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 complex targets IκB proteins for ubiquitination and degradation, allowing NF-κB to enter the nucleus and activate gene expression. CD40-mediated phosphorylation and degradation of IκBα in HOIP-deficient cells was dramatically impaired relative to that observed in parental A20.2J cells (Fig. 4). We also assayed activation of the stress-activated protein kinase JNK in response to CD40 engagement (Fig. 4). CD40-mediated JNK activation in HOIP-deficient cells was impaired as measured by phosphorylation of Thr183 and Tyr185 in JNK. CD40-induced activation of NF-κB and JNK in HOIP-reconstituted cells was normal, demonstrating that the defects observed in gene-deficient cells were due to the absence of HOIP expression.
10.1371/journal.pone.0023061.g004 Figure 4  CD40-induced activation of NF-κB and JNK is defective in HOIP-deficient cells.
CD40-mediated signaling was defective in HOIP-deficient cells (HOIP-/- + pMIP), but intact in parental cells transduced with an empty retroviral vector (A20.2J + pMIP) and in HOIP-deficient cells transduced with a retroviral vector encoding HOIP (HOIP-/- + HOIP). Cells were activated with CD154-expressing insect cells for the times indicated. As a negative control, cells were stimulated with insect cells lacking CD154 (5 minute time point only). Western blots of whole-cell lysates were probed with the indicated antibodies. Phospho-IκBα (pIκBα) and phospho-JNK (pJNK) blots were stripped and reprobed for total IκBα and total JNK, respectively (anti-JNK antibodies recognize p46 and p54 isoforms). IκBα blots were also reprobed for actin to demonstrate equal lane loading. Molecular weights are indicated at right. Similar results were obtained in a second experiment and in two additional experiments with a second HOIP-deficient clone.

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