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• W2554695763 abstract "Hepatocyte growth factor (HGF) signaling via c-Met is known to promote endothelial cell motility and angiogenesis. We have previously reported that HGF stimulates lamellipodia formation and motility of human lung microvascular endothelial cells (HLMVECs) via PI3K/Akt signal transduction and reactive oxygen species generation. Here, we report a role for HGF-induced intracellular sphingosine-1-phosphate (S1P) generation catalyzed by sphingosine kinase 1 (SphK1), S1P transporter, spinster homolog 2 (Spns2), and S1P receptor, S1P1, in lamellipodia formation and perhaps motility of HLMVECs. HGF stimulated SphK1 phosphorylation and enhanced intracellular S1P levels in HLMVECs, which was blocked by inhibition of SphK1. HGF enhanced co-localization of SphK1/p-SphK1 with actin/cortactin in lamellipodia and down-regulation or inhibition of SphK1 attenuated HGF-induced lamellipodia formation in HLMVECs. In addition, down-regulation of Spns2 also suppressed HGF-induced lamellipodia formation, suggesting a key role for inside-out S1P signaling. The HGF-mediated phosphorylation of SphK1 and its localization in lamellipodia was dependent on c-Met and ERK1/2 signaling, but not the PI3K/Akt pathway; however, blocking PI3K/Akt signaling attenuated HGF-mediated phosphorylation of Spns2. Down-regulation of S1P1, but not S1P2 or S1P3, with specific siRNA attenuated HGF-induced lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P1 that was blocked by inhibiting SphK1 activity with PF-543. Moreover, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2. Taken together, these results suggest that HGF/c-Met-mediated lamellipodia formation, and perhaps motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2-mediated extracellular transportation of S1P and its inside-out signaling via S1P1. Hepatocyte growth factor (HGF) signaling via c-Met is known to promote endothelial cell motility and angiogenesis. We have previously reported that HGF stimulates lamellipodia formation and motility of human lung microvascular endothelial cells (HLMVECs) via PI3K/Akt signal transduction and reactive oxygen species generation. Here, we report a role for HGF-induced intracellular sphingosine-1-phosphate (S1P) generation catalyzed by sphingosine kinase 1 (SphK1), S1P transporter, spinster homolog 2 (Spns2), and S1P receptor, S1P1, in lamellipodia formation and perhaps motility of HLMVECs. HGF stimulated SphK1 phosphorylation and enhanced intracellular S1P levels in HLMVECs, which was blocked by inhibition of SphK1. HGF enhanced co-localization of SphK1/p-SphK1 with actin/cortactin in lamellipodia and down-regulation or inhibition of SphK1 attenuated HGF-induced lamellipodia formation in HLMVECs. In addition, down-regulation of Spns2 also suppressed HGF-induced lamellipodia formation, suggesting a key role for inside-out S1P signaling. The HGF-mediated phosphorylation of SphK1 and its localization in lamellipodia was dependent on c-Met and ERK1/2 signaling, but not the PI3K/Akt pathway; however, blocking PI3K/Akt signaling attenuated HGF-mediated phosphorylation of Spns2. Down-regulation of S1P1, but not S1P2 or S1P3, with specific siRNA attenuated HGF-induced lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P1 that was blocked by inhibiting SphK1 activity with PF-543. Moreover, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2. Taken together, these results suggest that HGF/c-Met-mediated lamellipodia formation, and perhaps motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2-mediated extracellular transportation of S1P and its inside-out signaling via S1P1. Migration of vascular endothelial cells (ECs) 2The abbreviations used are: EC, endothelial cell; HGF, hepatocyte growth factor; HLMVEC, human lung microvascular endothelial cell; S1P, sphingosine-1-phosphate; SphK1, sphingosine kinase 1; SphK2, sphingosine kinase 2; Spns2, spinster homolog 2; S1PR, sphingosine-1-phosphate receptor; PI3K, phosphatidylinositol-3-kinase; EBM, endothelial basal