TNFα and IL-1β are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

A growing body of evidence recently suggests that glial cell activation plays an important role in several neurodegenerative diseases and neuropathic pain. Microglia in the central nervous system express toll-like receptor 4 (TLR4) that is traditionally accepted as the primary receptor of lipopolysaccharide (LPS). LPS activates TLR4 signaling pathways to induce the production of proinflammatory molecules. In the present studies, we verified the LPS signaling pathways using cultured highly aggressively proliferating immortalized (HAPI) microglial cells. We found that HAPI cells treated with LPS upregulated the expression of TLR4, phospho-JNK (pJNK) and phospho-NF-kappa B (pNF-kappa B), TNF alpha and IL-1 beta. Silencing TLR4 with siRNA reduced the expression of pJNK, TNF alpha and IL-1 beta, but not pNF-kappa B in the cells. Inhibition of JNK with SP600125 (a JNK inhibitor) decreased the expression of TNF alpha and IL-1 beta. Unexpectedly, we found that inhibition of Nod1 with ML130 significantly reduced the expression of pNF-kappa B. Inhibition of NF-kappa B also reduced the expression of TNF alpha, and IL-1 beta. Nod1 ligand, DAP induced the upregulation of pNF-kappa B which was blocked by Nod1 inhibitor. These data indicate that LPS-induced pJNK is TLR4-dependent, and that pNF-kappa B is Nod1-dependent in HAPI cells treated with LPS. Either TLR4-JNK or Nod1-NF-kappa B pathways is involved in the expression of TNF alpha and IL-1 beta. 2012 Elsevier Inc. All rights reserved.