 5 min. Thermo Scientific® Phusion High-Fidelity DNA Polymerase (New England BioLabs) was used as amplifying polymerase, according to the manufacturer’s instructions. The RT-PCR product of the expected size was gel purified and cloned into pcDNA3 expression vector (Addgene), provided with an amino-terminal HA or FLAG epitope using standard cloning methodologies and confirmed by sequencing. Three independent clones deriving from three different PCR amplifications gave the same nucleotides sequence, corresponding to that deposited in GenBank with access number XM_002660692.
The expression vectors used in this study have been previously described [
The zBcl10 protein sequence was analyzed by using the BLAST algorithm at the NCBI web site (
HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. The PAC2 fibroblast line, isolated from 24-h post-fertilization zebrafish embryos, was kindly provided by Dr. Isidoro [
Short hairpin RNAs targeting hBCL10 were the following:
shBCL10 #2: 5’-CCACCAGATCTACAGTTAGAACTCGAGTTCTAACTGTAGATCTGGTGGC-3’
shBCL10 #3: 5’-CCTTAAGATCACGTACTGTTTCTCGAGAAACAGTACGTGATCTTAAGG-3’
shBCL10 #4: 5’-CTTGTCGAACATCAAGTAGAACTCGAGTTCTACTTGATGTTCGACAAG-3’
shBCL10 #5: 5’-GTTGAATCTATTCGGCGAGAACTCGAGTTCTCGCCGAATAGATTCAAC-3’
Retroviral infections were carried out as previously described [
To assess for NF-κB activation, HEK293 were co-transfected in 6-well plates with 0.2 μg of pNF-κB-luc (Clontech), 0.1 μg of pRSV-βGal (Addgene) plus each expression plasmid. When necessary, the total amount of transfected plasmidic DNA (2 μg) was kept constant by adding empty vector. pNF-κB-luc encodes the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the NFκB transcriptional response element. The plasmid RSV-βGal, expressing β-galactosidase, was added to the transfection mixture in order to normalize for the efficiency of transfection. After transfection and treatments, luciferase activity was determined with Luciferase Assay System (Promega). For measurement of β-galactosidase activity, 20 μl of cell lysates diluted 100-fold with 0.1 M potassium phosphate buffer was mixed with 200 μl of Galactone (Tropix, Bedford, MA, USA) that was diluted 100-fold with 0.1 M potassium phosphate and 1 mM magnesium chloride, pH 7.8, for 1 hr at room temperature. Then, β-galactosidase activity was measured after addition of 300 μl of Emerald (Tropix). Luciferase activity was normalized on β-galactosidase activity and expressed in arbitrary units.
Cell lysates were made in lysis buffer (150 mM NaCl, 20 mM Hepes, pH 7.4, 1% Triton X-100, 10% glycerol) and a mixture of proteases inhibitors (Protease Inhibitor Cocktail, Roche) according to the manufacturer’s instructions. Proteins were separated by SDS–PAGE, transferred onto nitrocellulose membrane, and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). Blots were developed using the ECL system (Amersham Biosciences). For co-immunoprecipitation experiments, cells were lysed in lysis buffer and immunocomplexes were bound to protein A/G (Amersham Biosciences) for 2 hrs at 4°C. immunocomplexes were extensively washed, resolved by SDS–PAGE, and analyzed by immunoblot assay. Sources of antisera and monoclonal antibodies were the following: anti-FLAG, anti-β-Actin, Sigma; anti-HA and anti-BCL10 (H-197
6–9 months male zebrafish were euthanized and dissected. Proteic extracts from selected organs were prepared using Nonidet P-40 lysis buffer (1% v/v) Nonidet P-40, 150 mm NaCl, 50 mm Hepes, pH 7.4, 5 mm EDTA, 10% (v/v) glycerol, and complete protease inhibitor mixture (Roche). After homogenization and centrifugation (13,000 × g, 15 min, 4°C), protein concentration of supernatant was determined by BCA protein assay (Pierce). A 15 μg sample of whole cell extract was separated on SDS–polyacrylamide gel and transferred to membranes. Filters were blocked for 2 hrs in 3% nonfat dry milk in phosphate-buffered saline (PBS) with 0.3% Tween 20. Western blot analysis was performed using a rabbit anti-BCL10 antisera, followed by horseradish-peroxidase-conjugated mouse anti-rabbit antibody (Amersham Biosciences). Signal was developed using an enhanced chemiluminescence method (Amersham Biosciences) according to the manufacturer’s instructions.
1 x 104 HEK293 were grown to 50% confluence and transfected in six-well chamber slides (Falcon). Sixteen hours after transfection, cells were fixed in 4% paraformaldehyde for 15 min at room temperature and then permeabilized in PBS/0.1% Triton X-100. Cells were incubated for 30 min in 5% FCS–PBS with anti-FLAG antibody (Sigma-Aldrich) followed by several washes with 5% FCS–PBS, and then incubating for 30 min with secondary antibody in 5% FCS–PBS. All steps were done at room temperature.
