There is considerable evidence indicating that intracellular Ca2+ participates as a second messenger in TLR4-dependent signaling. However, how intracellular free Ca2+ concentrations ([Ca2+](i)) is increased in response to LPS and how they affect cytokine production are poorly understood. Here we examined the role of transient receptor potential (TRP), a major Ca2+ permeation pathway in non-excitable cells, in the LPS-induced cytokine production in macrophages. Pharmacologic experiments suggested that TRPV family members, but neither TRPC nor TRPM family members, are involved in the LPS-induced TNF alpha and IL-6 production in RAW264 macrophages. RT-PCR and immunoblot analyses showed that TRPV2 is the sole member of TRPV family expressed in macrophages. ShRNA against TRPV2 inhibited the LPS-induced TNF alpha and IL-6 production as well as I kappa B alpha degradation. Experiments using BAPTA/AM and EGTA, and Ca2+ imaging suggested that the LPS-induced increase in [Ca2+](i) involves both the TRPV2-mediated intracellular and extracellular Ca2+ mobilizations. BAPTA/AM abolished LPS-induced TNF alpha and IL-6 production, while EGTA only partially suppressed LPS-induced IL-6 production, but not TNF alpha production. These data indicate that TRPV2 is involved in the LPS-induced Ca2+ mobilization from intracellular Ca2+ store and extracellular Ca2+. In addition to Ca2+ mobilization through the IP3-receptor, TRPV2-mediated intracellular Ca2+ mobilization is involved in NF kappa B-dependent TNFa and IL-6 expression, while extracellular Ca2+ entry is involved in NF kappa B-independent IL-6 production. (C) 2010 Elsevier Inc. All rights reserved.