medium; IP, immunoprecipitation; ROS, reactive oxygen species; EGM-2, endothelial growth medium 2; PCC, Pearson–s correlation coefficient. is a complex process involving protrusion, adhesion, contraction, and retraction, which is important for a variety of physiologic and pathologic conditions such as vasculogenesis, angiogenesis, wound healing, and atherogenesis (1.Lamalice L. Le Boeuf F. Huot J. Endothelial cell migration during angiogenesis.Circ. Res. 2007; 100: 782-794Crossref PubMed Scopus (1042) Google Scholar, 2.Mudau M. Genis A. Lochner A. Strijdom H. Endothelial dysfunction: the early predictor of atherosclerosis.Cardiovasc. J. Afr. 2012; 23: 222-231Crossref PubMed Scopus (324) Google Scholar). Emerging evidence suggests that formation of protruding structures, termed lamellipodia, generated at the leading edge of migrating ECs in response to a variety of growth factors, are primarily involved in cell motility (3.Krause M. Gautreau A. Steering cell migration: lamellipodium dynamics and the regulation of directional persistence.Nat. Rev. Mol. Cell Biol. 2014; 15: 577-590Crossref PubMed Scopus (356) Google Scholar). Earlier studies have demonstrated the involvement of actin microfilaments, microtubules, and intermediate filaments in the generation of lamellipodia and EC motility (4.Fletcher D.A. Mullins R.D. Cell mechanics and the cytoskeleton.Nature. 2010; 463: 485-492Crossref PubMed Scopus (1773) Google Scholar). The driving force for EC motility is fueled by continuous growth of actin filaments and rearrangement of the actin binding protein cortactin in the lamellipodia. The lamellipodia formation is mediated by Rac and Cdc42, which regulate Arp2/3 complex through the Wiskott-Aldrich protein, while Rho GTPase regulates actomyosin contractility via stress actin fiber formation and focal adhesions (5.Nobes C.D. Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3729) Google Scholar). Hepatocyte growth factor (HGF), also known as scatter factor, promotes EC migration, barrier enhancement, and tumorigenesis through ligation to its receptor, c-Met (6.Bussolino F. Di Renzo M.F. Ziche M. Bocchietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1202) Google Scholar7.Cai W. Rook S.L. Jiang Z.Y. Takahara N. Aiello L.P. Mechanisms of hepatocyte growth factor-induced retinal endothelial cell migration and growth.Invest. Ophthalmol. Vis. Sci. 2000; 41: 1885-1893PubMed Google Scholar, 8.Ephstein Y. Singleton P.A. Chen W. Wang L. Salgia R. Kanteti P. Dudek S.M. Garcia J.G. Jacobson J.R. Critical role of S1PR1 and integrin β4 in HGF/c-Met-mediated increases in vascular integrity.J. Biol. Chem. 2013; 288: 2191-2200Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 9.Liu F. Schaphorst K.L. Verin A.D. Jacobs K. Birukova A. Day R.M. Bogatcheva N. Bottaro D.P. Garcia J.G. Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement: potential role of glycogen synthase kinase-3β.FASEB J. 2002; 16: 950-962Crossref PubMed Scopus (145) Google Scholar10.Webb C.P. Taylor G.A. Jeffers M. Fiscella M. Oskarsson M. Resau J.H. Vande Woude G.F. Evidence for a role of Met-HGF/SF during Ras-mediated tumorigenesis/metastasis.Oncogene. 1998; 17: 2019-2025Crossref PubMed Scopus (69) Google Scholar). c-Met is a receptor tyrosine kinase and ligand binding triggers autophosphorylation at multiple tyrosine sites that serves as a docking platform for recruitment of several adapter proteins such as Grb2, SHC, Crk/CrkL, and Gab1. These adapter proteins in turn recruit several signal transducing proteins to form an intricate signaling complex (11.Organ S.L. Tsao M.S. An overview of the c-MET signaling pathway.Ther. Adv. Med. Oncol. 2011; 3: S7-S19Crossref PubMed Scopus (542) Google Scholar). HGF binding to c-Met triggers downstream signaling cascade such as PI3K, Akt, and ERK1/2 activation and promotes EC migration (11.Organ S.L. Tsao M.S. An overview of the c-MET signaling pathway.Ther. Adv. Med. Oncol. 