Hck tyrosine kinase regulates TLR4-induced TNF and IL-6 production via AP-1.

The TLRs play a key role in host defense against infection and injury, and mounting evidence suggests that these receptors may also play a role in diseases such autoimmunity, atherosclerosis, and cancer. Activation of TLRs on macrophages results in the production of multiple soluble mediators including the key inflammatory cytokines, TNF and IL-6. Thus, the intracellular signaling mechanism by which TLRs signal is a subject of great interest. As well as activating the NF-κB and MAPK pathways, TLR engagement leads to tyrosine kinase activation within minutes. Src family kinases (SFKs) are the largest nonreceptor tyrosine kinase family with nine members: Src, Hck, Lyn, Fyn, Fgr, Blk, Lck, Yes, and Ylk. The role of the SFKs in TLR signaling has been an area of much controversy, with conflicting findings between studies using chemical inhibitors and knockout mice. Using primary human macrophages in combination with adenoviral overexpression and small interfering RNA knockdown studies, we show that the SFK, Hck, has a pre-eminent role in LPS/TLR4-induced TNF and IL-6 production. Hck kinase mediates TLR4-induced transcription of both TNF and IL-6 by a mechanism that involves neither the NF-κB nor the MAPK pathways, but rather leads to AP-1 binding with a complex of c-fos and JunD. These data highlight the importance of Hck as an active component in LPS-induced TLR signaling and suggest the possibility of targeting this kinase for the alleviation of excessive inflammation.

Tyrosine kinases and inflammatory signalling.

The activity of tyrosine kinases is central to many cellular processes, and accumulating evidence suggests that their role in inflammation is no less profound. Three main tyrosine kinase families, the Src, Tec and Syk kinase families are intimately involved in TLR signalling, the critical first step in cellular recognition of invading pathogens and tissue damage. Their activity results in changes in gene expression in affected cells. Key amongst these genes are the cytokines, which orchestrate both the duration and extent of inflammation. Tyrosine kinases also play important roles in cytokine function, and are implicated in signalling through both pro- and anti-inflammatory cytokines such as TNF, IL-6 and IL-10. Thus, strategies to modulate tyrosine kinase activity have significant therapeutic potential in combating the chronic inflammatory state that is typical of many major health issues that face us today, including Rheumatoid Arthritis, Cardiovascular disease and cancer. Here we review current knowledge of the role of tyrosine kinases in inflammation with particular emphasis on their role in TLR signalling.

Chemical inhibition of Src family kinases affects major LPS-activated pathways in primary human macrophages.

Understanding the signalling mechanisms controlling inflammatory cytokine production is pivotal to the research of both acute and chronic immune disorders. Tyrosine phosphorylation is one of the earliest events to occur in response to an immune challenge yet the role of specific tyrosine kinases in inflammatory cytokine production has been difficult to ascribe due to conflicting literature. Here we show that the pyrazolo pyrimidine compound PP2, a selective inhibitor of Src family kinases (SFK), can inhibit LPS-induced TNF production as well as a number of other inflammatory cytokines. In addition, we show similar effects of PP2 on cytokine production when induced by other TLRs, (1, 2 and 5-8), indicating that SFK are important common regulators of TLR signalling. PP2 suppressed the activity of both TNF and IL-10 driven reporter genes, suggesting that this activity is mediated at the level of transcription. Interestingly, however, PP2 had no significant effect on the activation of NF-kappaB, or on p42/44 ERK, p46/54 JNK or p38 MAPK phosphorylation. In contrast, PP2 did inhibit AP-1 nuclear accumulation in response to LPS. Taken together, these findings show that the Src kinases are able to control inflammatory cytokine production at the transcriptional level independently of NF-kappaB, and highlight the role of the AP-1 family of transcription factors as downstream mediators of Src kinase action.