UC San Diego

A key component of the innate immune system is the Toll- like Receptors (TLR), which identify invading microorganisms by recognizing pathogen associated molecular patterns. Toll like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) that is expressed on the cell wall of gram negative bacteria. TLR4 is unique in utilizing four adaptor proteins to specify the downstream transcriptional events. The MyD88 dependent pathway utilizes both MyD88 and TIRAP (TIR-associated protein) which lead to NF-[kappa]B activation. This pathway is critical in mediating the induction of pro- inflammatory cytokines. The MyD88 independent pathway signals though TRIF (TIR-doimain-containing adaptor protein-inducing IFN- [beta];) and TRAM (TRIF-related adaptor molecule) which activate IKK[epsilon]/TBK1 and this lead to Interferon regulatory factor 3 (IRF-3) activation. IRF-3 plays a pivotal role in up-regulating interferon stimulated genes in response to bacterial, viral infection and DNA damaging agents. In the present study we show that interferon stimulated gene 54 (ISG54) induction by LPS, which occurs in an IRF-3-dependent manner, requires the generation of ROS by the NADPH oxidase 4 (NOX4). Subsequent activation of ASK1 links LPS-induced ROS production to the activation of MKK6 and p38, two kinases that were previously identified as components of the LPS-induced IRF-3 activation cascade. Production of ROS occurs frequently concomitant with an increase in cytosolic calcium. Here, we reported that calcium mediates IRF-3 activation in response to LPS. In most eukaryotic cells, the release of calcium from the endoplasmic/sarcoplasmic to the cytosol is mediated by two receptors: inositol-1,4,5-triphosphate receptors (IP₃R) and Ryanodine receptors (RYRs).Phospholipase C gamma 2 (PLC[gamma]2) specifically mediates the cleavage of PIP₂ into IP₃ which regulates the release of calcium from ER into cytosol through the IP₃Rs. The calcium dependence is specific to LPS mediated ISG54 induction, and is not common to ISG54 induction by IFN[beta]. The expressions of two other IRF3 dependent genes, TRAIL and RANTES, were also abrogated at the presence of PLC[gamma]2 specific siRNA and the IP₃R antagonist. However, the expression of NF[kappa]B dependent genes such as TNF[alpha] and I[kappa]B remained intact. This further suggests that calcium is required for MyD88 independent but not MyD88 dependent pathway