2011; 3: S7-S19Crossref PubMed Scopus (542) Google Scholar). HGF-induced EC migration is also up-regulated by enhanced expression of inducible nitric-oxide synthase, but not endothelial NOS, and partially abrogated by PI3K inhibition (12.Purdie K.J. Whitley G.S. Johnstone A.P. Cartwright J.E. Hepatocyte growth factor-induced endothelial cell motility is mediated by the upregulation of inducible nitric oxide synthase expression.Cardiovasc. Res. 2002; 54: 659-668Crossref PubMed Scopus (35) Google Scholar). We have recently demonstrated that HGF stimulated c-Met phosphorylation at Tyr1234/1235, Tyr1349, Tyr1003, and Tyr1313, as well as Ser985 and Akt phosphorylation at Thr308 and Ser473, and potentiated lamellipodia formation in HLMVECs (13.Usatyuk P.V. Fu P. Mohan V. Epshtein Y. Jacobson J.R. Gomez-Cambronero J. Wary K.K. Bindokas V. Dudek S.M. Salgia R. Garcia J.G. Natarajan V. Role of c-Met/phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.J. Biol. Chem. 2014; 289: 13476-13491Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Further, HGF-stimulated NADPH oxidase-dependent reactive oxygen species (ROS) production in lamellipodia and inhibition of c-Met/PI3K/Akt signaling axis and NADPH oxidase attenuated lamellipodia formation and motility of lung ECs (13.Usatyuk P.V. Fu P. Mohan V. Epshtein Y. Jacobson J.R. Gomez-Cambronero J. Wary K.K. Bindokas V. Dudek S.M. Salgia R. Garcia J.G. Natarajan V. Role of c-Met/phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.J. Biol. Chem. 2014; 289: 13476-13491Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). During our investigation into mechanism(s) of EC lamellipodia formation, we observed that down-regulation of sphingosine kinase (SphK) 1 or sphingosine-1-phosphate (S1P) transporter, Spns2, partially abrogated HGF-induced lamellipodia formation. Further, inhibiting c-Met tyrosine kinase with SU11274 attenuated intracellular S1P levels in lung ECs. These results suggested a cross-talk and potential interaction between HGF/c-Met and SphK/S1P/S1P receptor (S1PR) signaling axis in lamellipodia formation and EC motility; however, the mechanism(s) of regulation of HGF-mediated lamellipodia formation and EC migration by SphK/S1P/S1PR signaling axis is unclear. Here, we determined the role of SphK1, Spns2, and S1PR in HGF-induced lamellipodia formation in lung ECs. Our results demonstrated that HGF/c-Met signaling stimulated phosphorylation of ERK1/2, which in turn phosphorylated SphK1. Both ERK1/2 and SphK1, but not SphK2, were localized in lamellipodia. Further, HGF stimulation of lung ECs increased intracellular S1P levels via activation of SphK1 and blocking SphK1 or Spns2 abrogated HGF-induced lamellipodia formation. Additionally, knocking down of S1P1, but not S1P2 or S1P3, with siRNA attenuated HGF-induced lamellipodia formation. Collectively, these results identify cross-talk between HGF/c-Met and SphK1/Spns2/S1P signaling as a novel pathway of lamellipodia formation and perhaps EC motility. In our recent study, we have shown a significant role for HGF-induced c-Met/PI3K/Akt signaling in NADPH oxidase activation and ROS generation in lamellipodia formation and motility of human lung ECs (13.Usatyuk P.V. Fu P. Mohan V. Epshtein Y. Jacobson J.R. Gomez-Cambronero J. Wary K.K. Bindokas V. Dudek S.M. Salgia R. Garcia J.G. Natarajan V. Role of c-Met/phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.J. Biol. Chem. 2014; 289: 13476-13491Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). HGF binding to c-Met triggers a cascade of signal transduction pathways including transactivation of G protein-coupled S1P receptors (8.Ephstein Y. Singleton P.A. Chen W. Wang L. Salgia R. Kanteti P. Dudek S.M. Garcia J.G. Jacobson J.R. Critical role of S1PR1 and integrin β4 in HGF/c-Met-mediated increases in vascular integrity.J. Biol. Chem. 2013; 288: 2191-2200Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar); however, the role of this transactivation in lamellipodia formation and cell motility is unclear. We therefore determined the role of HGF/c-Met signaling axis in SphK-mediated S1P generation in lamellipodia formation and EC motility. HLMVECs expressed both the isoforms