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Activation of the transcription factor NF-kB plays an important role in wound healing, inflammation, and apoptosis. Signaling pathways employed by G protein-coupled receptors to activate NF-kB, however, are poorly characterized. The "endothelial differentiation gene" (Edg) receptors Edg-3 and Edg-5, but not Edg-1, were found to mediate sphingosine 1-phosphate (S1P)-induced activation of an NF-kB -dependent reporter gene in HEK293 cells in a dose-dependent manner (EC50 = 100 nM). The activation was completely inhibited by the C. botulinum C3 exotoxin, Ro318220, and BAPTA, demonstrating requirements for Rho, protein kinase C, and Ca2+. Edg-3 and Edg-5, but not Edg-1, also activated the serum response factor (SRF), which is controlled by Rho. However, a receptor endogenous to HEK293 cells also promoted SRF, but not NF-kB, activation. Rho therefore appears to be required for activation of NF-kB, but is not necessarily sufficient. Suppression of Edg-3- and Edg-5-induced NF-kB activation by genistein, tyrphostin A25, and herbimycin A revealed the involvement of tyrosine kinases. Constitutively active GaqQ209L and, to a far lesser degree, Ga13Q226L (but not GaoQ189L or Gai2Q205L) activated NF-kB. Ga13QL, however, activated SRF more strongly than GaqQL, again demonstrating the lack of a tight correlation between SRF and NF-kB activation. Gbg weakly activated NF-kB, but potentiated activation achieved through Edg-3, Edg-5, and Ga13QL. In conclusion, our data demonstrate that Edg-3 and Edg-5 can promote activation of NF-kB, and suggest that these receptors activate Rho via Ga12/13 and/or Gaq, but that a coincident signal via Gq/PKC/Ca2+ is required to activate NF-kB. |