The Transcription Factor GLI3 Is a Novel Candidate Effector Of Toll-Like Receptor 4 (TLR4) Signaling In Monocytes

Conference Proceeding

Abstract

  • Abstract Monocytes play crucial supporting roles in a vast variety of malignancies ranging from solid tumors to hematological neoplasms. Monocytes contain repertoires of immune sensors on their cell surface mediating the inflammatory response in the tumor microenvironment. Of particular interest are Toll-like receptors (TLR), which have well-characterized and documented roles, in not only the innate immune response, but also in carcinogenesis. Our goal was to expand on these findings and further the understanding of the mechanisms mediating inflammation in the tumor microenvironment, especially, identification and characterization of pathways regulating pro-inflammatory cytokine genes in monocytes. Thus, we began our studies by identifying genes differentially regulated in monocyte cell lines treated with lipopolysaccharide (LPS), a known activator of TLR4 and subsequently the inflammatory response. After 3 hours, we found an increase in the expression of GLI3, an effector of the Hedgehog (HH) signaling pathway. Interestingly, treatment of the monocyte cell lines MonoMac6 (MM6) and THP-1 with the HH signaling inhibitor cyclopamine (10 μM) followed by LPS stimulation resulted in up-regulation of GLI3, suggesting that the regulation of GLI3 by TLR4 occurs in a HH-independent manner. TLR4 mediates signaling through MyD88-dependent or TRIF-dependent mechanisms. This is characterized by recruitment of TIRAP or TRAM to the Toll/Interleukin-1 Receptor (TIR) domain of the cytoplasmic portion of TLR4 and subsequent signaling through MyD88 or TRIF, respectively. To further characterize the mechanism of TLR4-GLI3 regulation, we examined the role of TLR signaling adaptors (TIRAP, MyD88, TRAM) in this process. Dominant negative (dn) forms of MyD88, TIRAP and TRAM were transfected into MM6 and THP-1 cells followed by LPS treatment for 3 hours. In response to LPS stimulation, GLI3 was up-regulated in samples transfected with dnMyD88 and dnTIRAP, while GLI3 expression was near basal levels in samples transfected with dnTRAM, the adaptor component for TRIF-mediated TLR4 signaling. This suggests TLR4 mediated GLI3 expression occurs in a TRIF-dependent manner. It has been previously shown that TLR4 signaling from endosomes can cause late NF-κβ and MAPK signaling in addition to classical TRIF-mediated IRF signaling. To further define the mechanism of regulation of GLI3, we utilized pharmacological inhibitors to target IRF, NF-κβ and MAPK signaling components. We found that inhibition of IRF and NF-κβ signaling had no effect on GLI3 up-regulation by LPS. However, p38 inhibition resulted in abolished GLI3 induction in response to LPS stimulation suggesting the p38 mediates TLR4-GLI3 regulation. Inhibition of Jnk did not have an effect on LPS-induced GLI3 expression, suggesting that TLR4-TRIF-p38 axis mediates increased GLI3 expression. Further analysis indicates that LPS stimulation increases the expression of pro-inflammatory chemokines CCL2 and CCL7. Interestingly, overexpression of GLI3 increases the expression of these chemokines suggesting GLI3 may be required for chemokine expression in response to LPS. Taken together, these results identify a novel interaction between TLR4 signaling and GLI3 transcription factor and show a novel signaling pathway that can modulate the expression of GLI proteins independent of HH signaling. Targeted inhibition of this signaling crosstalk may provide therapeutic efficacy for patients with inflammatory and/or HH-related malignant disorders. Disclosures: No relevant conflicts of interest to declare.
  • Authors

  • Boi, Shannon K
  • Zapico, Martin E Fernandez
  • Elsawa, Sherine
  • Status

    Publication Date

  • November 15, 2013
  • Has Subject Area

    Published In

  • Blood  Journal
  • Keywords

  • 2.1 Biological and endogenous factors
  • Biotechnology
  • Cancer
  • Inflammatory and Immune System
  • Digital Object Identifier (doi)

    Start Page

  • 2269
  • End Page

  • 2269
  • Volume

  • 122
  • Issue

  • 21