of SphK, SphK1 and SphK2; however, stimulation with HGF (20 ng/ml) resulted in a time-dependent phosphorylation of SphK1 (Ser225) with no detectable phosphorylation of SphK2 (Thr578) (Fig. 1, A and B). The HGF-induced activation of SphK1 resulted in enhanced accumulation of intracellular S1P levels, which was blocked by PF-543 (1 μm), a potent and specific inhibitor of SphK1 (14.Schnute M.E. McReynolds M.D. Kasten T. Yates M. Jerome G. Rains J.W. Hall T. Chrencik J. Kraus M. Cronin C.N. Saabye M. Highkin M.K. Broadus R. Ogawa S. Cukyne K. Zawadzke L.E. Peterkin V. Iyanar K. Scholten J.A. Wendling J. Fujiwara H. Nemirovskiy O. Wittwer A.J. Nagiec M.M. Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1.Biochem. J. 2012; 444: 79-88Crossref PubMed Scopus (209) Google Scholar) (Fig. 1C). HGF enhanced intracellular S1P generation as early as 2 min post-challenge and peaked at 10 min followed by a gradual decline in accumulation (Fig. 1C). Further, cell lysates from HGF-stimulated HLMVECs exhibited enhanced SphK1 activity compared with control cells in vitro, which was blocked by the SphK1 inhibitor PF-543 (Fig. 1D). These results suggest that phosphorylation and activation of SphK1 by HGF enhances accumulation of S1P in HLMVECs. We have earlier demonstrated that HGF stimulated reorganization and co-localization of actin and cortactin in lamellipodia of HLMVECs (13.Usatyuk P.V. Fu P. Mohan V. Epshtein Y. Jacobson J.R. Gomez-Cambronero J. Wary K.K. Bindokas V. Dudek S.M. Salgia R. Garcia J.G. Natarajan V. Role of c-Met/phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.J. Biol. Chem. 2014; 289: 13476-13491Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar); however, the role of SphK1 in HGF-mediated lamellipodia formation is not well defined. Therefore, we hypothesized that HGF-induced lamellipodia formation, in part, is dependent on SphK1 activation and its redistribution to lamellipodia and co-localization with actin and cortactin cytoskeleton at cell periphery. Cells challenged with vehicle revealed diffused SphK1, actin, and cortactin staining; however, HGF stimulated F-actin (red) and SphK1 (green) (Fig. 2, A and B) and cortactin (green) and SphK1 (red) (Fig. 2, C and D) redistribution and co-localization (merge, yellow) to cell periphery. A similar co-localization of p-SphK1 (Ser225) with actin and cortactin in lamellipodia after HGF treatment was observed in HLMVECs (Fig. 3, A–D). These results suggest that HGF-induced phosphorylation of SphK1 may play a role in lamellipodia formation in human lung ECs.FIGURE 3.HGF stimulates SphK1 phosphorylation and accumulation at lamellipodia. HLMVECs grown on slide chambers were treated with HGF (20 ng/ml) or PBS for 30 min and probed with anti-actin, anti-p-SphK1(ser225), and anti-cortactin antibodies, and lamellipodia was examined by immunofluorescence microscopy with 60× oil objective. A and C, co-localization of actin (red) and p-SphK1 (green) (A) or p-SphK1 (red) and cortactin (green) to lamellipodia (merge, yellow) (C) was visualized by immunofluorescent staining as described under “Experimental Procedures.” Shown are representative images from three independent experiments. Insets depict enhanced co-localization of actin and p-SphK1 or cortactin and p-SphK1 in lamellipodia caused by HGF treatment. A–D, the intensity or distribution of p-SphK1 with actin (C) or cortactin (D) was quantified from the upper panels (A and C) using ImageJ software and expressed as relative pixel intensity. At least 20 cells were analyzed for each condition, and the results are representative of three independent experiments. Con, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Binding of HGF to its receptor, c-Met, induces dimerization and autophosphorylation of specific tyrosine residues, resulting in enhanced kinase activity and activation of diverse intracellular signaling pathways including MAPKs, STAT3, Rac1, and Akt, which promote lamellipodia formation and cell motility. To determine whether phosphorylation of SphK1 by HGF and p-SphK1 co-localization in