The STK Receptor Tyrosine Kinase Regulates the Response of Primary Macrophages to LPS in a Ligand-Independent Manner.

We have shown previously that activation of the STK/RON receptor tyrosine kinase expressed on tissue resident macrophages, by it’s ligand macrophage stimulating protein (MSP), results in the inhibition of NFkB activation, inducible nitric oxide synthase (iNOS) expression and TNFa production, as well as the induction of arginase expression, suggesting a role for this receptor in the regulation of classical vs. alternative macrophage activation. Furthermore, mice with a targeted deletion in this receptor exhibit increased sensitivity to endotoxic shock and DTH responses. More recently, we have demonstrated that MSP stimulation of primary peritoneal macrophages inhibits the production of IL-12. In order to map the domains of STK responsible for the inhibition of classical macrophage activation by MSP, we generated mutant forms of the receptor and expressed wild-type and mutant receptors in primary bone marrow derived macrophages by retroviral transduction. Expression of wild-type STK in these primary cells resulted in the ligand-independent reduction in IL-12p40 production in response to LPS stimulation, which was further inhibited by MSP treatment. This is consistent with the lack of a requirement for MSP in regulating responses to endotoxin in vivo. Surprisingly, a kinase dead receptor, which fails to signal in 293T cells, was fully functional in this assay, suggesting that the kinase activity of the receptor is not required for the inhibition of IL-12p40 under these conditions. However, the docking site tyrosines in the c-terminal tail of the receptor are essential for the inhibition of IL-12p40 by STK, suggesting that STK may be phosphorylated by an another kinase in this system. STK/RON has been shown to associate both physically and functionally with a number of other cell-surface receptors including EpoR, IL-3R b_c, EGFR, MET as well as a number of integrins and cadherins. We have shown previously that STK regulates the activity of the a_Mb₂ integrin (CR3) in peritoneal macrophages in a PI3K, PKCz-dependent manner. Here we show that STK also physically associates with CR3, as well as CD14, in RAW264.7 cells in the absence of ligand. Both CR3 and CD14 are capable of directly binding to LPS. Thus, we speculate that STK may exist as part of a receptor complex in macrophages and that signalling through STK might be induced directly by LPS. This would provide a means by which STK could temper the response of tissue-resident macrophages to LPS thereby preventing damage to host tissues.