perifosine synthesis Several models show the MAPK pathway in
Several models show the MAPK pathway involving p38 as the perifosine synthesis pathway in pro-inflammatory responses, with the ERK pathway involvement primarily devoted to cell growth and differentiation events (Lai et al., 2001, Widmann et al., 1999). The results of the current study would contradict those models, as stimulation of thrombocytes with LPS resulted in increased expression of IL-6 mRNA, which was inhibited by treatment with the ERK inhibitor (Fig. 1B). The model for signaling in the thrombocyte (Fig. 3) is now expanded from that proposed by Scott and Owens (2008). Our current model includes two MAPK and the NFκB pathways for expression of genes encoded for pro-inflammatory molecules. The inhibition of IKKB or MEK1/2 alone was not enough to completely inhibit IL-6 production (Fig. 3). ERK can be activated through MEK1/2 directly or through IKKB (indirectly through NFκB1 and Tpl2, which can activate MEK1/2) to activate AP1 and thus upregulated production of IL-6. Therefore, inhibiting one of those molecules allowed production of some IL-6 through the other molecule. However, inhibiting molecules downstream of IKK, MEKs or MKKs such as p38 and ERK effectively lowered IL-6 production. LPS has been shown to stimulate iNOS to produce NO through common signaling pathways involving activation of protein kinase C (PKC), MAPKs (p38 MAPK and MEK1/2) and NFκB in avian macrophages (He and Kogut, 2003, Peroval et al., 2013). Previously, we have not detected nitric oxide (NO) release nor increased gene expression of inducible nitric oxide synthase (iNOS) with one-hour stimulation of thrombocytes with LPS (Ferdous and Scott, 2015). Based on this information, we examined NO production of the control, inhibitor treated, and untreated LPS stimulated thrombocyte supernatants (data not shown) and still were not able to detect any NO production and release. Analysis of Western blots of thrombocyte lysates showed differential presence of activated phosphorylated forms of ERK1/2, p38 and NFκB. LPS simultaneously induces gene expression and subsequent activation of a MAPK Phosphatase (DUSP 5/8) as p38 and others are activated in the cell. Lower signal of P-ERK1/2 (Fig. 2B) can possibly be explained by the presence of MAPK phosphatases that catalyze dephosphorylation of activated MAPKs and deactivates them through a regulatory feedback mechanism. We have performed additional investigation of P-ERK1/2 at 15 min and 30 min (not shown). P-ERK1/2 signal is detected in LPS only stimulated cell lysates at 15 min and is stronger in lysates at 30 min. However, P-ERK1/2 signal decreases by 60 min indicating activity of MAPK phosphatases. In murine macrophages, LPS triggered transient activation of three MAP kinase subfamilies, ERK, JNK, and p38 (Chen et al., 2002, Zhao et al., 2005). However, LPS also rapidly induced MKP-1, which correlated with dephosphorylation of these MAP kinases in a relatively short time post-stimulation (Zhao et al., 2005). Although P-ERK1/2 should be low in samples treated with PD98059 (inhibitor of MEK1 upstream of ERK), we detected a relatively higher amount of P-ERK1/2 relative to control (-LPS). This may be attributed to the fact that ERK1/2 was activated through other MEK activation possibly through IKKB and NFκB1 and Tpl2. Unlike murine macrophages stimulated with LPS, where P-p38 was detected at earlier timepoints such as at 15–30 min (Chen et al., 2002, Zhao et al., 2005), we were not able to detect P-p38 in any thrombocyte lysate before 60 min (not shown). In the case of P-p38 detection, low P-p38 in LPS treated samples without inhibitor can be explained by MKPs as described above for P-ERK1/2. We attempted to detect MKPs or Dual Specificity Protein Phosphatases (DUSPs) in our thrombocyte lysates, however despite trying different available antibodies for MKPs we were not able to find one that worked with our chicken thrombocyte lysates. However, we have transcriptome data (NCBI GenBank Sequence Read Archive under the following SRA Accessions: SAMN05818716-SAMN05818721 under the BioProject Accession: PRJNA34407), which shows that 1 h of LPS stimulation upregulates DUSP5/8 gene transcripts in chicken thrombocytes. DUSP5 is known to be specific for ERK1/2 while DUSP8 for p38 and JNK (Kondoh and Nishida, 2007). PD98059 and BMS345541 were not able to affect activation of p38 as expected and relative to these two samples. It is not clear why the ERK inhibitor affected expression of P-p38 and may be due to crosstalk of other MAPK molecules. The essential cross-talk between MAPKs was shown by blockade of ERK signaling by the MEK-1/2 inhibitor PD98059 and reversed inhibitory effects of molecules such as TGF-β (Xiao et al., 2002). We also attempted to detect the active form of the transcription factor, AP1 activated by MAP kinases with different available antibody reagents. We did not find one that could detect AP1(P-c-fos/P-c-jun) in chicken thrombocytes.