Investigation, K

Investigation, K.M., Y.H., H.O., and Y.S. These results suggest that PKC could be involved in this process. Furthermore, crosslinking-induced MHC-II endocytosis was suppressed by inhibitors of clathrin-dependent endocytosis. Our results indicate that the crosslinking of MHC-II could stimulate Ca2+ mobilization and induce the clathrin-dependent endocytosis of MHC-II in murine DCs. < 0.01 are regarded as significant. 3.2. Intracellular Ca2+ Mobilization Induces Endocytosis of MHC-II Next, we investigated whether intracellular Ca2+ mobilization accelerates the endocytosis of MHC-II by examining the effects of a reagent that induces intracellular Ca2+ mobilization. An endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, is known to induce Ca2+ mobilization in many cell types and we confirmed that thapsigargin induced intracellular Ca2+ mobilization in BMDCs (Figure 2A). We then investigated the effects of thapsigargin on MHC-II endocytosis and found that thapsigargin treatment induced significant levels of endocytosis of MHC-II (Figure 2B). These results suggest Harmine that intracellular Ca2+ mobilization can induce MHC-II endocytosis. Open in a separate window Figure 2 Intracellular Ca2+ mobilization induces endocytosis of MHC-II. (A) BMDCs were stimulated with or without thapsigargin and cytosolic Ca2+ mobilization was detected using Fura-2/AM. Bars = 1 min. Relative changes in the cytosolic Ca2+ concentrations are expressed as the ratio of fluorescence intensity at 340/380 nm. (B) BMDCs were incubated with an anti-MHC-II antibody for 30 min at 4 C. The cells were washed and stimulated with thapsigargin for 30 min at 37 C. The remaining cell surface anti-MHC-II antibody was detected using a PE-labeled anti-mouse IgG antibody. Values of * < 0.05, ** < 0.01 are regarded as significant. 3.3. Crosslinking-Induced Endocytosis of MHC-II is Syk and Phospholipase C (PLC)-Dependent Activation of an immunoreceptor tyrosine-based activation motif (ITAM) containing C-type lectin Harmine receptor, Dectin-1, was found to induce intracellular Ca2+ mobilization, which was mediated by Syk tyrosine kinase-mediated PLC activation, in BMDCs [19]. It was also reported that MHC-II interacted with the ITAM-containing adaptor Harmine protein, FcR, in BMDCs, and that MHC-II crosslinking induces tyrosine phosphorylation of cellular proteins [7]. Therefore, we examined the contribution of Syk and phospholipase C (PLC) using inhibitors against these molecules. Crosslinking-induced endocytosis of MHC-II was suppressed by Syk inhibitors, (piceatannol and R406), and a PLC inhibitor (U73122) (Figure 3). We then examined the effect of piceatannol and U73122 on intracellular Ca2+ mobilization, and found that both inhibitors suppressed Ca2+ mobilization induced by MHC-II crosslinking (Figure 4A,B). These results suggest that Syk and PLC are potentially involved in intracellular Ca2+ mobilization and MHC-II endocytosis induced by crosslinking of MHC-II. Open in a separate window Figure 3 Effects of Syk and phospholipase C (PLC) inhibitors on crosslinking-induced MHC-II endocytosis. BMDCs were pre-incubated with (A) piceatannol (50 M), (B) R406 (indicated concentrations), or (C) U73122 (10 M) for 30 min at 37 C. The cell surface MHC-II was crosslinked with an anti-MHC-II antibody Rock2 for 30 min. The remaining cell surface MHC-II was detected by flow cytometry. Values of * < 0.05, ** < 0.01 are regarded as significant. Open in a separate window Figure 4 Effects of Syk and PLC inhibitors on MHC-II crosslinking-induced intracellular Ca2+ mobilization. BMDCs were pre-incubated with Harmine (A) piceatannol (50 M) or (B) U73122 (10 M) for 30 min at 37 C. The cytosolic Ca2+ mobilization after MHC-II crosslinking was measured using Fura-2/AM. Relative changes in the cytosolic Ca2+ concentrations are expressed as the ratio of fluorescence intensity at 340/380 nm. Bars = 1 min. Values of * < 0.05, ** < 0.01 are regarded as significant. 3.4. Protein Kinase C (PKC) Activation is Involved in MHC-II Endocytosis One of the signaling molecules activated by intracellular Ca2+ mobilization is PKC. We, therefore, examined the effects of Ca2+ ionophore, A23,187, and a phorbol ester, TPA, which are known activators of conventional PKC isoforms, on.