Fig 1.
α7 nAChR expression and GTS-21 stimulation do not block TNF-induced NFκB-mediated signaling in GH4C1 cells.
(A) 125I-αBGT binding to WT GH4C1 cells compared to α7 nAChR transfected cells (α7). Concentrations and error bars are as described in methods. *** P< 0.0001 compared to WT. (B) Left: Western blot data showing a time-dependent TNF-induced IκB phosphorylation levels. Right: Different concentrations of GTS-21 added 30 min prior to a 1 h TNF exposure (20 ng/ml), had no effect on pIκB levels in α7 nAChR transfected GH4C1 cells. In contrast, the NFκB blocker PDTC decreased the levels of pIκB. GAPDH is the loading control. (C) Effects of different concentrations of GTS-21 on TNF-induced SEAP. PDTC, an NFκB inhibitor, blocked TNF-induced SEAP, confirming that inflammatory signaling is intact in GH4C1 cells and that these cells can be used to study NFκB-mediated signaling. GTS-21 was unable to block TNF-induced SEAP in GH4C1 cells transfected with α7 nAChR and NFκB-SEAP plasmids.
Fig 2.
Confirmation of α7 nAChR surface expression on mouse macrophages.
(A) PCR analysis showing a band of 264 bp size confirming the presence of α7 nAChR mRNA in macrophages isolated from WT but not α7 nAChR knockout mice. The 100 base pair ladder was from New England Biolabs. No RT: No reverse transcriptase control. (B) 125I-αBGT binding data comparing α7 nAChR expression on macrophages isolated from WT and α7 nAChR knockout murine model. α7 nAChR transfected GH4C1 cells were used as a positive control. Macrophages from WT mouse showed a small but significant αBGT specific binding site (detected with 100 nM 125I-αBGT). Bars in the figure represent specific binding. *** P< 0.0001 compared to α7 nAChR knockout mouse macrophages. (C) Left: Western blot data suggested LPS induced maximal pIκB levels in WT mouse macrophages at 1 h (circled). Right: GTS-21 attenuated LPS-stimulated pIκB levels in mouse macrophages in a dose-dependent manner. GAPDH was the loading control.
Fig 3.
GTS-21 attenuates IL6 and TNF secretion in macrophages from WT and α7 nAChR knockout mice.
Macrophages from WT and α7 nAChR knockout mice were isolated and activated by LPS before assaying the amount of IL6 and TNF release measured by ELISA as indicated in the methods section. Quadruplicate responses are normalized to cytokine release by LPS in the absence of GTS-21. A repetition of this experiment yielded similar results. One-way ANOVA (analysis of variance) with post-hoc Tukey HSD (honestly significant difference) test used to compare differences between multiple treatment groups. ** P< 0.01, *** P< 0.001 for the a7 KO TNF curve, other curves had similar P values (not shown).
Fig 4.
α7 nAChR-independent anti-inflammatory effects of GTS-21 in WT and Chrna7-/- mouse macrophages.
(A) 50 μM GTS-21 pre-treatment significantly blocked endotoxin-stimulated TNF and IL6 secretions and this concentration was selected to further study the effects of α7 nAChR antagonists, αBGT and MLA. ELISA data showing approx. 10% reversal of GTS-21 block on TNF secretion by 1.25 μM αBGT. No reversal was observed with MLA. ELISA data in panel (B) demonstrates 40% reversal of GTS-21 block on IL6 secretion by 1.25 μM αBGT and 30% reversal by 1 μM MLA. Neither αBGT nor MLA produced significant changes in the GTS-21 block of LPS-induced (C) TNF or (D) IL6 secretion in α7 nAChR absence. One-way ANOVA (analysis of variance) with post-hoc Tukey HSD (honestly significant difference) test used to compare differences between multiple treatment groups. * P< 0.01, ** P< 0.001, *** P< 0.0001. A duplicate of this experiment showed similar effects.
Fig 5.
Schematic comparing cell-dependent effects of GTS-21 in GH4C1 cells and primary mouse macrophages.
Top panel: GTS-21 was unable to suppress TNF-induced secreted alkaline phosphatase in a α7 nAChR and NFκB-SEAP expressing GH4C1 cell system. Bottom panel: GTS-21 reduced LPS-induced TNF and IL6 levels through both an α7 nAChR-dependent and an α7 nAChR-independent mechanism in primary mouse macrophages. In both cases, activation of the NFkB pathway leads to phosphorylation of IkB, and dimerization and translocation of two subunits of cytoplasmic NFκB (usually p50 and p65) to the nucleus to act as a transcription factor (Lawrence et al., 2009). In spite of the common NFkB pathway, the immediate effects of TNF and LPS differ in terms of the receptors and linkage to NFkB signaling, where GTS-21 non-nicotinic receptor actions may be taking place.