Laura Nicolucci

Canada

Investigating the Effects on Targeting the TRAF1/PKN-1 Signalling Axis in Inflammation

Laura Nicolucci1, Ali. Abdul-Sater1
1. Department if Kinesiology and Health Science, York University, Toronto, Canada

Abstract

Background

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a signaling adaptor protein in lymphocytes and monocytes. In monocytes, TRAF1 attenuates inflammatory signaling by sequestering the linear ubiquitin chain assembly complex (LUBAC), which limits the activation of proteins involved in pro-inflammatory pathways. TRAF1 is particularly notable due to its overexpression in 48% of B-cell-related cancers. In lymphomas, TRAF1 promotes survival signaling by recruiting cellular inhibitors of apoptosis (cIAP2). Protein Kinase 1 (PKN-1) phosphorylates TRAF1 to protect it from degradation by cIAP2, and PKN1 knockdown or inhibition destabilizes TRAF1, leading to reduced lymphoma survival. Recent evidence implicates TRAF1/cIAP2 axis in promoting inflammatory signaling in monocytes downstream of TLRs, which prompted us to investigate whether targeting PKN1 would alter inflammation in monocytes. We hypothesized that removing TRAF1 phosphorylation would lower inflammation downstream of the TLR/TRAF1/CIAP2 signaling complex.

Methods

PKN-1 knockouts were generated via CRISPR/Cas9 technology on THP1 human monocytic cells. Inflammatory signalling and gene expression were assessed via flow cytometry and qPCR. Further, wild-type THP1 cells were treated with PKN-1 inhibitor, OTSSP167. The effects of the drug on inflammatory gene and protein expression were assessed via qPCR and western blotting.

Results

We found that inhibition of PKN-1 using OTSSP167 led to reduced TRAF1 phosphorylation and protein levels along with a notable decrease in inflammatory gene expression. PKN-1-/- cells exhibited reduced inflammatory signaling and gene expression compared to WT cells.

Conclusions

This study represents a critical step towards advancing our understanding of inflammatory signalling in monocytes. This strategy may facilitate the development of novel therapies that target the PKN1/TRAF1 axis to prevent the onset of inflammatory and autoimmune conditions.