<<<<<< << <<< "DC.Date" content="2014-05-01" /> d in Highlights of This Issue, [p. 1019][1] [1]: /lookd in Highlights of This Issue, [p. 1019][1] [1]: /lookup/volpage/d in Highlights of This Issue, [p. 1019][1] [1]: /lookup/vold in Highlights of This Issue, [p. 1019][1] [1]: /lookup/vod in Highlights of This Issue, [p. 1019][1] [1]: /lood in Highlights of This Issue, [p. 1019][1] [1]d in Highlights of This Issue, [p. 1019][1] [1]: /lood in Highlights of This Issue, [p. 1019][1] [1]: /lookup/vod in Highlights of This Issue, [p. 1019][1] [1]: /ld in Highlights of This Issue, [p. 1019][1] [1]: /lookup/vd in Highlights of This Issue, [p. 1019][1] [1]: /lookup/d in Highlights of This Issue, [p. 1019][1] [1]: /lookupd in Highlights of This Issue, [p. 1019][1] [1]: /lookup/volpage/13/1019?iss=5" /> brane, and the clinically relevant mouse orthotopic floor-of-brane, and the clinically relevant mouse orthotopic floor-ofbrane, and the clinically relevant mouse orthotopic floor-of-mouth models. GALR2 brane, and the clinically relevant mouse orthotopic floor-of-mouth models. GALR2 induced angiogenesis via p38-MAPK?mediated brane, and the clinically relevant mouse orthotopic floor-of-mouth models. GALR2 induced angiogenesis via p38-MAPK?mediated secretion of proangiogenic cytokines, VEGF, and interleukin-6 (IL-6). Moreover, GALR2 activated small-GTP-protein, RAP1B, thereby inducing p38-mediated inactivation of tristetraprolin (TTP), which functions to destabilize cytokine transcripts. This resulted in enhanced secretion of proangiogenic cytokines and angiogenesis in vitro and in vivo . In SCCHN cells overexpressing GALR2, inactivation of TTP increased secretion of IL-6 and VEGF, whereas inhibition angiogenesis assays. Chemical inhibitor and genetic knockdown strategies were used to understand the key regulators. The <i>in vivo</i> impact of GALR2 on angiogenesis was investigated in mouse xenograft, chick chorioallantoic membrane, and the clinically relevant mouse orthotopic floor-of-mouth models. GALR2 induced angiogenesis via p38-MAPK?mediated secretion of proangiogenic cytokines, VEGF, and interleukin-6 (IL-6). Moreover, GALR2 activated small-GTP-protein, RAP1B, thereby inducing p38-mediated inactivation of tristetraprolin (TTP), which functions to destabilize cytokine transcripts. This resulted in enhanced secretion of proangiogenic cytokines and angiogenesis <i>in vitro</i> and <i>in vivo</i>. In SCCHN cells overexpressing GALR2, inactivation of TTP increased secretion of IL-6 and VEGF, whereas inhibition of p38 activated TTP and decreased cytokine secretion. Here, we report that GALR2 stimulates tumor angiogenesis in SCCHN via p38-mediated inhibition of TTP with resultant enhanced cytokine secretion. Given that p38 inhibitors are in clinical use for inflammatory disorders, GALR2/p38-mediated cytokine secretion may be an excellent target for new adjuvant therapy in SCCHN. <i>Mol Cancer Ther; 13(5); 1323?33. 2014 AACR</i>.</p>" /> angiogenesis assays. Chemical inhibitor and genetic knockdown strategies were angiogenesis assays. Chemical inhibitor and genetic knockdown strategies were used to angiogenesis assays. Chemical inhibitor and genetic knockdown angiogenesis assays. Chemical inhibitor and genetic knockdow angiogenesis assays. Chemical inhibitor and gen angiogenesis assays. Chemical inhibitor and genetic knockdown strategies were used to under angiogenesis assays. Chemical inhibitor and genetic knockdown strategies were used to understand the key re angiogenesis assays. Chemical inhibitor and genetic knockdown strategies were used to understand th angiogenesis assays. Chemical inhibitor and genetic knockdown strategies were used to underst /> /> dicine, University of Michigan School of Dentistry; Depadicine, University of Michigan School of Dentistry; Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan; Department of Craniofacial Biology, The Medical University of South Carolina, Columbia, South Carolina" /> cs and Oral Medicine, University of Michigan School of Dentistcs and Oral Medicine, University of Michigan School of Dentistry; Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan; Department of Craniofacial Biology, The Medical University of South Carolina, Columbia, South Carolina" /> on_author_institution" content="Authors' Affiliations: