PMC:3148254 / 4668-13422 JSON TXT

Annnotations TAB JSON ListView MergeView

bionlp-st-ge-2016-reference

bionlp-st-ge-2016-reference-tees

events-check-again

pmc-enju-pas

2_test

{"project":"2_test","denotations":[{"id":"21829693-20614026-89653758","span":{"begin":456,"end":457},"obj":"20614026"},{"id":"21829693-12958312-89653759","span":{"begin":1272,"end":1273},"obj":"12958312"},{"id":"21829693-17878362-89653760","span":{"begin":1277,"end":1278},"obj":"17878362"},{"id":"21829693-14981114-89653761","span":{"begin":1282,"end":1283},"obj":"14981114"},{"id":"21829693-7681471-89653762","span":{"begin":4311,"end":4313},"obj":"7681471"},{"id":"21829693-7681587-89653763","span":{"begin":5695,"end":5697},"obj":"7681587"},{"id":"21829693-1701824-89653764","span":{"begin":5701,"end":5703},"obj":"1701824"},{"id":"21829693-12949496-89653765","span":{"begin":6185,"end":6187},"obj":"12949496"},{"id":"21829693-12958312-89653766","span":{"begin":8180,"end":8181},"obj":"12958312"},{"id":"21829693-17878362-89653767","span":{"begin":8185,"end":8186},"obj":"17878362"},{"id":"21829693-17633023-89653768","span":{"begin":8306,"end":8307},"obj":"17633023"},{"id":"T45718","span":{"begin":456,"end":457},"obj":"20614026"},{"id":"T92919","span":{"begin":1272,"end":1273},"obj":"12958312"},{"id":"T70074","span":{"begin":1277,"end":1278},"obj":"17878362"},{"id":"T10373","span":{"begin":1282,"end":1283},"obj":"14981114"},{"id":"T72061","span":{"begin":4311,"end":4313},"obj":"7681471"},{"id":"T18517","span":{"begin":5695,"end":5697},"obj":"7681587"},{"id":"T73302","span":{"begin":5701,"end":5703},"obj":"1701824"},{"id":"T513","span":{"begin":6185,"end":6187},"obj":"12949496"},{"id":"T39552","span":{"begin":8180,"end":8181},"obj":"12958312"},{"id":"T34158","span":{"begin":8185,"end":8186},"obj":"17878362"},{"id":"T9898","span":{"begin":8306,"end":8307},"obj":"17633023"}],"text":"IκB kinase (IKK) complex with CD40 was undetectable in HOIP-deficient cells. Together, our results indicate that HOIP plays a critical role in the activation of signaling pathways that regulate cellular responses to CD40 engagement.\n\nResults\n\nGeneration of HOIP-deficient B cells via targeted disruption of Rnf31, the gene encoding HOIP\nIn our previous study, we demonstrated that HOIP is recruited to the CD40 signaling complex in two mouse B cell lines [6]. Similar results were obtained with mouse splenocytes, indicating that the interaction of CD40 with HOIP is not limited to transformed B cell lines (unpublished observations). We also found that the recruitment of HOIP to CD40 was TRAF2-dependent and that overexpression of a truncated HOIP mutant partially inhibited CD40-mediated NF-κB activation. These results support the hypothesis that HOIP plays a role in CD40 signal transduction. To further define the role and evaluate the importance of HOIP in CD40 signaling, we used somatic cell gene targeting to disrupt the gene encoding HOIP in the mouse B cell line A20.2J. This cell line has been particularly useful in the characterization of CD40 signaling mechanisms due to the relative ease with which its genome can be modified by homologous recombination [7], [8], [9]. We used a targeting vector capable of undergoing homologous recombination with Rnf31 (the gene encoding HOIP) to disrupt the coding sequence of the gene in exon 5 (Fig. 1A). Following introduction of the vector, the neomycin-resistant clones that arose were screened by PCR amplification of genomic DNA to identify cells containing a disrupted