FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023356119> ?p ?o ?g. }

• W2023356119 endingPage "50454" @default.
• W2023356119 startingPage "50446" @default.
• W2023356119 abstract "The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38α/γ-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression. The migration of endothelial cells in response to various stimulating factors plays an essential role in angiogenesis. The p38 MAPK pathway has been implicated to play an important role in endothelial cell migration because inhibiting p38 MAPK activity down-regulates vascular endothelial growth factor (VEGF)-stimulated migration. Currently, the signaling components in the p38 MAPK activation pathway and especially the mechanisms responsible for p38 MAPK-regulated endothelial cell migration are not well understood. In the present study, we found that p38 MAPK activity is required for endothelial cell migration stimulated by both VEGF and nongrowth factor stimulants, sphingosine 1-phosphate and soluble vascular cell adhesion molecule. By using dominant negative forms of signaling components in the p38 MAPK pathway, we identified that a regulatory pathway consisting of MKK3-p38α/γ-MAPK-activated protein kinase 2 participated in VEGF-stimulated migration. In further studies, we showed that a minimum of a 10-h treatment with SB203580 (specific p38 MAPK inhibitor) was needed to block VEGF-stimulated migration, suggesting an indirect role of p38 MAPK in this cellular event. Most interestingly, the occurrence of SB203580-induced migratory inhibition coincided with a reduction of urokinase plasminogen activator (uPA) expression. Furthermore, agents disrupting uPA and uPA receptor interaction abrogated VEGF-stimulated cell migration. These results suggest a possible association between cell migration and uPA expression. Indeed, VEGF-stimulated migration was not compromised by SB203580 in endothelial cells expressing the uPA transgene; however, VEGF-stimulated migration was inhibited by agents disrupting uPA-uPA receptor interaction. These results thus suggest that the p38 MAPK pathway participates in endothelial cell migration by regulating uPA expression. The formation of new blood vessels, known as angiogenesis, is necessary for the growth and metastasis of many tumors (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7218) Google Scholar, 2Blagosklonny M.V. Cancer Cells. 2004; 5: 13-17Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Angiogenesis involves the activation, proliferation, migration, and reorganization of endothelial cells (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7218) Google Scholar). The migration process is promoted by angiogenic stimulating factors such as vascular endothelial growth factor (VEGF), 1The abbreviations used are: VEGF, vascular endothelial growth factor; S-1-P, sphingosine 1-phosphate; VCAM, vascular cell adhesion molecule; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; MK, MAPK-activated protein kinase; PRAK, p38-activated kinase; uPA, urokinase plasminogen activator; uPAR, uPA receptor; HUVEC, human umbilical vascular endothelial cell; mAb, monoclonal antibody; RIPA, radioimmunoprecipitation assay; Ad, adenovirus. fibroblast growth factor, sphingosine 1-phosphate (S-1-P), and soluble vascular cell adhesion molecule (VCAM) (3Zachary I. Biochem. Soc. Trans. 2003; 31: 1171-1177Crossref PubMed Google Scholar, 4Prager G.W. Breuss J.M. Steurer S. Mihaly J. Binder B.R. Blood. 2004; 103: 955-962Crossref PubMed Scopus (109) Google Scholar, 5Panetti T.S. Biochim. Biophys. Acta. 2002; 1582: 190-196Crossref PubMed Scopus (94) Google Scholar, 6Nakao S. Kuwano T. Ishibashi T. Kuwano M. Ono M. J. Immunol. 2003; 170: 5704-5711Crossref PubMed Scopus (71) Google Scholar). Therapeutic approaches aimed at intercepting angiogenic factor-mediated signaling pathways show promising potential for the inhibition of tumor growth and metastasis (2Blagosklonny M.V. Cancer Cells. 