Deparon_author_institution" content="Authors' Affiliations: Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry; Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan; Department of Craniofacial Biology, The Medical University of South Carolina, Columbia, South Carolina" /> nahan;citation_author=J. Folkman;citation_title=Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.;citation_pages=353-364;citation_volume=86;citation_year=1996;citation_issue=3;citation_pmid=8756718;citation_doi=10.1016/S0092-8674(00)80108-7" /> view of anticancer therapy;citation_journal_abbrev=Expert Rev Anticancer Ther;citation_author=R. Pries;citation_author=S. Nitsch;citation_author=B. Wollenberg;citation_title=Role of cytokines in head and neck squamous cell carcinoma.;citation_pages=1195-1203;citation_volume=6;citation_year=2006;citation_issue=9;citation_pmid=17020454;citation_doi=10.1586/14737140.6.9.1195" /> ontent="citation_journal_title=New England Journal of Medicine;citation_journal_abbrev=NEJM;citation_author=RB. D'Agostino;citation_title=Changing end points in breast-cancer drug approval--the Avastin story.;citation_pages=e2-e2;citation_volume=365;citation_year=2011;citation_issue=2;citation_issn=0028-4793;citation_pmid=21707384;citation_doi=10.1056/NEJMp1106984" /> ce" content="citation_journal_title=Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research;citation_journal_abbrev=J Interferon Cytokine Res;citation_author=W. Zhao;citation_author=M. Liu;citation_author=NJ. D'Silva;citation_author=KL. Kirkwood;citation_title=Tristetraprolin regulates interleukin-6 expression through p38 MAPK-dependent affinity changes with mRNA 3' untranslated region.;citation_pages=629-637;citation_volume=31;citation_year=2011;citation_issue=8;citation_pmid=214ce" content="citation_journal_title=Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research;citation_journal_abbrev=J Interferon Cytokine Res;citation_author=W. Zhao;citation_author=M. Liu;citation_author=. Roussou;citation_title=Relationship between circulating serum soluble interleukin-6 receptor and the angiogenic cytokines basic fibroblast growth factor and vascular endothelial growth factor in multiple myeloma.;citation_pages=19-23;citation_volume=82;citation_year=2003;citation_issue=1;citation_pmid=12574959" /> . Roussou;citation_title=Relationship between circulating serum soluble interleukin-6 receptor and the angiogenic cytokines basic fibroblast growth factor and vascular endothelial growth factor in multiple myeloma.;citation_pages=19-23;citation_volume=82;citation_year=2003;citation_issue=1;citation_pmid=12574959" /> ;citation_doi=10.1016/S0165-6147(00)01446-2" /> ted extracellular activation of progalanin in angiogenesis;citation_pages=999-1004;citation_volume=430;citation_year=2013;citation_pmid=23261456;citation_doi=10.1016/j.bbrc.2012.11.124" /> citation_author=EA. Van Tubergen;citation_author=R. Banerjee;citation_author=M. Liu;citation_author=R. Vander Broek;citation_author=E. Light;citation_author=S. Kuo;citation_title=Inactivation or Loss of TTP Promotes Invasion in Head and Neck Cancer via Transcript Stabilization and Secretion of MMP9, MMP2, and IL-6;citation_volume=19;citation_year=2013;citation_pmid=23349315;citation_doi=10.1158/1078-0432.CCR-12-2927" /> ibits tumor growth in oropharyngeal squamous cell carcinoma.;citation_pages=585-596;citation_volume=168;citation_year=2006;citation_issue=2;citation_issn=0002-9440;citation_pmid=16436672;citation_doi=10.2353/ajpath.2006.050132" /> by cytokine-stimulated human retinal pigment epithelial cells.;citation_pages=361-368;citation_volume=54;citation_year=1992;citation_issue=3;citation_pmid=1381679;citation_doi=10.1016/0014-4835(92)90048-W" /> ation_year=2006;citation_issue=3;citation_pmid=16611409;citation_doi=10.1593/neo.05691" /> ation_issue=16;citation_pmid=16931911;citation_doi=10.4161/cc.5.16.3018" /> en-activated protein kinase activation.;citation_pages=883-887;citation_volume=20;citation_year=2007;citation_issue=6;citation_pmid=17982698" /> edunskas;citation_author=P. Amornphimoltham;citation_title=Decreased lymphangiogenesis and lymph node metastasis by mTOR inhibition in head and neck cancer;citation_volume=71;citation_year=2011;citation_pmid=21975930;citation_doi=10.1158/0008-5472.CAN-10-3192" /> liferation in squamous carcinoma cells: activation of the extracellular signal regulated kinase pathway and induction of