Rnf31 allele. To remove the selectable marker gene cassette from the disrupted Rnf31 allele, recombinant cell lines were transiently transfected with a plasmid that encodes Cre recombinase. This step allowed us to perform a second round of targeting and drug selection, generating cells in which both copies of Rnf31 were disrupted. Two independent clonal cell lines were chosen for further analysis. HOIP protein expression was undetectable in both cell lines, as determined by Western blot analysis of cell lysates (Fig. 1B), demonstrating that the targeting process was successful. In the text that follows, the two gene-targeted cell lines are referred to as HOIP-deficient cells.\n10.1371/journal.pone.0023061.g001 Figure 1 HOIP gene targeting.\n(A) Regions of Rnf31 gene sequence (bottom) used in the HOIP gene targeting vector (top) are shown. Arrows (F and R) indicate approximate positions of sequences homologous to oligonucleotides used for PCR-mediated detection of homologous recombination. Homologous recombination of the vector with Rnf31 resulted in a small chromosomal deletion and the in-frame insertion of neomycin phosphotransferase (NeoR) into the amino-terminal HOIP coding sequence. A diphtheria toxin (DT) cassette in the vector facilitates the negative selection of cells in which random chromosomal insertion of the vector takes place. LoxP sequences allow the Cre-mediated deletion of the NeoR coding sequence. The SV40pA sequence helps to ensure disruption of gene expression after deletion of the NeoR sequence. (B) Anti-HOIP and anti-FLAG Western blots of cell lysates from A20.2J cells and HOIP-deficient (HOIP-/-) clones. A partial decrease in HOIP protein expression is evident in cells following disruption of one copy of Rnf31 (HOIP−/+). HOIP expression in clones reconstituted with an empty retroviral vector (pMIP) or a retroviral vector encoding FLAG-tagged HOIP is also shown. Approximate molecular weight of HOIP is 120 kD. To enable us to confirm that any signaling or functional defects observed in HOIP-deficient cells were due to disruption of the Rnf31 gene, both HOIP-deficient cell lines were transduced with a retrovirus encoding FLAG-tagged wild-type HOIP, thus restoring HOIP protein expression (Fig. 1B). These cells are referred to as HOIP-reconstituted cells. In the experiments that follow, responses by these cells were compared to those of A20.2J cells and HOIP-deficient cells transduced with a retroviral vector (pMIP) lacking a cDNA insert (empty vector).\n\nHOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nHOIP mediates CD40-stimulated NF-κB and JNK activation\nThe results described above show that HOIP plays an important role in CD40-mediated effector functions of B cells. It follows that HOIP is likely a key mediator of CD40 signaling. To test this hypothesis, we stimulated A20.2J and HOIP-deficient cells with CD154 (CD40 ligand) expressed by HI5 insect cells [7], [8] and measured activation of the NF-κB and JNK pathways, two of the major transcriptional regulators activated by CD40 [4]. Cell-associated CD154 was used as the stimulus in these experiments as it typically provides more robust, and therefore more readily detected, activation signals than does anti-CD40 antibody. Activation of the canonical NF-κB pathway is initiated with the phosphorylation of IκB proteins by the IκB kinase complex (IKK). In resting cells, IκB proteins are responsible for sequestering NF-κB subunits in the cytoplasm. Phosphorylation by the IKK"}

