However, Syk shRNA transduced cells lost the effect of IgE. PDGF consistently showed highly significant thymi dine incorporation in inhibitor DZNeP both scramble and Syk inhibited HASM cells. These results suggest that IgE induced proliferation requires the function of Syk, a key signaling pathway in Fc��RI activation. IgE activates multiple signaling pathways in HASM cells To understand the downstream molecular signaling path ways involved in IgE induced HASM cell proliferation, we assessed the phosphorylation of MAPK and Akt by performing Western blot analysis on HASM cell lysates stimulated with IgE for 0 120 min. Western blotting re vealed a significant JNK phosphorylation at 20 30 min, Erk1 2 at 60 min, p38 at 120 min, and Akt at 60 min. In summary, IgE phosphorylates MAPK and Akt kinases in HASM cells which may play a role in IgE induced cell proliferation.

MAPK inhibitors abrogate the IgE induced HASM cell proliferation We then confirmed the involvement of different MAPKs in IgE induced HASM cell proliferation by using specific MAPK inhibitors. The dose of various inhibitors was first optimized to find the dose that inhibits IgE induced cell proliferation without inducing a noticeable cytoto icity. Figure 4 shows that IgE induced HASM cell proliferation was inhibited signifi cantly upon pre incubation for one hour with inhibitors of Erk1 2, JNK, p38, and Akt. DMSO vehicle control did not show any ef fect on HASM cell proliferation. In con clusion, IgE induced HASM cell proliferation involves the activation of Erk1 2, p38, JNK MAPK, and Akt kinases.

STAT3 is critical in IgE induced HASM cell proliferation STAT3 activation is indispensable in HASM cell prolifer ation in response to PDGF. Interestingly, monomeric IgE induces STAT3 phosphorylation in murine bone marrow derived mast cells and rat basophilic leukemia cells, and induce the transcription of genes important in cell survival. With these reports in consideration, we first sought to determine whether IgE is able to phos phorylate STAT3 in HASM cells. A representative blot in Figure 5A and summary of 4 e periments in Figure 5B show that IgE indeed induced STAT3 phosphorylation in HASM cells. To confirm its role in HASM cell proliferation, we employed lentiviral vector mediated STAT3 silencing approach. HASM cells were stably transduced with pseudotyped lentiviral vector encoding specific STAT3 shRNA.

Mock and scramble sequence served as controls. More than 95% of HASM cells were transduced as observed by turbo GFP signal by FACS analysis. Lentiviral STAT3 shRNA transduction resulted in a noticeable decrease in STAT3 Carfilzomib e pression compared to WT or scramble shRNA trans duction controls. Both scramble shRNA and STAT3 shRNA transduced HASM cells were stimulated with IgE and PDGF to analyze thymi dine incorporation. Since PDGF induced mitogenic sig naling requires STAT3 e pression, 10% FBS was used as an additional positive control in this e peri ment.