cyclin-dependent kinase inhibitors.;citation_pages=5762-5771;citation_volume=26;citation_year=2007;citation_issue=39;citation_pmid=17384686;citation_doi=10.1038/sj.onc.1210384" /> icson;citation_title=Galanin in pituitary adenomas.;citation_pages=127-139;citation_volume=117;citation_year=2004;citation_issue=2;citation_pmid=14700749;citation_doi=10.1016/j.regpep.2003.10.022" /> iiiiicicson;citation_title=Galanin in pituitary adenomas.;citation_pages=127-139;citation_volume=117;citation_year=2004;citation_issue=2;citation_pmid=14700749;citation_doi=10.10115cDWjsK6KrFlQVXQix9YgNeYysf22XZHj-Y-c__unwnZ7RrqytOjdCtj4wVGbOpirsyh1KHZtJLYZw1QVM.css" media="all" /> 11515cDWjsK6KrFlQVXQix9YgNeYysf22XZHj-Y-c__unwnZ7RrqytOjdCtj4wVGbOpirsyh1KHZtJLYZw1QVM.css" media="all" /> 115cDWjsK6KrFlQVXQ15cDW)))k kkk kk k kkaa aaaaass ss px) anpx) apx) and (min-width: 768px) and (orientation:landscapecode.com/svn/trunk/html5.js"> ecode.com/ecode.com/svn/trunk/html5.js"> ecode.com/svn/trunk/ecode.comecode.com/secode.com/svn/trunk/html5.js"> ecode.com/svn/trunk/html5.js"> ecode.com/svnecodecode.com/svn/trecode.com/svn/trunk/html5.js"> ecode.com/svn/trecodecode.ecode.com/svn/trunk/html5.js"> data-hide-link-title="0" target="_blank" class="datadata-hdata-hide-link-title="0" target="_blank" class="" data-icon-position="">Cancer Epidemiodata-hide-link-title="0" tdatadata-hdata-hide-link-title="0" target="_blank" class="" data-icon-position="">Cancer Epidemiology, Biodata-hide-link-title="0" tdatadata-hdata-hide-link-title="0" target="_blank" class="" data-icon-position="">Cancer Edata-hide-link-datadata-hdata-hide-link-title="0" target="_blank" class="" data-icon-position="">Cancer Epidemdata-hide-link-title="0"datadata-hdata-hide-link-title="0" target="_blank" class="" data-icon-position="">Cadata-hide-link-title="0" datadata-hdata-hide-link-title="0" target="_blank" class="" data-icon-position="">Cancer data-hide-link-title="0" targdatadata-hdata-data-hdata-data-hide-ldata-hidedata-hidata-hdata-hide-link-title="0" target="_blank" class="" data-icon-position="">Cancer Epidemiology, Biomarkers & Prevention
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    lina, Columbia, South Carolinalina, Columbia, South Carolinalina, Columbia, South Carolinalina,lina, Columbia, South Carolinalina, Columbia, South Carolina
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    Joel P. Lints
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  • Keith L. Kirkwood
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    enesis via p38-MAPK?mediated secretion of proangiogenic cytokines, VEGF, and interleukin-6 (IL-6). Moreover, GALR2 activated small-GTP-protein, RAP1B, thereby inducing p38-mediated inactivation of tristetraprolin (TTP), which functions to destabilize cytokine transcripts. This resulted in enhanced secretion of proangiogenic cytokines and angiogenesis enesis via penesis viaenesis via enesis via p38-MAPK?mediated secretion of proangiogenic cytokines, VEGF, and interleukin-6 (IL-6). Moreover, GALR2 activated small-GTP-protein, RAP1B, thereby inducing p38-mediated inactivation of tristetraprolin (TTP), which functions to destabilize cytokine transcripts. This resulted in enhanced secretion of proangiogenic cytokines and angiogenesis in vitro and in vivo. In SCCHN cells overexpressing GALR2, inactivation of TTP increased secretion of IL-6 and VEGF, whereasenesis via p38-MAPK?mediated secretion of proanenesisenesenesisenesis via p38-MAPK?enesis via p38-MAPK?mediated secretion of proangiogenic cytokines, VEGF, and interleuenesis enesenesienesisenesis via p38-MAPK?mediated secretion enesis via p38-Menesisenesis via p38-MAPK?mediated secretion of proangiogenic cytokines, VEGF, and interleukin-6 (IL-6). Moreover, GALR2 activated small-GTP-protein, RAP1B, thereby inducing p3eenesis via p38-MAPK?mediated secretion of proangiogenic cytokines, VEGF, and interleukin-6 (IL-6).eenesis via p38-MAPK?mediated secretion of proangiogenic cytokines, VEGF, and interleukin-6 (IL-6). Moreover, GALR2 activated small-GTP-protein, RAP1B, thereby inducing p38-mediated inactivationenesenesis via p38-MAPK?mediated secretion of proangiogenic cytokines, VEGF, and interleukin-6 (IL-6). Moreover, GALR2 activated small-GTP-enesisenesis veenesis via p38-MAPK?mediated secretion of proans tumor growth and spread (ss tumos tums tumor growth and spread (2).

