The innate immune response to pathogens involves the expression and secretion of inflammatory cytokines that act to facilitate eradication of the infecting pathogen. The dysregulation of cytokine production (known as chronic inflammation) can have negative effects on the host. Therefore, understanding the regulation of inflammatory cytokines is important for our understanding of their role in promoting various diseases such as autoimmune and malignant diseases. We have identified the transcription factor GLI3 as a novel target of TLR4 signaling pathway. Stimulation of TLR4 with LPS rapidly increases GLI3 expression. Inhibition of TLR4 signaling reduces GLI3 expression. The regulation of GLI3 by TLR4 signaling is independent of hedgehog (HH) signaling and appears to occur through TRIFmediated downstream signaling. TLR4 signaling and GLI3 regulate a similar set of inflammatory cytokine genes suggesting GLI3 may mediate TLR4-induced inflammation. Indeed GLI3 knockdown in human monocytes reduces inflammatory cytokine (CCL2, CCL7 and IL-6) expression. Furthermore, mice with conditional loss of one GLI3 allele in monocytes (GLI3Mdel+/-) have reduced cytokine expression in macrophages. Upon stimulation of macrophages from GLI3Mdel+/- mice with LPS, there is a significant reduction in LPS/TLR4-induced inflammation. Therefore, our preliminary studies suggest that GLI3 acts as a transcriptional effector of TLR4 signaling in a HH-independent manner. We also identified a novel interaction between GLI3 and p300. In this application, we propose to: 1) characterize the mechanism by which TLR4 signaling regulates GLI3 expression; 2) characterize the mechanism by which GLI3 cooperates with p300 to regulate inflammatory cytokine genes and 3) determine the role of GLI3 in vivo using mice with a conditional loss of GLI3 (both alleles) in monocytes (GLI3Mdel-/-).