lamellipodia with actin is mediated by c-Met signaling axis, we used SU11274, an inhibitor of c-Met tyrosine phosphorylation (15.Wang X. Le P. Liang C. Chan J. Kiewlich D. Miller T. Harris D. Sun L. Rice A. Vasile S. Blake R.A. Howlett A.R. Patel N. McMahon G. Lipson K.E. Potent and selective inhibitors of the Met [hepatocyte growth factor/scatter factor (HGF/SF) receptor] tyrosine kinase block HGF/SF-induced tumor cell growth and invasion.Mol. Cancer Ther. 2003; 2: 1085-1092PubMed Google Scholar). As demonstrated earlier (13.Usatyuk P.V. Fu P. Mohan V. Epshtein Y. Jacobson J.R. Gomez-Cambronero J. Wary K.K. Bindokas V. Dudek S.M. Salgia R. Garcia J.G. Natarajan V. Role of c-Met/phosphatidylinositol 3-kinase (PI3K)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells.J. Biol. Chem. 2014; 289: 13476-13491Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), HGF induced c-Met phosphorylation at residues Tyr1234/1235 in HLMVECs (Fig. 3A), and pretreatment of cells with SU11274 (1 μm) for 1 h prior to HGF treatment (20 ng/ml, 15 min) significantly attenuated HGF-induced c-Met phosphorylation at residues Tyr1234/1235, SphK1 phosphorylation at Ser225, and ERK1/2 phosphorylation (Thr202/Tyr204) (Fig. 4, A and B). As a result of inhibition of SphK1, S1P generation induced by HGF was significantly attenuated by SU11274 (Fig. 4C). Furthermore, inhibition of c-Met phosphorylation by SU11274 attenuated phospho-SphK1 (green) co-localization with actin (red) in lamellipodia (merge, yellow) (Fig. 4D). Taken together, these results establish a key role for HGF/c-Met signaling in SphK1 phosphorylation and co-localization with actin in lamellipodia of HLMVECs. Having established that SphK1/p-SphK1 is localized in lamellipodia in response to HGF treatment, we sought to determine whether SphK1 activity and/or protein expression is required for lamellipodia formation. Intrinsic SphK1 activity in HLMVECs was inhibited by PF-543, a specific inhibitor of SphK1, or SphK1 expression was down-regulated by siRNA transfection. As shown in Fig. 5 (A and B), pretreatment of HLMVECs with PF-543 (1 μm) for 1 h significantly attenuated HGF-induced lamellipodia formation as indicated by co-localization of actin (red) and cortactin (green) in the cell periphery (merge, yellow) by immunofluorescent staining. Similarly, knockdown of SphK1, but not SphK2, using specific siRNA significantly attenuated HGF-induced co-localization of actin (red) and cortactin (green) in lamellipodia (Fig. 5, C and D). Knockdown of SphK1 and SphK2 was confirmed by Western blotting (Fig. 5E). Furthermore, intrinsic SphK1 activity was inhibited by transfection of cells with catalytically inactive FLAG-tagged adenoviral SphK1 plasmid. In control cells, there was no difference between transfected and non-transfected cells in terms of cortactin localization; however, cells transfected with SphK1 mutant adenovirus did not show cortactin localization in lamellipodia after stimulation with HGF (data not shown). These results show a potential role for SphK1 activity and protein expression in HGF-induced lamellipodia formation in lung ECs. ERK1/2 has been shown to be phosphorylated in response to growth factors and localized to lamellipodial protrusion and adhesions (16.Mendoza M.C. Er E.E. Zhang W. Ballif B.A. Elliott H.L. Danuser G. Blenis J. ERK-MAPK drives lamellipodia protrusion by activating the WAVE2 regulatory complex.Mol. Cell. 2011; 41: 661-671Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Also, there is evidence that ERK1/2 mediates phosphorylation of SphK1 at Ser225 and other Ser/Thr residues in mammalian cells in response to external stimuli (17.