    s tus tumor growth and spread (ss tumor growth and spread (ss tumor growth and spread (2).

    In SCCHN, angiogenesis is activated by cytoss tumor growth and spread (2).

    In SCCHN, angiogenesis is activated by cytokines, including interleukin-6 (IL-6) and VEGF (p38-MAPK is a critical signaling molecule t"p-8""p-8">p38-MAPK is a critical signaling molecule that activates cytokines. In SCCHN and multiple myeloma, p38 is constitutively active and promotes tumor gro"p"p-8">p38-MAPK is a critical signaling molecule that activates cytokines. In SCCHN and multiple myeloma"p-8">"p-8">p38-MAPK is a critical signaling molecule that activates cytokines. In SCCHN and multiple myeloma, p38 is constitutively ac"p"p-8">p38-MAPK is a critical signaling molecule that activates cytokines. In SCCHN a"p"p-8">p38-MAPK is a critical signaling molecule that activates cytokines. In SCCH"p"p-8">"p"p-8">p38-MAPK is a critical signaling molecule that activates c"p"p-8">p38-MAPK is a critical signaling molecule that activates cytokines. In SCCHN and multipl"p"p-8">"p"p-8">p38-MAPK is a critical signaling molecule that activates cytokines. In SCCHN and multiple myeloma, p38 is constitutively a"p"p-8">"p-8""p-8">p38-MAPK is a critical signaling molecule that activates cytokines. In SCCHN and mult"p"p-8">p38-MAPK is a critical signaling molecule that activates cytokines. In SCCHN and multis, Yamamoto and colleagues (s,s, Yamamoto and colleagues (23) recently showed that GAL stimulates angiogenesis but the signaling cascade was uncharacterized. In the present study, we show that GAL induces angiogenesis via GALR2-induced, RAP1B-p38?mediated IL-6 and VEGF secretion. RAP1B, a ras-like protein, shuttles between inactive GDP- and active GTP-bound forms. Given the importance of proangiogenic cytokines such as IL-6 and VEGF in tumor progression, inhibition of secretion of these factors by targeting a common upstream s, Ys, Yams, Yamamoto and colleagues (16).

