Macrophages are central to the inflammatory response and have key functions in the initiation, course and termination of inflammation. As professional sentinel phagocytes, macrophages engulf invading bacteria and are potently activated by lipopolysaccharide (LPS) to generate NO and cytokines. Although NO pathway effector cGMP-dependent protein kinase 1 (PKG) is important for cytoskeletal events and is implicated in transcriptional activity, its roles during macrophage phagocytosis and gene expression have remained undefined. This work is the first to show by RT-PCR, in vitro phosphate transfer, and by Western blotting directly for PKG and for phosphorylated substrate VASP that PKG is expressed in primary murine peritoneal macrophages (PM) but not macrophage-like murine leukemic RAW264.7. Furthermore, we found that macrophage PKG expression is 25-fold lower than in vascular smooth muscle cells (VMSC). PKG and its substrates such as VASP, Rho and others are extensively reported to be important to cytoskeletal events in VMSC. Additionally, VASP, Rho and Rac are necessary for phagocytosis and we have previously shown that PKG can indirectly activate Rac1. Here we demonstrate that the specific, membrane permeable PKG inhibitor DT-2 abrogates uptake of E.coli and serum-opsonized polystyrene beads by PM but not by RAW264.7. However, when PKG was exogenously expressed in RAW264.7, phagocytic ability is enhanced and DT-2 could abolish this effect. Additionally, we show LPS-induced PKG substrate phosphorylation in RAW264.7 exogenously expressing PKG, suggesting that PKG may have roles in LPS-activated macrophages. By cDNA gene array and RT-PCR in PM, we show that DT-2 decreased early LPS-induced transcript for Cxcl2, IL-1β and TNF-α. We show here by Western bict with a phospho-specific p38 MAPK antibody that LPS stimulation resulted in increased phosphorylation of p38 MAPK in PM. This effect was blocked by DT-2. This finding suggests that PKG mediates activation of p38 MAPK in LPS-stimulated macrophages, and we propose that p38 MAPK may be an effector in the mechanism behind PKG mediated cytokine gene expression in LPS-activated macrophages. The recognition that PKG is essential for phagocytosis and production of LPS induced pro-inflammatory molecules suggests PKG as a putative therapeutic drug target in macrophage-mediate inflammatory diseases and warrants further investigation.

INDEX WORDS: cGMP-dependent protein kinase 1, Phagocytosis, LPS, Cytokine Expression