bionlp-st-ge-2016-spacy-parsed

GO-BP

GO-MF

GO-CC

sentences

ICD10

{"project":"ICD10","denotations":[{"id":"T15255","span":{"begin":2837,"end":2847},"obj":"http://purl.bioontology.org/ontology/ICD10/A36.9"},{"id":"T15256","span":{"begin":2837,"end":2847},"obj":"http://purl.bioontology.org/ontology/ICD10/A36"}],"text":"IκB kinase (IKK) complex with CD40 was undetectable in HOIP-deficient cells. Together, our results indicate that HOIP plays a critical role in the activation of signaling pathways that regulate cellular responses to CD40 engagement.\n\nResults\n\nGeneration of HOIP-deficient B cells via targeted disruption of Rnf31, the gene encoding HOIP\nIn our previous study, we demonstrated that HOIP is recruited to the CD40 signaling complex in two mouse B cell lines [6]. Similar results were obtained with mouse splenocytes, indicating that the interaction of CD40 with HOIP is not limited to transformed B cell lines (unpublished observations). We also found that the recruitment of HOIP to CD40 was TRAF2-dependent and that overexpression of a truncated HOIP mutant partially inhibited CD40-mediated NF-κB activation. These results support the hypothesis that HOIP plays a role in CD40 signal transduction. To further define the role and evaluate the importance of HOIP in CD40 signaling, we used somatic cell gene targeting to disrupt the gene encoding HOIP in the mouse B cell line A20.2J. This cell line has been particularly useful in the characterization of CD40 signaling mechanisms due to the relative ease with which its genome can be modified by homologous recombination [7], [8], [9]. We used a targeting vector capable of undergoing homologous recombination with Rnf31 (the gene encoding HOIP) to disrupt the coding sequence of the gene in exon 5 (Fig. 1A). Following introduction of the vector, the neomycin-resistant clones that arose were screened by PCR amplification of genomic DNA to identify cells containing a disrupted Rnf31 allele. To remove the selectable marker gene cassette from the disrupted Rnf31 allele, recombinant cell lines were transiently transfected with a plasmid that encodes Cre recombinase. This step allowed us to perform a second round of targeting and drug selection, generating cells in which both copies of Rnf31 were disrupted. Two independent clonal cell lines were chosen for further analysis. HOIP protein expression was undetectable in both cell lines, as determined by Western blot analysis of cell lysates (Fig. 1B), demonstrating that the targeting process was successful. In the text that follows, the two gene-targeted cell lines are referred to as HOIP-deficient cells.\n10.1371/journal.pone.0023061.g001 Figure 1 HOIP gene targeting.\n(A) Regions of Rnf31 gene sequence (bottom) used in the HOIP gene targeting vector (top) are shown. Arrows (F and R) indicate approximate positions of sequences homologous to oligonucleotides used for PCR-mediated detection of homologous recombination. Homologous recombination of the vector with Rnf31 resulted in a small chromosomal deletion and the in-frame insertion of neomycin phosphotransferase (NeoR) into the amino-terminal HOIP coding sequence. A diphtheria toxin (DT) cassette in the vector facilitates the negative selection of cells in which random chromosomal insertion of the vector takes place. LoxP sequences allow the Cre-mediated deletion of the NeoR coding sequence. The SV40pA sequence helps to ensure disruption of gene expression after deletion of the NeoR sequence. (B) Anti-HOIP and anti-FLAG Western blots of cell lysates from A20.2J cells and HOIP-deficient (HOIP-/-) clones. A partial decrease in HOIP protein expression is evident in cells following disruption of one copy of Rnf31 (HOIP−/+). HOIP expression in clones reconstituted with an empty retroviral vector (pMIP) or a retroviral vector encoding FLAG-tagged HOIP is also shown. Approximate molecular weight of HOIP is 120 kD. To enable us to confirm that any signaling or functional defects observed in HOIP-deficient cells were due to disruption of the Rnf31 gene, both HOIP-deficient cell lines were transduced with a retrovirus encoding FLAG-tagged wild-type HOIP, thus restoring HOIP protein expression (Fig. 1B). These cells are referred to as HOIP-reconstituted cells. In the experiments that follow, responses by these cells were compared to those of A20.2J cells and HOIP-deficient cells transduced with a retroviral vector (pMIP) lacking a cDNA insert (empty vector).