ERK negatively regulates TLR2 induced transcription of TNFα, IL-6, iNOS, Cox2, and IL1β in bone marrow derived macrophages

Abstract

Extracellular signal-regulated kinase (ERK) was recently identified as a type of mitogen-activated protein kinase involved in Toll-like Receptor (TLR) downstream signaling and its corresponding cytokine production in bone marrow derived macrophages. Previous papers focused on the correlation between TLR4 induced transcription and ERK when introduced to Lipopolysaccharide (LPS). Here, I evaluate the relationship between ERK and TLR2 induced transcription when Pam3CSK4 is applied. Specifically, I assessed ERK dependent regulation of pro-inflammatory cytokines such as TNFα, IL-6, iNOS, Cox2, and IL1β in order to illustrate the pathway in which ERK negatively regulates TLR2 induced transcription. Since no direct inhibitor of ERK exists, I used a well-defined inhibitor (U0126) of Mitogen-activated protein kinase kinase (MEK1/2), an upstream factor known to directly regulate ERK, to test ERK-dependent negative regulation. In order to detect protein and mRNA level of the cytokines, I used Western Blotting and quantitative Real-time Reverse Transcription Polymerase Chain Reaction(qRT-PCR), respectively. Furthermore, to locate the point in which negative regulation of TLR2 downstream transcription by ERK occurs, I evaluated potential pathways suggested by previous studies involving MEK1/2 and phosphorylated Signal Transducers and Activators of Transcription (p-STAT) using Western Blotting. Results showed that activation by Pam3CSK4 took a distinct pathway from LPS, leading to diagnosis of other potential pathways involving phosphorylation of p38, JNK, IκBα, and CREB. In addition, I tested macrophage-like cell lines: J774A.1, Immortalized bone marrow derived macrophage (iBMDM), and Raw264.7 to create a MEK1/2 knockout cell line using the CRISPR-CAS9 system for substitution of in vivo experimentation with knockout mice. In final, to confirm that the negative regulation effect is TLR2 specific, TLR2 specific ligands: Pam3CSK4 and Lipoteichoic Acid (LTA) are contrasted with ligands of TLR3, TLR4, and TLR9. Collectively, these data suggest that ERK negatively regulates TLR2-induced transcription of pro-inflammatory cytokines: TNFα, IL-6, iNOS, Cox2, and IL1β. These findings may contribute in the development of drugs that modulate TLR activity to treat inflammatory diseases and cancer.