After 30 minutes, ear punches (8 mm) of both ears were collected and were used for extraction of the Evan’s blue dye followed by measurement of absorbance at 620 nm. Fasudil protected mice against IgE-mediated challenge. Our results identify ROCK/LIMK pathway as a novel therapeutic target for Eng treating allergic diseases involving mast cells. mice. Figure ?Figure1A1A shows deletion of Rock1 and expression Cipargamin of Rock2 in bone marrow derived mast cells (BMMC). BM cells from WT and mice were cultured for 4 weeks in the presence of IL-3 and their maturation into mast cells was assessed by staining the cells for the co-expression of KIT and FcR1 receptors followed by flow cytometric analysis. While WT BM cells fully matured into KIT and IgE receptor double positive mast cells by three weeks of culture, Rock1-deficient BMMCs showed reduced maturation at early time points, however their maturation was complete by 4 weeks as assessed by the presence of close to 100% KIT and IgE receptor double positive cells (Figure ?(Figure1B).1B). Thus, although BM cells lag behind their WT counter parts with respect to the acquisition of KIT and IgE receptor double positive BMMCs, their expression is completely attained by the end of the 4th week of culture. We next analyzed the growth of WT and BMMC’s in response to IL-3 by thymidine incorporation. As seen in Figure ?Figure1C,1C, no difference in the growth of WT and BMMCs was observed in the presence of IL-3. These results suggest that Rock1 may play a minor role in the differentiation of BMMCs from its precursors in the BM. Open in a separate window Figure 1 Deficiency of Rock1 results in impaired early maturation of bone marrow derived mast cell (BMMC)(A) Expression of ROCK isoforms in WT and mice were cultured in the presence of IL-3 (10 ng/mL) for 4 weeks. At indicated time points, maturation was analyzed by staining the cells with antibodies that recognize KIT and Cipargamin IgE receptor followed by flow cytometry. Shown is dot blot profile from one of three independent experiments. (C) Rock1 deficiency have no effect on IL-3 mediated growth of BMMCs. BMMCs from WT and mice were starved for 6 hours in serum- and Cipargamin cytokine-free media and cultured in the presence or absence of IL-3 (10 ng/mL). After 48 hours, proliferation was evaluated by [3H] thymidine incorporation. Bars represent the mean [3H] thymidine incorporation in BMMCs (CPM + SD) from one representative experiment performed in quadruplicate. Similar results were observed in three independent experiments. Rock1 deficient BMMCs show reduced growth in response to SCF We next performed studies to analyze the role of Rock1 in KIT receptor mediated growth of BMMCs. BMMCs derived from WT and mice at the end of week 1, week 2 and week 3 were starved and cultured in the presence or absence of SCF for 48 hours, and proliferation was analyzed by thymidine incorporation. While WT BMMCs demonstrated a significant increase in thymidine incorporation in the presence of SCF relative to un-stimulated cells, deficiency of Rock1 resulted in a significant loss of SCF-mediated growth (Figure ?(Figure2A).2A). The reduced SCF-mediated growth was noted at the end of week 1, week 2 and week 3. Since Rock1 deficient cells show reduced maturation at early times during the culture period, we further assessed if the reduced growth in response to SCF is either due to reduced KIT expression or due to a Cipargamin cell intrinsic defect as a result of Rock1 deficiency. We sorted WT and BMMCs based on KIT expression and measured growth in response to SCF stimulation by thymidine incorporation. As seen in Figure ?Figure2B,2B, sorted KIT positive BMMCs also showed reduced growth in response to SCF suggesting a critical role for Rock1 in normal growth of mast cells. Open in a separate window Figure 2 Rock1 deficient cells show altered SCF-mediated growth(A) Deficiency of Rock1 alters the SCF-mediated growth of BMMCs. LDMNCs from WT and mice were cultured in the presence of IL-3 (10 ng/mL) for 3 weeks. At indicated time points, proliferation was evaluated by [3H] thymidine incorporation. BMMCs from WT and mice were starved for 6 hours in serum- and cytokine-free media and cultured in the presence or absence of SCF (50 ng/mL). After 48 hours, proliferation was evaluated by [3H] thymidine incorporation. Bars represent the mean [3H] thymidine incorporation in BMMCs (CPM + SD) from one representative experiment.