The determination and the analysis of the zebrafish genome revealed the existence, in zebrafish, of a sequence similar to that encoding for the human protein BCL10 [
A) Alignment of zBcl10 and human BCL10 with the consensus sequence generated by aligning the BCL10 sequences of fish, birds and reptiles, and mammals. The Xenopus tropicalis sequence is the only amphibian BCL10 sequence available. The alignment was done using ClustalW. Printout from multiple-aligned sequences and consensus sequences calculation were done with BOXSHADE. The black background designates identical amino acids, the gray background conservative substitutions. The red rectangles indicate amino acids conserved in all sequences examined. At the top of the alignment, the six alpha helix regions of the CARD are shown. The sequences used for alignment and generation of the consensus sequence are available in Supplementary Material. B) Phylogenetic tree analysis of BCL10 proteins. Phylogenetic analyses with bootstrapping (100 replicates) were obtained by the Neighbor-joining method using complete deletion and the p-distance amino acid model in MEGA [
zBcl10 | 241 | 112/241 (46%) | 178/241 (74%) | 28/241 (12%) | |
hBCL10 | 233 | ||||
zBcl10 6–113 | 108 | 67/108 (62%) | 90/108 (83%) | 0/108 (0%) | |
hBCL10 8–115 | 108 |
When analyzed in immunoblot assay, HA-tagged zBcl10 expressed in mammalian cells migrates as a 35 kDa protein (
A) Immunoblot analysis of lysates from HEK293 cells transfected with and expression vector empty (
In mammals, BCL10 has an essential role in the signal transduction pathway that leads to activation of the transcription factor NF-κB [
A) HEK293 cells were transiently cotransfected with FLAG-tagged and HA-tagged version of zBcl10 and hBCL10 or empty vector (
Next, we tested whether zBcl10 is able to activate NF-κB in mammalian cells using an NF-κB luciferase-based reporter plasmid, in which the firefly luciferase reporter gene is placed under the control of a minimal (m)CMV promoter and tandem repeats of the NFκB transcriptional response element (see
A) HEK293 cells were transiently cotransfected with an expression vector empy (
To exclude the possibility that NF-κB activation mediated by zBcl10 was due to its interaction and subsequent oligomerization of hBCL10, we abolished expression of hBCL10 in the human cell line HEK293 through retrovirus-mediated trasduction of short hairpin RNAs (shRNA) targeting hBCL10. As shown in
A) HEK293 cells were infected with recombinant retrovirus expressing shRNAs targeting hBCL10. 72 hrs later, hBCL10 expression was monitored by immunoblot assay. B) NF-κB-driven luciferase activity in HEK293 cells silenced for hBCL10 and stimulated with PMA. C) NF-κB-driven luciferase activity in HEK293 cells silenced for hBCL10 and transfected with zBcl10.
Fluorescence microscopy experiments and structural studies have shown that the NF-κB activity produced by hBCL10 is regulated through formation of cytosolic filamentous structures [
A) HEK293 cells were transfected with mammalian FLAG-tagged vector, empty (vector) or expressing zBcl10. 16 hrs after transfection, cells were stained with anti-FLAG mAb, followed by FITC-conjugated anti-mouse IgG. B) The same experiment described in A) was carried out in HEK293 cells infected with recombinant retrovirus expressing a shRNA targeting hBCL10. All photographs were taken at a 100X magnification.
Finally, we examined whether zBcl10 is able to activate NF-κB in zebrafish cells. For this, we used the the same NF-κB-luciferase assay we have used for mammalian cells (Figs.
A) PAC2 cells were transiently cotransfected with expression vectors encoding for the indicated polypeptides, together with pNF-κB-luc and pRSV-βgal reporter vectors. The total amount of transfected plasmidic DNA was maintained constant by adding empty vector. 24 hrs after transfection, cell lysates were prepared and luciferase activity was measured. Data shown (mean + SEM, n = 9) represent relative luciferase activity normalized on β-galactosidase activity and is representative of six independent experiments done in triplicate. Statistical analysis was performed by Student's t test; a p value of <0.05 was considered significant, and is indicated with the symbol *. B, C) HEK293 and PAC2 cells were transiently cotransfected with expression vectors encoding for wt CARMA2
There are several reasons that make particularly interesting the work here presented. The first one is represented by the possibility of using zebrafish as a model system to study the signal transduction pathways that modulate the activation state of the transcription factor NF-κB. Given the importance of this transcription factor in both normal cell biology and autoimmune, immunoproliferative and tumoral disorders, the possibility of using a model organism so flexible and informative such as zebrafish, certainly represents a field to massively explore further. This possibility is further supported by the extensive genomic knowledge on zebrafish we are acquiring in these days. Recently, one of these studies has revealed the presence of genes encoding for several putative CARD-containing proteins in the zebrafish genome, including the three CARMA proteins [
Secondly, it is interesting to note that the serine residues with inhibitory function present in hBCL10 (S136, S141 and S144) [
Finally, the data presented here demonstrate a perfect conservation of the mechanisms through which BCL10 regulates the activation of NF-κB state, and thus concretely open the possibility of using zebrafish as a model system. Regarding this, it is certainly intriguing to note that the ubiquitination mechanisms required by hBCL10 to activate NF-κB are as well conserved in zBcl10 (
In summary, the present study has clearly demonstrated that the NF-κB signalling pathways regulated by BCL10 are conserved among vertebrates. Data presented will support further investigation of this intricate and fascinating pathway in zebrafish, which could represent a valuable
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