    ibedibed (16).

    For stable knockdown of TTP, UM-SCC-ibibed (16).

    For stable knockdown of TTP, UM-SCC-1 and -81B cells wibed (ibed (icicate in 150 ?L of conditioned medium from cells transfected with nontarget (NT), sip38, siTTP, or siIL-6. VEGF (1 ng/mL) was used as a positive control in initial experiicaticate iicate in 150 ?L of conditioned mediumicate in 150 ?L of conditiicate icate in 150 ?L of conditioned medium from cells transfected with nontarget (NT), sip38, siTTP, or siIL-6. VEGF icaticate iicate in 150 ?L of conditioned mediumicaticate in 15icate in 150 ?L of conditioned medium icate icate in 150 ?L oicate in 15icate in 150 ?L of conditioned medium from cells transfected with nontarget (NT), sip38, siTTP, or siIL-6. VEGF (1 ng/mL) was used as a positive control in initial expericate icate in 150 ?L of conditioned medium icateicate in 150 ?L of conditioned medium from cells transfected with nontarget (NT), sip38, siTTP, or siIL-6. VEGF (1 ng/mL) was used as a icicate in 150 ?L of conditioned medium from cells transfected with nontarget (NT), sip38, siTTP, or siIL-6. VEGF (1 ng/mL) was used as a positive control in initial experiments (not shown). DMEM served as a negative control. Digital images of endothicate icate in 150 ?L of conditioned medium from cells transfected with nontargeticicate in 150 ?L of hemistry was performed on tissue sections for pancytokeratin (Millipore; ref. hehemistry was performed on tishemihemistrhemistry was performed on tissue secthemistry was pehemisthemistry was performed on tissue sections for pancytokeratin (Millipore; ref. hehemistry was perfohemistry was performed on tissue sections for pancytohemistry was performed on tissue sections fhemihemistrhemistry was performed on tissue secthemistry was performed ohemisthemistry was performed on tissue sections for pancytokeratin (Mihemishemistry washemishemistry was performed on tissue sections for pancytokeratin (Millipore; ref. hehemistry was performed on tissue sections for pancytokeratin (Millipore; ref. 29) and Facthemistrhemistry was performed on tissue sections for pancytokeratin (Millipore; ref. 29) and Factor-VIII (DAKO).