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation.EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (457) Google Scholar). To further investigate the role of ERK1/2 and PI3K in SphK1 phosphorylation and lamellipodia formation, we used PD98059, an ERK1/2 inhibitor, and LY294002, a PI3K specific inhibitor. PD98059 significantly attenuated HGF-induced co-localization of p-SphK1 (green) with cortactin (red) in lamellipodia (merge, yellow) and redistribution of p-SphK1 in cell periphery (Fig. 6, A and B), as well as phosphorylation of ERK1/2 and SphK1 (Fig. 7C). Furthermore, immunofluorescent staining showed enhanced co-localization of phospho-SphK1 with phospho-ERK1/2 at lamellipodia in response to HGF stimulation (Fig. 7, A and B). However, the PI3K inhibitor LY294002 had no effect on HGF-induced SphK1 or ERK1/2 phosphorylation (Fig. 7, E and F). These results indicate that ERK1/2, but not PI3K, mediates phosphorylation of SphK1 and its localization in lamellipodia.FIGURE 7.HGF stimulates co-localization of p-SphK1 and p-ERK in lamellipodia and SphK1 phosphorylation is mediated by ERK and not PI3K in HLMVECs. A, HLMVECs grown on slide chambers to ∼90% confluence were treated with HGF (20 ng/ml, 30 min) or PBS and probed with anti-p-SphK1 (Ser225) and anti-p-Erk (Thr/Ser204) antibodies, and lamellipodia formation was examined by immunofluorescence microscopy with 60× oil objective. Co-localization of phospho-SphK1 and phospho-ERK was performed as described under “Experimental Procedures.” Co-localization of phospho-SphK1 (red) and phospho-ERK (green) in lamellipodia (merge, yellow) was visualized by immunofluorescent staining. Shown are representative images from three independent experiments. Insets depict enhanced co-localization of p-SphK1 and p-Erk in lamellipodia. B, the co-localization of phospho-SphK1 and phospho-ERK in lamellipodia was quantified from A using ImageJ software and expressed as relative pixel intensity. At least 20 cells were analyzed for each condition. C, HLMVECs grown to ∼90% confluence in 35-mm dishes were pretreated with PD98059 (1 μm, 30 min) followed by HGF (20 ng/ml, 30 min) treatment. The cell lysates were subjected to 10% SDS-PAGE and probed with anti-phospho-ERK1/2, anti-phospho-SphK1, anti-ERK1/2, and anti-SphK1 antibodies. Shown are representative blots from three independent experiments. D, phosphorylation of SphK1 and ERK was analyzed from C by ImageJ software. The values are the means ± S.E. *, significantly different compared with cells not stimulated with HGF (p < 0.01); #, significantly different in cells pretreated with PD98059 and exposed to HGF as compared cells exposed to HGF without the inhibitor (p < 0.05). E, HLMVECs grown to ∼90% confluence in 35-mm dishes were pretreated with LY294002 (1 μm), a PI3K inhibitor, for 30 min followed by HGF (20 ng/ml, 30 min) treatment. The cell lysates were subjected to 10% SDS-PAGE and probed with anti-phospho-Akt, anti-phospho-ERK1/2, anti-phospho-SphK1, anti-Akt, anti-ERK1/2, and anti-SphK1 antibodies. Shown is a representative blot from three independent experiments. F, phosphorylation of SphK1, ERK, and Akt was analyzed by image analysis of Western blots from E. The values are the means ± S.E. *, significantly different compared with HGF non-treated control cells (p < 0.01); #, significantly different compared with HGF challenged cells (p < 0.05); **, not significant compared with LY294002 treated cells exposed to HGF (p > 0.05). Con, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Having established a role for SphK1 in HGF-induced lamellipodia formation, we next investigated whether Spns2, the S1P transporter, plays a role in HGF-induced lamellipodia formation and cell motility. As shown in Fig. 8 (A and B), vehicle-treated HLMVECs revealed a diffused cytosolic distribution of Spns2 (green) and HGF treatment induced redistribution of Spns2 to cell periphery and co-localization with actin (red) in lamellipodia (merge, yellow). To determine the role of Spns2 in lamellipodia formation and cell motility, Spns2 was down-regulated (>80%) by Spns2 specific siRNA for 72 h prior to HGF stimulation (Fig. 8E). Down-regulation of Spns2 with siRNA significantly inhibited HGF-induced lamellipodia formation, as determined by actin (red) and cortactin (green) co-localization in cell periphery (Fig. 8, C and D). We next investigated whether HGF-induced S1P generated inside the cell signals via inside-out mechanism using a specific anti-S1P antibody (18.Berdyshev E.V. Gorshkova I. Usatyuk P. Kalari S. Zhao Y. Pyne N.J. Pyne S. Sabbadini R.A. Garcia J.G. Natarajan V. Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase.PLoS One. 2011; 6: e16571Crossref PubMed Scopus (48) Google Scholar). Exogenous addition of anti-S1P monoclonal antibody (150 μg/ml) to the medium blocked HGF-induced lamellipodia formation compared with control HLMVECs treated with IgG (150 μg/ml) (Fig. 8, F and G). Intracellular S1P levels in scrambled RNA- and Spns2 siRNA-transfected cells did not show significant difference in the absence of HGF; however, a statistically significant increase in intracellular S1P levels was observed between scrambled RNA-transfected and Spns2 siRNA-transfected cells after HGF treatment (Fig. 9A), indicating that HGF-induced intracellular S1P could not be effectively transported outside. No difference in S1P levels was seen in the medium in cells treated with scrambled or Spns2 siRNA in the absence or presence of HGF (data not shown). To investigate the role of Spns2 in cell motility, wound healing assay was performed with scrambled and Spns2 siRNA-transfected HLMVECs. As shown in Fig. 9 (B and C), HGF enhanced the process of wound closure compared with control cells, and knockdown of Spns2 significantly attenuated HGF-mediated wound healing. These results show an essential role of Spns2 in HGF-induced lamellipodia formation and cell motility in human lung ECs. S1P, generated in cells by SphK1/2, can signal intracellularly or extracellularly (inside-out mechanism) by ligation to cell surface G protein-coupled S1P1–5. Human lung ECs exhibit high expression of S1P1 and S1P3 (19.Waeber C. Blondeau N. Salomone S. Vascular sphingosine-1-phosphate S1P1 and S1P3 receptors.Drug News Perspect. 2004; 17: 365-382Crossref PubMed Scopus (125) Google Scholar). To further characterize the type of S1P receptor(s) involved in HGF-mediated lamellipodia formation, S1P1, S1P2, and S1P3 were down-regulated by specific siRNA, and knockdown of these receptors was confirmed by Western blotting (Fig. 10C). As expected, HGF stimulated redistribution of actin (red) and cortactin (green) to cell periphery and enhanced co-localization of actin and cortactin in lamellipodia (merge, yellow) (Fig. 10A). Furthermore, HGF-induced lamellipodia was inhibited in cells transfected with S1P1, but not S1P2 or S1P3, siRNA (Fig. 10B). These results suggest a role for S1P1 in mediating HGF-induced lamellipodia formation in human lung ECs. Given our results indicating that both Spns2 and SphK1 are localized in lamellipodia in response to HGF, we next investigated potential association between SphK1, S1P1, and Spns2 before and after HGF stimulation. HLMVECs were infected with control adenovirus or FLAG-tagged SphK1 adenovirus prior to HGF stimulation, cell lysates were subjected to immunoprecipitation with anti-FLAG antibody, and the immunoprecipitates were analyzed for co-immunoprecipitation of Spns2, S1P1, or FLAG tag. As shown in Fig. 11A, HGF challenge enhanced SphK1 interaction with S1P1 and Spns2 compared with vehicle treated cells. Similarly, HGF challenge increased co-immunoprecipitation of Spns2 and p-SphK1 in S1P1 immunoprecipitates (Fig. 11C) and S1P1 and p-SphK1 in Spns2 immunoprecipitates (Fig. 11E). A similar increase in co-localization of SphK1 with Spns2 and SphK1 with S1P1 in lamellipodia was observed by immunofluorescence after HGF stimulation of HLMVECs (Fig. 11, G and I). These data suggest that Spns2, SphK1, and S1P1 may be part of a protein platform in lamellipodia of human lung" @default.
• W2554695763 created "2016-11-30" @default.
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• W2554695763 date "2016-12-01" @default.
• W2554695763 modified "2023-10-14" @default.
• W2554695763 title "Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium" @default.
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• W2554695763 doi "https://doi.org/10.1074/jbc.m116.758946" @default.
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