Scientific Report 2005

Immunology

Cellular Activation Mechanisms in the Innate Immune System

J. Chen, M. Cheung, Y. Kang, S. Lee, Y. Li, J. Mols, A. Seit-Nebi, M. Otsuka, C.-C. Wu, Y. Xu, T. Zarubin, H. Zhou, J. Han

Bacterial pathogens and inflammatory cytokines are important players in the innate immune response. Our interests are the signal transduction pathways activated by pathogens and cytokines. We study the signaling events at different cellular levels.

Signaling at the Cell Surface

Toll-like receptors (TLRs) are an important point of first contact between host and microbe. Once activated, the receptors generate signals that culminate in the induction of genes important for host defense. TLRs often need to cooperate with other proteins on the cell surface to function. For example, CD14 and MD2 are involved in TLR4-mediated activation by lipopolysaccharide.

We found that 4-1BB (CD137) ligand is another protein that interacts with TLRs on the cell surface. Unlike CD14 and MD2, 4-1BB ligand affects intracellular signaling rather than ligand recognition. 4-1BB ligand influences ligand-induced MAP kinase activation but has no effect on NF-κB activation. Studies are under way to define the mechanisms that selectively activate MAP kinase and NF-κB.

Intracellular Signaling

The p38 MAP kinase pathway is important in TLR-induced cellular changes. One of our interests is to determine how the signal is transduced from TLRs to the p38 pathway. We evaluated the contribution of MAP kinase kinase (MKK)–dependent and MKK-independent p38 activation. We found that both MKK-dependent and MKK-independent pathways contribute to p38 activation; the MKK-dependent pathway is the primary one in most situations. We also found that MKK-independent p38 activation can occur with or without dependence on TAB1, the activating protein for transforming growth factor-β–activated kinase 1. MyD88, TRIF, and TRAF6 are clearly the signaling molecules between the TLRs and p38 activation. However, which molecules link these adaptors and the p38 pathway is still not clear; we are identifying these proteins. We think that information on the proteins will bridge the gap in our knowledge of how the p38 pathway is activated by TLRs.

Regulation of Gene Expression

The p38 pathway plays a key role in posttranscriptional regulation of cytokine gene expression. We used Drosophila melanogaster S2 cells to screen a number of genes for their requirement in mRNA decay mediated by adenine-uridine–rich elements (AREs). We found that proteins in the Dicer and Ago families are required for ARE-mediated mRNA decay. We further confirmed the requirement of Dicer in mammalian cells. Because Dicer and Ago family members are required for processing microRNA, we evaluated whether any microRNA is required for ARE-mediated mRNA degradation. Sequence analysis revealed that miR16, a human microRNA containing a sequence complementary to the ARE sequence, has complementary sequence to miR16, and we confirmed that miR16 is required for the quick ARE-mediated decay of mRNA. Because miR16 and ARE can only form a maximum of 8 pairs, most likely miR16 alone is not sufficient to guide the targeting of AREs by the RNA-induced gene-silencing complex (RISC).

We hypothesized that ARE-binding proteins may have a role and further determined that the ARE-binding protein tristetraprolin is required for miR16 to target ARE-containing mRNA. Tristetraprolin does not directly interact with miR16, but via association with an Ago family member forms a complex with miR16.

Thus, the initiation of ARE-induced mRNA decay requires miR16-containing RISC and tristetraprolin. Currently, we are focusing on the signaling mechanism between the p38 pathway and the involvement of RISC and tristetraprolin in ARE-mediated mRNA decay.

Macrophage Death

The activation and life span of macrophages are important in innate immunity. Although macrophage death in vivo has not been well evaluated, it can be induced by different stimuli, such as a combination of cytokines and lipopolysaccharide or anthrax lethal toxin. We used 2 systems, macrophage death induced by lipopolysaccharide plus the caspase inhibitor vZAD and macrophage death induced by anthrax lethal toxin, to study the death process of macrophages.

Previously, we showed that induction of the orphan nuclear receptor Nur77 plays a role in macrophage death induced by lipopolysaccharide plus vZAD. We recently found that macrophage death induced by this combination had a phenotype of autophagy and depends on reactive oxygen species and poly(ADP-ribose) polymerase. We also found that macrophage death induced by lipopolysaccharide plus vZAD can be effectively blocked by serine protease inhibitors. Thus, multiple cellular events may be involved in this type of macrophage death. To study macrophage death induced by anthrax lethal toxin, we used random mutagenesis to identify genes that are required for lethal toxin–induced death. The identified genes are being characterized.

Publications

Cao, J., Semenova, M.M., Solovyan, V.T., Han, J., Coffey, E.T., Courtney, M.J. Distinct requirements for p38α and c-Jun N-terminal kinase stress-activated protein kinases in different forms of apoptotic neuronal death. J. Biol. Chem. 279:35903, 2004.

Jing, Q., Huang, S., Guth, S., Zarubin, T., Motoyama, A., Chen, J., Di Padova, F., Lin, S.-C., Gram, H., Han, J. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120:623, 2005.

Li, J., Li, Q., Xie, C., Zhou, H., Wang, Y., Zhang, N., Shao, H., Chan, S.C., Peng, X., Lin, S.-C., Han, J. β-Actin is required for mitochondria clustering and ROS generation in TNF-induced caspase-independent cell death. J. Cell Sci. 117(Pt. 20):4673, 2004.

Qi, X., Tang, J., Pramanik, R., Schultz, R.M., Shirasawa, S., Sasazuki, T., Han, J., Chen, G. p38 MAPK activation selectively induces cell death in K-ras-mutated human colon cancer cells through regulation of vitamin D receptor. J. Biol. Chem. 279:22138, 2004.

Rui, Y., Xu, Z., Lin, S., Li, Q., Rui, H., Luo, W., Zhou, H.M., Cheung, P.Y., Wu, Z., Ye, Z., Li, P., Han, J., Lin, S.C. Axin stimulates p53 functions by activation of HIPK2 kinase through multimeric complex formation. EMBO J. 23:4583, 2004.

Sakai, A., Han, J., Cato, A.C., Akira, S., Li, J.D. Glucocorticoids synergize with
IL-1β to induce TLR2 expression via MAP kinase phosphatase-1-dependent dual Inhibition of MAPK JNK and p38 in epithelial cells. BMC Mol. Biol. 5:2, 2004.

Shen, G., Hebbar, V., Nair, S., Xu, C., Li, W., Lin, W., Keum, Y.S., Han, J., Gallo, M.A., Kong, A.N. Regulation of Nrf2 transactivation domain activity: the differential effects of mitogen-activated protein kinase cascades and synergistic stimulatory effect of Raf and CREB-binding protein. J. Biol. Chem. 279:23052, 2004.