    Chicken CAM

    Cells were seeded on the chick chorioallantoic membrane (CAM), an in vivo model of angiogeneshemistrhemistry was performed on tissue sections for pancytokeratin (Millipore; ref. 29) and Factor-VIII (DAKO).controls were appropriately negative (not shown).contrcontrols were appropriately negative (nocontrols were appropriately ncontrols were appropriately negativecontrols were appropriately HMEC-1 cells and photographed atFigure 1.HMEC-1 celFigure 1.HMEC-1 cellHMEC-1 HMECHMEC-1HMEC-1HMEC-1 cells and photographed at 24 hoursHMEC-1 cells and photographed aHMEC-1 cells anHMECHMEC-HMEC-1 cells and phoHMEC-1 cells anHMECHMEC-HMEC-1 cells and photographed HMEC-1 cells and phHMECHMEC-HMEC-HMEC-1HMEC-1 cells and photographed at 24 hours (middle left). Both averagHMEC-1 cells and photographed at HMEC-1 ceHMEC-1 cells and photographed at 24 hours (middle left). Both average number of tubes and length of tubes were calculated from 10 representative fields (*, <em>P</em> < 0.01HMEC-HMEC-1 cells and photographed at 24 hours (middle left). Both average number of tubes and lengtrol is shown (bar, 1cm). The neovasculature was quantified and graphed (middle left; *, rol irol is shown (bar, 1cm). The neovasculature was quantified and graphed (middle left; *, P < 0.007). Both control and UM-SCC-1-GALR2 tumor sections were stained with hematoxylin and eosin (H&E), Factor-VIII, and cytokeratin (middle right). Number of vessels from endothelialrol irol is shown (bar, 1cm). The neovasculature was quantified and graphed (middle left; *, PP < 0.007). Both control and UM-SCC-1-GALR2 tumor sections were stained with hematoxylin and eosin (H&E), Factor-VIII, and cytokeratin (middle right). Number of vessels from endothelial cells was quantified from each group from five representative fields and gPem>P < 0.012). Histology of the corresponding tumors shows vasculature in UM-SCC-1-GALR2 (arrowheads) and control tumors (right;em>PP < 0.012). Histology of the corresponding tumors shows vasculature in UM-SCC-1-GALR2 (arrowheads) and control tumors (right; *, P < 0.008). C, UM-SCC-1-GALR2 and pcDNA cells were immunoblotted with GALR2 and GAPDH antibodies. Band intensities were quantified by ImageJ software and expressed as arbitrary densitometric unit (DU) normalized to control (left). Conditioned media from UM-SCC-1-GALR2 and pcDNA cells were normalized to equal cell number and incubated overnight on Matrigel seeded with HMEC-1 cellem>PP < 0.012). Histology of the corresponding tumors shows vasculature in UM-SCC-1-GALR2 (arrowheads) and control tumem>PP < 0.012). Histology oem>PPPP < 0.012). Histology of the corresponding tumors shows vasculature in UM-SCC-1-GALR2 (arrowhemem>P < 0.012). Histology of the corresponding tumors shows vasculature in UM-SCC-1-GALR2 (arrowheads) and controem>PP < 0.012). Histology of the correm>PP &emem>P < 0.012). Histology of the coem>PP < 0.012). Histology of the corresponding tumors shows vasculature in UM-SCC-1-GALR2 (arrowheads) and control tumors (right; *, P < 0.008). C, UM-SCC-1-Gem>PP < 0.012). Histology of the corresponding tumors shows vasculature in UM-SCC-1-GALR2 (arrowheads) and control tumors (rigem>PP < 0.012). Histology of the corresponding tumors shows vasculature in UM-SCC-1-GALR2 (arrowheads) and control tumors (right; *, P < 0nsistent with a role for GALR2 in angiogenesis, the GALR2-specific inhibitor M871 reduced vascularity nsistent winsistent with a role for GALR2 in angiogenesis, the GALR2-specific inhibitor M871 reduced vascularity in vivo (Supplensistent witnsistent with a role for GALR2 in angiogenesis, the GALR2-specific inhibitor M871 reduced vascularity in vivo (Supplementary Fig. S2). To verify whether the enhanced vascularity is mediated by cytokines secreted from tumor cells, in vitro sprouting assays were perfonsistennsistent with a role for GALR2 in angiogenesis, the GALR2-specific inhibitor M871 reduced vasculnsistennsistent with a role for GALR2 in angiogenesis, the GALR2-specific inhibitor Mnsistent witnsistent wnsistent winsistent with a role for GALR2 in angiogenesis, the GALR2-specific inhibitor M871 reducnsisnsistennsistent with a role for GALR2 in angnsistent with a role for GALR2 in angiogenesis, the GALR2-specific inhinsistensistent with a role for GALR2 in angiogenesis, the GALR2-specific nnsistent with a role for GALR2 in angiogenesis, the GALR2-specific inhibitor M871 reduced vascularity in vivo (Supplementary Fig. S2). To verify whether the enhanced vascularity is mediated by cytokines secreted from tumor cells, in vitro sprouting assays were performed. Endothelial cells (HMEC-1) were incubated with conditioned media from UM-SCC-1-GALR2 or control cells. The number and length of tubes (arrows) were increased in HMEC-1 cellnsistent withnsistent with a role for GALR2 in angiogenesis, the GALR2-specific inhibitor M871 reduced vascularity in vivo (Supplementary Fig. S2). To verify whether the enhanced vascularity is mediated by cytokines secretednsistennsistent witnsistnsistent with a role for GALR2 in angiog

