Regulation and functional impact of lipopolysaccharide induced Nod2 gene expression in the murine epididymal epithelial cell line PC1.

Epididymitis represents a serious threat to male fertility and usually develops following secondary bacterial infection of the epididymis such as urinary tract infections or sexually transmitted diseases. Surprisingly, very little is known about the innate host response triggered by bacterial infection in the male reproductive tract. In this study we investigated the regulation and function of Nod2 in epididymal epithelial cells following lipopolysaccharide (LPS) stimulation. The immortalized epididymal epithelial cell line PC1 (proximal caput 1) constitutively expressed Toll-like receptor 4, MD-2, CD-14 but not Nod2 messenger RNA. Lipopolysaccharide (LPS; 0.5 microg/ml) rapidly induced I kappaB phosphorylation and degradation, RelA nuclear translocation and phosphorylation, which correlated with enhanced transcriptional activity (four-fold) in PC1 cells. The LPS and lipid A rapidly (1 hr) induced Nod2 messenger RNA accumulation in a dose-dependent manner. RelA and RNApolII recruitment to the Nod2 gene promoter was enhanced in LPS-stimulated cells. Molecular blockade of nuclear factor-kappaB signalling with adenovirus 5 (Ad5) I kappaB AA or adenovirus 5 double-negative (Ad5dn) IKK beta prevented LPS-induced Nod2 gene expression. Functionally, Nod2 upregulation enhanced muramyl dipeptide (MDP) -induced tumour necrosis factor messenger RNA accumulation in PC1 cells. We conclude that epididymal epithelial cells mount an innate response following LPS exposure which leads to upregulation of Nod2 and enhanced responsiveness to the microbial product MDP. The rapid Nod2 upregulation in epididymal epithelial cells is probably part of a complex innate host response aimed at protecting the male reproductive tract from the deleterious impact of bacteria.

Development of a high-throughput assay for identifying inhibitors of TBK1 and IKKε.

IKKε and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKε has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKε. This information enabled the design of an optimal TBK1/IKKε substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 µM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.