1. A pyrrole-based microtubule-depolymerizing compound reduces pro-inflammatory
signaling in RAW264.7 macrophages
Samuel P. Gilmorea, Anna Gonyea, Santiago Espinosa de los Reyesa, Elizabeth Lia, John T.
Guptonb, Omar Quinteroa, Krista Fischer-Stengera
aDepartment of Biology, University of Richmond, Richmond, VA 23173, United States
bDepartment of Chemistry, University of Richmond, Richmond, VA 23173, United States
Activation of the Nuclear Factor-kappa B (NF-κB) signaling pathway is required for the
production of pro-inflammatory mediators by macrophages during an inflammatory response.
The pathway is activated by lipopolysaccharide (LPS), a cell wall component of gram-negative
bacteria, binding to the Toll-like receptor 4 (TLR4) complex on the surface of macrophages.
Initiation of the pathway triggers the phosphorylation and subsequent degradation of IκB
inhibitory proteins that are bound to the NF-κB transcription factor. NF-κB is then released from
this inactive state and translocates into the nucleus where it interacts with the promoter region of
pro-inflammatory genes. Nuclear translocation of NF-κBis influenced by the polymerization
state of the microtubule network, although the exact role of microtubules in this translocation has
yet to be identified. Previous work from our laboratory indicated that exposure to a pyrrole-based
microtubule-depolymerizing compound, JG-03-14, delays the degradation of the inhibitory
protein, IκB, in activated macrophages which may prevent the release of NF-κB and decrease the
translocation of NF-κB into the nucleus. This study, along with our present work using the new
pyrrole-based compound, NT-07-16, did not find evidence that the delay in IκB degradation was
a result of a decrease in the phosphorylation of one of the IκB proteins but suggest that the
degradation delay may result from a decreased ability of the transcription factor to bind
microtubules. To further understand the mechanism behind the anti-inflammatory effects of these
compounds RAW264.7 macrophages were exposed to NT-07-16 for one hour prior to LPS
activation, and then using immunofluorescence microscopy the location of the p65 subunit of
NF-κB was analyzed over time. These studies indicate that less NF-κBmoved from the
cytoplasm into the nucleus when the macrophages were exposed to NT-07-16 prior to LPS
activation. Furthermore, studies using immunoprecipitation with an anti-tubulin antibody suggest
that treatment with NT-07-16 reduced the interaction of the p65 subunit with the microtubules
which may explain why less NF-κB is observed in the nucleus after NT-07-16 treatment. These
findings provide evidence that the anti-inflammatory effects of NT-07-16 on macrophages may
be explained at least in part by the depolymerization of the microtubules which results in less
NF-κB reaching the nucleus and reducing the transcription of pro-inflammatory cytokines.