    class="highwire-figure">
    Figure 2.

    GALR2 stimulates cytokine secretion and angiogenesis via RAP1B-p38-MAPK. A, stable mixed clonal population of UM-SCC-1-GALR2 or control pcDNA cells were serum starved for 4 hours and treated with 10 nmol/L Galanin for 0, 2, 5, or 10 minutes. Whole-cell lysates were immunoblotted for phospho-p38 and p38 antibodies, quantified (DU), and expressed as percentage of control (left). UM-SCC-1-GALR2 stable cells were treated with NT-siRNA or si-RAP1B. After 68 hours of transfection, cells were serum starved for 4 hours and were either stals.oals.org/content/molcanther/13/5/1323/F2.large.jpg" class="highwire-figure-link highwire-figure-link-newtab" target="_blank" data-icon-position="" data-hide-link-title="0">Open in new tab

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    We previously showed that p38 phosphorylates TTP, which destabilizes transcripts including matrix metalloproteinases and IL-6 cretioncretion

    We previously showed that p38 phosphorylates TTP, which destacretioncretion

    We previously showed that p38 phosphorylates TTP, which descrcretiocreticretion

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    Webated with corresponding conditiFigure 3.bated withFigure 3.bated with bated wbatebated bated bated with corresponding conditioned medibated with corresponding conditbated with corrbatebatedbated with corresponbated with corrbatebatedbated with corresponding condibated with correspobatebatedbatedbated bated with corresponding bated with corresponding conditiobated witbated with corresponding conditioned medium collected from cells. tion of TTP to enhance angiogenesis. A, schematic representation of signaling pathway showing GALR2-mediated phosphorylation and activation of p38 that inhibits TTP via phosphorylation. Inactivation or genetic loss (by siRNA) of TTP prevents cytokine degradation giving rise to overall steady state increase in cytokines and acts as angiogenic switch to tumor growth. Chemical inhibitor (SB203580) to p38 kinase activity can dephosphorylate TTP to its active form and degrade cytokines. Alternatively targeting individual cytokines by siRNA could be used to control this angiogenic signal. Upstream inhibitors to angiogenesis might be a better treatment option (left). UM-SCC-1-GALR2 cells were serum starved for 4 hours and then treated with 10 ?mol/L of SB203580 for 2 hours and were then stimulated with 10 nmol/L galanin for 10 minutes or vehicle control. Clarified cell lysates were immunoprecipitated with TTP antibody, and blotted with anti-TTP and anti-phosphoserine antibodies (right). B, UM-SCC-1-GALR2 cells were transfected with siTTP or NT-siRNA and were treated with 10 ?mol/L of SB203580 or vehicle control. Cell lysates were immunoblotted with VEGF, IL-6, TTP, and actin antibodies (left). Conditioned medium was collected from each of the treatment groups and ELISA was performed for VEGF and IL-6 (right) and was quantified as pg/mL/million cells and was finally normalized to control. C, UM-SCC-1-GALR2 cells were transduced with shVEGF and control shRNA lentiviral particles and immunoblotted with VEGF antibody and GAPDH. tion of TTP tion of TTP to enhance angiogenesis. A, schematic representation of signaling pathway showing GALR2-mediated phosphorylation and activation of p38 that inhibits TTP via phosphorylation. Inactivation or genetic loss (by siRNtion tion of TTP to enhance angiogenesis. A, schematic representation of signaling pathway showing GALR2-mediated phosphorylation and acttion of TTP tion of TTP to enhance angiogenesis. A, schematic representation of signaling pathway showing GALR2-mediated phosphorylight; *, ight;ight; *, P < 0.02). Data are representative of three identical experiments ight;ight; *, P < 0.02). Daight; ight; ight; *ight; *, P < 0.02). Data are representative of three identical experiments in triplicate.

