Recombinant TAT-CD137 Ligand Cytoplasmic Domain Fusion Protein Induces the Production of IL-6 and TNF-α in Peritoneal Macrophages.

BACKGROUND: The ligand for CD137 (CD137L; also called 4-1BBL) is mainly expressed on activated APCs such as dendritic cells, B cells and macrophages. Even though CD137L functions as a trigger of the CD137 signaling pathway for T cell activation and expansion, engagement of CD137L can deliver a signal leading to the production of proinflammatory cytokines in macrophages. METHODS: We generated cell-permeable TAT-CD137L cytoplasmic domain fusion protein (TAT-CD137Lct) and examined its ability to initiate the CD137L reverse signaling pathway. RESULTS: Treatment of TAT-CD137Lct induced the production of high levels of IL-6 and TNF-α mRNAs and proteins in peritoneal macrophages. TAT-CD137Lct increased phosphorylation of Erk, p38 MAPK and Jnk, and activated transcription factors C/EBP and CREB. However, TAT-CD137Lct did not visibly affect the degradation of the inhibitor of NF-kB (IkBα). We further demonstrated that JNK activation was required for TAT-CD137Lct-induced production of TNF-α, while activation of Erk and p38 MAPK were involved in IL-6 and TNF-α production. CONCLUSION: Our results suggest that TAT-CD137Lct is an effective activator for the CD137L reverse signaling pathway.

A novel mouse PKCδ splice variant, PKCδIX, inhibits etoposide-induced apoptosis.

Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδin vivo.