LPS Stimulated MAPK Signaling T L R 4 LBP CD14 Rac1 Review: Endotoxin LPS (Lipopolysaccharide) is a component of the outer membrane of Gram-negative bacteria that potently promotes the activation of macrophages and microglia cells, which are important sensors of infection by bacteria, fungi, and viruses, in both the periphery and the CNS (Central Nervous System). There are several known LBP (LPS-Binding Proteins) present on macrophage membranes, including CD14, CD11b/18, and TLR (Toll-like Receptors) (Ref.1). TLRs are mammalian homologs of the Drosophila Toll receptor and are involved in initiating innate immune defense against bacteria and fungi. CD14 lacks a transmembrane domain and is unable to initiate signals on its own. Thus, the TLR4 has emerged as a potential signaling partner for LPS/CD14 interactions. The key downstream pathway for LPS-induced signaling events is the MAPK (Mitogen Activated Protein Kinase) cascade that leads to several functional responses. Although distinct in their activation, there is considerable co-operation between these kinases and many substrates are shared between pathways (Ref.2). The MAPK family is composed of the ERK1/2 (Extracellular Signal-Regulated Kinase), p38 and SAPK (Stress-Activated Protein Kinase)/JNK (Jun N-terminal kinase) pathways. Three isoforms of JNK exist (JNK1, JNK2, and JNK3), and each has multiple alternatively spliced subtypes. The primary substrates for JNK are c-Jun, c-Fos and ATF2 (Activating Transcription Factor-2), which induce gene expression by binding to the Activator Protein-1 binding site in the promoters. MEKK1/4 (MAP/ERK Kinase Kinase) or Raf activates (through phosphorylation) a MEK1, 2 (MAP/ERK Kinase) or MKK3/4/7 (MAP Kinase Kinase), which in turn phosphorylates a specific tyrosine and threonine residue on a MAPK (Ref.3) At least three members of the MKK superfamily are capable of activating p38 MAPK. When over expressed in transfected cell lines MKK3, MKK4, and MKK6 activate p38 MAPK and MKK7 activates JNK. This family of kinases is important in a wide spectrum of cell functions including proliferation, apoptosis, cytokine biosynthesis, and cytoskeletal reorganization (Ref.4). The activated MAPK are responsible for phosphorylating and activating numerous transcription factors which function to stimulate the synthesis of various inflammatory proteins including the cytokines TNF-a, IL-1b and IL-6. The MAPK are also involved in the transcriptional regulation of NOS2 (Nitric Oxide Synthase-2) and COX2 (Cyclooxygenase-2) (Ref.5). LPS-mediated signaling also activate various small molecular weight GTPases including Rac, CDC42 (Cell Division Cycle protein-42), PAK1 (p21 Activated Kinase-1) and Ras. Also occurring early after LPS stimulation is the activation of the PI-3K (Phosphoinositide-3-Kinase) and subsequent activation of PKC (Protein Kinase-C). PC-PLC (Phosphatidylcholine-specific Phospholipase-C) may also stimulate PKC activation. Subsequently activation of the MAPK kinase kinases including MEKK1/4, Raf1, ASK1 (Apoptosis Signal-regulating Kinase-1) and TAK1 (TGF-Beta Activated Kinase-1) and the activation of MAPK kinases such as the MKKs and MEK1/2 takes place. In the nucleus these lead to the recruitment of the transcriptional coactivators CREB (cAMP Response Element Binding Protein) and Elk1. Elk1 is a part of a TCF (Ternary Complex Factor) that binds together with SRF (Serum Response Factor) to the SRE (Serum Response Element). CREB can form homodimers or heterodimers with other members of the ATF family, including ATF1 by binding to CRE (cAMP Response Element). The MAP kinase can directly phosphorylate various transcription factors or can work through activation of kinases like MSK1 (Mitogen and Stress Activated Kinase-1). TAK1 activates two kinase cascades, one leads to the activation of JNK, and the other cascade leads to the activation of the IKK (I-KappaB Kinases) through NIK (NF-KappaB-Inducing Kinase), which phosphorylate I-KappaB (Inhibitor of Kappa Light Chain Gene Enhancer in B-Cells). This phosphorylation triggers ubiquitination and subsequent degradation of I-KappaB, resulting in the release of NF-KappaB subunits p50 and p65 that translocate to the nucleus and induce the transcriptional activation of a wide variety of inflammatory and immune response genes. Although LPS is a potent activator of several cell types, such as macrophages and microglial cells, these cells often play unique tissue-specific roles and exhibit different sensitivities to LPS. These types of biological differences may be inherent in variations within LPS signaling pathways. Stimulation of neutrophils with LPS evokes several "immediate" functional responses, including actin assembly, adherence, activation of NF-kappaB, and the ability to prime for an enhanced secretory response. As a single stimulus, LPS is ineffective in evoking chemokinesis, chemotaxis, or the release of superoxide anion or granular enzymes. References: 1. Fenton MJ, Golenbock DT LPS-binding proteins and receptors. J. Leukoc Biol. 1998 Jul; 64(1): 25-32. Review. PubMed ID: 9665271 2. Tamemoto H, Kadowaki T, Tobe K, Ueki K, Izumi T, Chatani Y, Kohno M, Kasuga M, Yazaki Y, Akanuma Y. Biphasic activation of two mitogen-activated protein kinases during the cell cycle in mammalian cells. J. Biol. Chem. 1992 Oct 5; 267(28): 20293-7. PubMed ID: 1400347 3. Cobb MH, Goldsmith EJ. How MAP kinases are regulated. J. Biol. Chem. 1995 Jun 23; 270(25): 14843-6. PubMed ID: 7797459 4. Meja KK, Seldon PM, Nasuhara Y, Ito K, Barnes PJ, Lindsay MA, Giembycz MA. p38 MAP kinase and MKK-1 co-operate in the generation of GM-CSF from LPS-stimulated human monocytes by an NF-kappa B-independent mechanism. Br. J. Pharmacol. 2000 Nov; 131(6): 1143-53. PubMed ID: 11082122 5. Chan ED, Morris KR, Belisle JT, Hill P, Remigio LK, Brennan PJ, Riches DW. Induction of inducible nitric oxide synthase-NO* by lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1-ERK, MKK7-JNK, and NF-kappaB signaling pathways. Infect. Immun. 2001 Apr; 69(4): 2001-10. PubMed ID: 11254551 PC-PLC PI3K CDC42 Ras PKC PAK1 Raf1 ASK1 TAK1 NIK MEK1/2 MEKK 1/4 MKK 3/4/6 P IKKs IkBs ERK1/2 NF-kB p38 JNK1/2 IkBs Degradation P P P P Elk1 SRF P CREB NF-kB P ATF1 SRE CRE P P P P c-Jun c-Fos c-Jun ATF2 Gene Expression