Tie2-R849W Mutant in Venous Malformations Chronically Activates a Functional STAT1 to Modulate Gene Expression  Hsiao-Tang Hu, Yi-Hsien Huang, Yi-Ann Chang, Chien-Kuo Lee, Meei-Jyh Jiang, Li-Wha Wu  Journal of Investigative Dermatology  Volume 128, Issue 9, Pages 2325-2333 (September 2008) DOI: 10.1038/jid.2008.89 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Tie2-bearing adenoviral infection increased the Tie2 or its variant expression on cell surface. (a) Lysates from adenovirus-infected A549 cells were harvested at 24-hours post infection for immunoprecipitation with anti-Tie2 antibody, followed by western blot analysis using anti-phosphotyrosine (p-Tie2) antibody. (b) Lysates from adenovirus-infected HUVECs were harvested for western blot analyses using anti-p-Tie2 or anti-Tie2 antibodies. (c) The infected cells were harvested at 24-hours post adenoviral infection for cell-surface Tie2 protein labeling using flow cytometry. The values represent mean±SD of three independent experiments (bottom panel). MFI, mean fluorescence intensity; **P<0.01 versus LacZ; NS, not significant versus Tie2-WT. Journal of Investigative Dermatology 2008 128, 2325-2333DOI: (10.1038/jid.2008.89) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effect of ectopic Tie or its variants on HUVEC proliferation, apoptosis, migration, and tube formation. (a) Following adenovirus infection, serum-starved HUVECs were subjected to proliferation assay for 48hours. (b) The infected cells were serum-deprived for 24hours to induce apoptosis for apoptosis assay. (c) Adenovirus-infected HUVECs were seeded on Matrigel for tube formation assay. Images of tube-like structures were taken at 6 (i–v) and 36hours (vi–x) post-seeding. Quantitative data at 36-hours post-seeding is shown at the bottom. *P<0.05 or **P<0.01 versus LacZ; §P<0.05 versus Tie2-WT. Journal of Investigative Dermatology 2008 128, 2325-2333DOI: (10.1038/jid.2008.89) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Tie2-R849W induced STAT1 tyrosine phosphorylation by direct association with STAT1. (a, b) Total proteins from the indicated adenoviral infected HUVECs were analyzed by western blot analysis using anti-p-STAT1(Y701), STAT1, p-STAT3(Y705), and STAT3. STAT1 and STAT3 serve as loading controls. (c, d) Protein lysates from the indicated infected HUVECs were immunoprecipitated with anti-STAT1 antibody followed by western blotting with anti-myc or immunoprecipitated with myc-agarose beads, and blotting with anti-STAT1 antibody. Journal of Investigative Dermatology 2008 128, 2325-2333DOI: (10.1038/jid.2008.89) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Subcellular localization of Tie2-R849W-mediated phosphorylation of STAT1, and its effect on IRF1-promoter activity. (a) Western blot analysis of whole-cell lysate (WCL), nuclei, and cytosol harvested from the indicated adenovirus-infected cells using anti-p-STAT1(Y701), STAT1, c-Jun, and tubulin antibodies. c-Jun served as nuclear loading control, whereas α-tubulin served as cytosol loading control. (b) Immunofluorescence microscopy of the indicated virus-infected cells. Green, p-STAT1(Y701); blue, 4′6-Diamidino-2-phenylindole; merge, superimposed STAT1 and 4′6-Diamidino-2-phenylindole; bar=100μm. (c) HUVECs (left panel) or HEK293 cells (right panel) were individually transfected with pJFE vector, Tie2-WT, Tie2-K855A, or Tie2-R849W together with IRF1-driven promoter constructs. Promoter activity was expressed as relative luciferase unit following normalization with β-galactosidase. *P<0.05 or **P<0.01 versus vector control. (d) Tie2-expressing HUVECs were treated for 4hours with LPS (100ngml−1) before total RNA isolation for semi-quantitative reverse transcriptase-PCR. GAPDH served as a loading control. The top panel represents one independent experiment and the bottom panel represents mean±SD of three experiments using densitometry. NS, not significant versus null; ***P<0.001 versus null. Journal of Investigative Dermatology 2008 128, 2325-2333DOI: (10.1038/jid.2008.89) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The role of p38MAPK and JNK activation in Tie2-R849W-mediated STAT1 tyrosine phosphorylation and IRF-promoter activity. (a) Following adenoviral infection, cells were incubated in the growth medium for 22hours. In the final hour of incubation, infected cells were treated for 1hour with DMSO (0.1%), U0126 (10μM), SB203580 (10μM), wortmannin (200nM), and SP600125 (10μM) before western blotting using anti-p-Y701-STAT1 and STAT1 antibodies. U0126, SB203580, wortmannin, and SP600125 are inhibitors, respectively, for ERK1/2, p38MAPK, phosphoinositide-3-kinase, and JNK activation. (b) The status of STAT1 tyrosine phosphorylation in infected cells treated for 24hours with the indicated inhibitor. (c) Following transient transfection of IRF1-promoter constructs with Tie2 vectors for 12hours, HUVECs were treated with DMSO or the indicated inhibitor for 24hours before cell lysate collection for luciferase assay. *P<0.05 versus pJFE vector control; #P<0.05 versus DMSO-treated Tie2-R849W cells. Journal of Investigative Dermatology 2008 128, 2325-2333DOI: (10.1038/jid.2008.89) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions