PubAnnotation

http://purl.obolibrary.org/obo/GO_0004842

denotes

E3

T2391

http://purl.obolibrary.org/obo/GO_0019787

denotes

E3

T2392

http://purl.obolibrary.org/obo/GO_0061630

denotes

E3

T2393

http://purl.obolibrary.org/obo/GO_0061659

denotes

E3

T2394

http://purl.obolibrary.org/obo/GO_0061660

denotes

E3

T2395

http://purl.obolibrary.org/obo/GO_0061661

denotes

E3

T2396

http://purl.obolibrary.org/obo/GO_0061662

denotes

E3

T2397

http://purl.obolibrary.org/obo/GO_0061663

denotes

E3

T2398

http://purl.obolibrary.org/obo/GO_0061664

denotes

E3

T2399

http://purl.obolibrary.org/obo/GO_0061665

denotes

E3

T2400

http://purl.obolibrary.org/obo/GO_0061666

denotes

E3

T2401

http://purl.obolibrary.org/obo/GO_0061667

denotes

E3

T2402

3126-3135

http://purl.obolibrary.org/obo/GO_0031386

denotes

ubiquitin

T2403

3485-3494

http://purl.obolibrary.org/obo/GO_0031386

denotes

ubiquitin

T2404

3660-3668

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T2407

4235-4239

http://purl.obolibrary.org/obo/GO_0005136

denotes

IL-4

T2405

4291-4305

http://purl.obolibrary.org/obo/GO_0003823

denotes

immunoglobulin

T2406

4356-4370

http://purl.obolibrary.org/obo/GO_0003823

denotes

immunoglobulin

T2408

4516-4547

http://purl.obolibrary.org/obo/GO_0016909

denotes

stress-activated protein kinase

T2389

4523-4547

http://purl.obolibrary.org/obo/GO_0030295

denotes

activated protein kinase

T2409

4533-4549

http://purl.obolibrary.org/obo/GO_0004697

denotes

protein kinase c

T2410

4533-4549

http://purl.obolibrary.org/obo/GO_0005080

denotes

protein kinase c

T2411

4562-4565

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T8304

4668-13422

http://purl.obolibrary.org/obo/GO_0008384

denotes

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. Results Generation of HOIP-deficient B cells via targeted disruption of Rnf31, the gene encoding HOIP In 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with mouse splenocytes, indicating that the interaction of CD40 with HOIP is not limited to transformed B cell lines (unpublished observations).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. 10.1371/journal.pone.0023061.g001 Figure 1 HOIP gene targeting. (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). HOIP is required for CD40-mediated CD80 upregulation and activation of germline epsilon transcription Engagement 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. 10.1371/journal.pone.0023061.g002 Figure 2 CD40-mediated CD80 upregulation is defective in HOIP-deficient cells. Cells 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. 10.1371/journal.pone.0023061.g003 Figure 3 GLε transcription is defective in CD40-stimulated HOIP-deficient cells. A20.2J 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. HOIP mediates CD40-stimulated NF-κB and JNK activation The 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

T2412

4680-4683

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T15597

9791-9799

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T15598

9820-9828

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T15599

9927-9935

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T15600

9988-9996

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T5086

10329-10343

http://purl.obolibrary.org/obo/GO_0003823

denotes

immunoglobulin

T5087

10397-10411

http://purl.obolibrary.org/obo/GO_0003823

denotes

immunoglobulin

T5088

10550-10558

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T5092

10664-10668

http://purl.obolibrary.org/obo/GO_0005136

denotes

IL-4

T5093

11020-11024

http://purl.obolibrary.org/obo/GO_0005136

denotes

IL-4

T5089

11109-11117

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T5094

11153-11157

http://purl.obolibrary.org/obo/GO_0005136

denotes

IL-4

T5090

11340-11348

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T5095

11372-11376

http://purl.obolibrary.org/obo/GO_0005136

denotes

IL-4

T5091

11477-11485

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T5096

11509-11513

http://purl.obolibrary.org/obo/GO_0005136

denotes

IL-4

T16042

12019-12027

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T16043

12051-12055

http://purl.obolibrary.org/obo/GO_0005136

denotes

IL-4

T6123

12526-12529

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T6129

12809-12815

http://purl.obolibrary.org/obo/GO_0005488

denotes

ligand

T6124

12897-12900

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T6130

13160-13168

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T6131

13293-13296

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T6132

13419-13422

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T6133

13760-13784

http://purl.obolibrary.org/obo/GO_0030295

denotes

activated protein kinase

T6125

13785-13788

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T6126

13844-13847

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T6127

13951-13954

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T6128

13993-13996

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T16571

14226-14229

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T16575

14785-14795

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibodies

T16572

14830-14833

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T16573

14899-14902

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T16574

14923-14926

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T16576

14927-14937

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibodies

T8306

16201-16209

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T8307

16476-16484

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T17753

16975-16978

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T17755

17532-17540

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T17756

18131-18139

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T17757

18163-18171

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T17754

18327-18330

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T17758

18971-18981

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibodies

T17759

19081-19092

http://purl.obolibrary.org/obo/GO_0016791

denotes

phosphatase

T17760

19176-19187

http://purl.obolibrary.org/obo/GO_0016791

denotes

phosphatase

T8305

20302-20305

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T8308

21102-21110

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T8309

21441-21452

http://purl.obolibrary.org/obo/GO_0016791

denotes

phosphatase

T8310

21769-21780

http://purl.obolibrary.org/obo/GO_0016791

denotes

phosphatase

T11345

22365-22379

http://purl.obolibrary.org/obo/GO_0003823

denotes

immunoglobulin

T11346

22450-22458

http://purl.obolibrary.org/obo/GO_0003823

denotes

antibody

T11347

22663-22666

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T11351

22844-22847

http://purl.obolibrary.org/obo/GO_0004705

denotes

JNK

T11348

24183-24186

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T11349

24332-24335

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T11350

24420-24423

http://purl.obolibrary.org/obo/GO_0008384

denotes

IKK

T11355

24454-24463

http://purl.obolibrary.org/obo/GO_0031386

denotes

ubiquitin

T11357

http://purl.obolibrary.org/obo/GO_0004842

denotes

ubiquitin ligase activity

T11359

http://purl.obolibrary.org/obo/GO_0050372

denotes

ubiquitin ligase activity

T11361

http://purl.obolibrary.org/obo/GO_1990757

denotes

ubiquitin ligase activity

T11363

http://purl.obolibrary.org/obo/GO_0061630

denotes

ubiquitin ligase activity

T11365

http://purl.obolibrary.org/obo/GO_0034450

denotes

ubiquitin ligase activity

T11367

24464-24479

http://purl.obolibrary.org/obo/GO_0016874

denotes

ligase activity

T11369

24572-24579

http://purl.obolibrary.org/obo/GO_0005488

denotes

binding

T11372

24572-24600

http://purl.obolibrary.org/obo/GO_1990450

denotes

binding linear polyubiquitin

T11356

25108-25117

http://purl.obolibrary.org/obo/GO_0031386

denotes

ubiquitin

T11358

http://purl.obolibrary.org/obo/GO_0004842

denotes

ubiquitin ligase activity

T11360

http://purl.obolibrary.org/obo/GO_0050372

denotes

ubiquitin ligase activity

T11362

http://purl.obolibrary.org/obo/GO_1990757

denotes

ubiquitin ligase activity

T11364

http://purl.obolibrary.org/obo/GO_0061630

denotes

ubiquitin ligase activity

T11366