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 A. 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

MT depolymerizing drugs inhibit nuclear localization of NF-κBIntroduction Microtubules and NF-κB interact biochemicallyNF-κB is a transcription factor that stimulates the inflammatory immune response in macrophages. In non-activated macrophages, NF-κB is predominantly found in the cytoplasm. The binding of pathogenic markers to the Toll-Like Receptor 4 (TLR4) complex on the surface of macrophage cells stimulates a pathway that phosphorylates IκB, which is bound to and suppresses NF-κB translocation. Phosphorylation of IκB leads to it’s degradation, allowing NF-κB to make it’s way into the nucleus of the cell, where it binds to DNA - stimulating the transcription of pro-inflammatory genes and the subsequent release of cytokines. Our study looks at the effects of a novel microtubule depolymerizer on the phosphorylation of IκB, the interaction of NF-κB with tubulin, as well as the translocation of NF-κB from the cytoplasm into the nucleus. NT-07-16 is a novel polysubstituted pyrrole shown to have microtubule binding abilities. It is our hypothesis that, by significantly depolymerizing the microtubule network, we can reduce the levels of NF-κB translocation into the nuclei of macrophage cells. Our previous work has shown that treatment with microtubule depolymerizers 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. However, our results indicate that the delay in degradation is a result of NF-kB’s failure to translocate into the nucleus – implicating it’s failure to bind to microtubules in the presence of NT-07-16. Furthermore, we were able to show, using fluorescence microscopy, that treatment of macrophages with NT-07-16 significantly decreased translocation of NF-kB from the cytoplasm into the nucleus.

Figure 1: The canonical NF-κB signaling pathway. In the canonical NF-κB signaling pathway lipopolysaccharide (LPS), tumor necrosis factor α (TNFα) or interleukin-1 (IL-1) activates a variety of transmembrane receptors. A signaling pathway is triggered and this leads to the activation of IKKβ kinase, which phosphorylates IκB. This phosphorylation results in the subsequent polyubiquitination of the IκB complex, which is then degraded by a proteasome. NF-κB then translocates to nucleus and activates transcription of pro-inflammatory genes.Conclusions and Future Directions• Delay in IκB degradation after exposure to a microtubule-depolymerizer is not a result of a decrease in the phosphorylation of the IκB-α proteins but instead, the degradation delay may result from a decreased ability of NF-κB to bind microtubules• Results suggest NT-07-16 and Nocodazole both cause a decrease in nuclear localization of NF-κB into the nucleus; suggesting involvement of microtubules in NF-kB localization

Figure 3. The effect of NT-07-16 on the association between NFᴋB and α-Tubulin in RAW 264.7 cells. 1 x 106 cells were exposed to NT-07-16 for 1 hour followed by 0.5 and 1 hours of LPS activation. Total cell lysates were collected and immunoprecipitation was used to isolate α-tubulin. The amount of NFᴋB complexed to it was visualized through (A) western blotting and (B) densitometry was used to quantify this value.

Figure 2. Effect of NT-07-16 on the phosphorylation of IκB-α over time. RAW264.7 macrophages were activated with LPS for the indicated length of time and cell lysates were prepared and analyzed by Western blotting using antibodies specific for IκB-α and p-IκB-α. Macrophages were either (A) not exposed to or (B) were exposed to NT-07-16 for one hour prior to LPS activation.

CTRL LPS

LPS + Noc. LPS + NT-07-16

20 μm

Figure 4: Quantification of NF-κB localization. All treatment groups showed lower nuclear localization of NF-κB than the LPS activated cells (p < 0.05). Treating LPS-activated cells with nocodazole or NT-07-16 resulted in increased nuclear NF-kB localization compared to cells that were not activated with LPS (p < 0.05). Treating unactivated cells with either nocodazole or NT-07-16 did not alter nuclear NF-kB localization, compared to control cells (Tukey Analysis, ncells > 100 per treatment, ndays = 3 replicates).

Yellow areas represent an example of the regions of interest (ROI) selected. ROI used to measure average pixel intensity in the NF-κB channel. To account for day-to-day staining variability, results were normalized against each day’s “no LPS” control cell ratio.Hypothetical

RAW 264.7 Cell

Analysis:

Translocation Ratio (TR) = IN / IC Normalized per day = TRS / TRCTRL

CTRL LPS

LPS + Noc. LPS + NT-07-16

20 μm

Figure 5: Possible quantification for MT depolymerization by measuring the standard deviation of pixel intensity. Cells treated with LPS+Noc demonstrated lower standard deviation relative to control cells, LPS-activated cells or LPS-activated cells treated with NT-07-16. Samples were normalized to control cells for that day to account for day-to-day variation in staining. (Tukey Analysis p<0.01, ncells > 80 per treatment, ndays = 3 replicates).

Future studies: Is the NT-07-16 effect solely microtubule dependent?

Approach:

Cells cultured on coverslips overnight

~18hr

Pre-treat 1hr1 μM Nocodazole

or 1 μM NT-07-16

500 ng/ml LPS treatment 1hr, in presence

of drug

Fix and stain cells with: • DAPI• α-NF-κB

antibody• Phalloidin• α-tubulin

(DM1A)

References & Acknowledgements

NT-07-16

Nocodazole

• Ciemniecki JA, Lewis CP, Gupton JT, Fischer-Stenger K. (2016). Effects of a pyrrole-based, microtubule-depolymerizing compound on RAW 264.7 macrophages. Chem Biol Interact 25;246:63-8. doi: 10.1016/j.cbi.2016.01.009. •   • Noursadeghi M, Tsang J, Haustein T, Miller RF, Chain BM, Katz DR. (2008). Quantitative imaging assay for NF-kappaB nuclear translocation in primary human macrophages. J Immunol Methods 329(1-2):194-200.• Rai A, Kapoor S, Singh S, Chatterji PB, Panda D (2015). Transcription factor NF-kappaB associates with microtubules and stimulates apoptosis in response to suppression of microtubule dynamics in MCF-7 cells. Biochemical Pharmacology 93:277-289. •   • Rohena CC, Telang NS, Da C, Risinger AL, Sikorski JA, Kellogg GE, Gupton JT, Mooberry SL. (2016). Biological Characterization of an Improved Pyrrole-Based Colchicine Site Agent Identified through Structure-Based Design. Mol Pharmacol 89(2):287-96. doi: 10.1124/mol.115.101592.Thank you to HHMI and University of Richmond School of Arts & Sciences for funding this work.

Abstract: 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-κB is 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-κB moved 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.