\n\nHOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription\nEngagement of CD40 on B cells upregulates expression of CD80 [10], a cell surface protein that promotes activation of T cells interacting with B cells and other APC. To begin evaluating potential contributions of HOIP to CD40 signaling, we assayed the upregulation of CD80 induced by engagement of CD40 on parental A20.2J, HOIP-deficient, and HOIP-reconstituted cells (Fig. 2). Compared to the parental cell line, the CD40-stimulated upregulation of CD80 was dramatically reduced in all HOIP-deficient cells tested. In contrast, upregulation of CD80 was restored in HOIP-reconstituted cells, confirming that the defect in CD80 upregulation in the HOIP-deficient cells was due to the disruption of Rnf31.\n10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells.\nCells were incubated for 72 hrs with an isotype control antibody (left) or anti-CD40 antibody (right) and then stained for expression of CD80 (filled profiles indicate staining with anti-CD80 antibody; open profiles are staining with an isotype control antibody). Transduction of both HOIP-deficient (HOIP-/-) clones with a retrovirus encoding FLAG-HOIP reversed defective CD80 upregulation, while transduction with an empty vector (pMIP) did not. Similar results were obtained in a second experiment. CD40 signals in B cells can also contribute to the activation of DNA recombination in the immunoglobulin heavy chain locus [11], [12]. This process, known as immunoglobulin class switching (or isotype switching), allows B cells, initially expressing IgM, to switch to IgA, IgG, or IgE depending on the class of antibody most appropriate for a particular immune response. Switching to IgE can be induced by the combination of IL-4 and CD40 signals. The gene rearrangement necessary for IgE production is preceded by the production of non-coding RNA transcripts from germline sequences in the IgE heavy chain locus [13]. To determine whether HOIP is required in the initiation of the class switching process, the relevant B cell lines were stimulated through CD40 in the presence of IL-4 and tested for production of germline epsilon (GLε) transcripts (Fig. 3). Anti-CD40 antibody stimulation alone or together with IL-4 induced GLε transcription in parental A20.2J cells as expected. In contrast, only low levels of GLε expression were detected in HOIP-deficient cells stimulated with either anti-CD40 antibody alone or anti-CD40 and IL-4. Normal levels of GLε expression were induced in HOIP-reconstituted cells stimulated with anti-CD40 antibody alone or together with IL-4. Collectively, these data demonstrate that HOIP has a critical role in cellular responses to CD40 signaling.\n10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells.\nA20.2J and HOIP-deficient cells transduced with an empty retroviral vector (A20.2J + pMIP and HOIP-/- + pMIP, respectively), or HOIP-deficient cells transduced with a HOIP-encoding retrovirus (HOIP-/- + HOIP) were cultured overnight with agonistic anti-CD40 or an isotype control antibody (iso), with or without IL-4. RNA was isolated from the cells, reverse-transcribed, and then subjected to quantitative PCR to determine levels of GLε transcripts. Results were normalized to the levels of Hprt1 transcripts in each sample. Symbols indicate the values from duplicate cultures (a line indicates the mean of the two values). Similar results were obtained in a second experiment and in an additional experiment with a second HOIP-deficient clone.\n\nHOIP mediates CD40-stimulated NF-κB and JNK activation\nThe results described above show that HOIP plays an important role in CD40-mediated effector functions of B cells. It follows that HOIP is likely a key mediator of CD40 signaling. To test this hypothesis, we stimulated A20.2J and HOIP-deficient cells with CD154 (CD40 ligand) expressed by HI5 insect cells [7], [8] and measured activation of the NF-κB and JNK pathways, two of the major transcriptional regulators activated by CD40 [4]. Cell-associated CD154 was used as the stimulus in these experiments as it typically provides more robust, and therefore more readily detected, activation signals than does anti-CD40 antibody. Activation of the canonical NF-κB pathway is initiated with the phosphorylation of IκB proteins by the IκB kinase complex (IKK). In resting cells, IκB proteins are responsible for sequestering NF-κB subunits in the cytoplasm. Phosphorylation by the IKK"}

bionlp-st-ge-2016-uniprot

test2

PMC:3148254 / 4668-13422 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:3148254 / 4668-13422 JSON TXT