    igight; *, P < 0.02). Data are representative of three identical experiments inight; *ight; *, P < 0.02). Data are representative of thr were incubated with SB203580 or vehicle. Compared with baseline, the p38 inhibitor, which increases functional TTP, reduced expression and secretion of VEGF in cell lysate (e incube incubated with SB203580 or vehicle. Compared with baseline, the p38 inhibitor, which increases functional TTP, reduced expression and sece incube incubated with SB203580 or vehicle. Compared with baseline, the p38 inhibitor, which increases functional TTP, reduced expression and secretion of VEGF in cell lysate (Fig. 3B, left, lanes 1 and 2) and conditioned medium (middle graph, bars 1 and 2), respectively. In contrast, siTTP enhanced VEGF expression (e incube incubated with SB203580 or vehicle. Comparee incube incubated with SB203580 or vehicle. Compared with baseline, the p38 inhibitor, whie e incubated with SB203580 or vehicle. e incube incubated e ince incubated with SB203580 or vehicle. Compared with be incue incubated with SB203580 or vehicle. Compared with e incue incubated with SB203580 or vehicle. Compared with baseline, the p38 inhibitor, which increases functional TTP, reduced expression and secretion of VEGFe incube incubated with SB203580 or vehicle. Compared with baseline, the p38 inhibitor, which increases functional TTP, reduced expression ane incube incubated with SB203580 or vehicle. Compared with baseline, the p38 inhibitor, which increases functional TTP, reduced expression and secretion e incube incubated with SB20358unction experiments with siIL-6 in UM-SCC-1-GALR2 had a similar effect and also decreased endothelial sprouting (unctionunctiounctunctionunction experiments with siIL-6 in UMunction experiments with siIL-6 in UM-SCC-1-unctiounction experiments with siIL-6 in UM-SCC-1-GALR2 had a similar effect and also decreased enunction experunction experiments with siIL-6 in UM-SCC-1-GALR2 had a similar effect and also decreased endothelial sprouting (Fig. 3D).

    unctionunction experiments with siIL-6 in UM-SCC-1-GALR2 had a similar effect and also decreased endothelial sprounctionunction experiments with siIL-6 in UM-SCC-1-GALR2 had a similar effect and also decreased endothelial sprouting (Fig. 3D).

    unctionunction experiments with siIL-6 in UM-SCC-1-GALR2 had a similar effect and also decreased endothelial sprouting (unctionunction experiments with siIL-6 in UM-SCC-1-unctiunction experiments with siIL-6 in UM-SCunction experiments with siILunction experiments with siIL-6 in Uunction experiments with siIresentative fields (middle and rFigure 4.resentativFigure 4.resentativeresentareseresentresentresentative fields (middle and right, resresentative fields (middle and resentative fiereseresenresentative fields (-icon-position=-ico-icon-icon-position="" data-hide-li-icon-position="" d-ico-icon-icon-icon--icon-position="" data-hi-icon-position="" data-hide-link--icon-pos-icon-position="" data-hide-link-title="0">Open in new tab
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