2004; 5: 13-17Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 7Huang X. Wong M.K. Yi H. Watkins S. Laird A.D. Wolf S.F. Gorelik E. Cancer Res. 2002; 62: 5727-5735PubMed Google Scholar, 8Pavco P.A. Bouhana K.S. Gallegos A.M. Agrawal A. Blanchard K.S. Grimm S.L. Jensen K.L. Andrews L.E. Wincott F.E. Pitot P.A. Tressler R.J. Cushman C. Reynolds M.A. Parry T.J. Clin. Cancer Res. 2000; 6: 2094-2103PubMed Google Scholar). The p38 mitogen-activated protein kinase (MAPK) family contains four members, namely p38α,-β,-γ, and -δ MAPKs (9Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar). p38 MAPKs are activated by upstream MKK3 and MKK6 (9Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar, 10Martin-Blanco E. BioEssay. 2000; 22: 637-645Crossref PubMed Scopus (172) Google Scholar). The effects of p38 MAPKs are mediated by various p38 MAPK substrates including MAPK-activated protein kinase 2 (MK2), MK3, and p38 MAPK-activated kinase (PRAK) (9Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar, 10Martin-Blanco E. BioEssay. 2000; 22: 637-645Crossref PubMed Scopus (172) Google Scholar). In addition to the more defined role in inflammation and cell stress responses (11Nebreda A.R. Porras A. Trends Biochem. Sci. 2000; 25: 257-260Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar), p38 MAPK has also been implicated in the cytoskeleton reorganization and the cellular migration of various cell types (12Heuertz R.M. Tricomi S.M. Uthayashanker R. Ezekiel R. Webster R.O. J. Biol. Chem. 1999; 274: 17968-17974Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 13Ray A.K. Jones A.C. Carnes D.L. Cochran D.L. Mellonig J.T. Oates Jr., T.W. J. Periodontol. 2003; 74: 1320-1328Crossref PubMed Scopus (17) Google Scholar, 14Li W. Nadelman C. Henry G. Fan J. Muellenhoff M. Medina E. Gratch N.S. Chen M.E. Woodley D. J. Investig. Dermatol. 2001; 117: 1601-1611Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 15Kavurma M.M. Khachigian L.M. J. Cell. Biochem. 2003; 89: 289-300Crossref PubMed Scopus (95) Google Scholar, 16Sharma G.-D. He J. Bazan H.E.P. J. Biol. Chem. 2003; 278: 21989-21997Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 17Denes L. Jednakovits A. Hargital J. Penzes Z. Balla A. Talosi L. Krajcsi P. Csermely P. Br. J. Pharmacol. 2002; 136: 597-603Crossref PubMed Scopus (20) Google Scholar, 18Mudgett J.S. Ding J. Guh-Siesel L. Chartrain N.A. Yang L. Gopal S. Shen M.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10454-10459Crossref PubMed Scopus (329) Google Scholar). The use of specific inhibitors has demonstrated the importance of p38 MAPK in endothelial cell migration stimulated by angiogenic factors such as VEGF (19Rousseau S. Houle F. Kotanides H. Witte L. Waltenberger J. Landry J. Huot J. J. Biol. Chem. 2000; 275: 10661-10672Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). However, the signaling components and the mechanisms of the p38 MAPK pathway involved in endothelial cell migration are not well defined. Urokinase plasminogen activator (uPA) is a serine protease and, when bound to its receptor, uPAR, initiates the activation of metalloproteinases as well as the conversion of plasminogen to plasmin (20Collen C. Thromb. Haemostasis. 1999; 82: 259-270Crossref PubMed Scopus (354) Google Scholar, 21Blasi F. Thromb. Haemostasis. 1999; 82: 298-304Crossref PubMed Scopus (171) Google Scholar). In addition, it also stimulates the migration of various cell types including smooth muscle and epithelial and endothelial cells (22Degryse B. Resnati M. Rabbani S.A. Villa A. Fazioli F. Blasi F. Blood. 1999; 94: 649-662Crossref PubMed Google Scholar, 23Kusch A. Tkachuk S. Haller H. Dietz R. Gulba D.C. Lipp M. Dumler I. J. Biol. Chem. 2000; 275: 39466-39473Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 24Nguyen D.H.D. Webb D.J. Catling A.D. Song Q. Dhakephalkar A. Weber M.J. Ravichandran K.S. Gonias S.L. J. Biol. Chem. 2000; 275: 19382-19388Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 25Mazar A.P. Henkin J. Goldfarb R.H. Angiogenesis. 1999; 3: 15-32Crossref PubMed Scopus (155) Google Scholar, 26Koolwijk P. Kapiteijn K. Molenaar B. van Spronsen E. van der Vecht B. Helmerhorst F.M. van Hinsbergh V.W.M. J. Clin. Endocrinol. Metab. 2001; 86: 3359-3367Crossref PubMed Scopus (21) Google Scholar). Blocking uPA or uPAR function with antagonists or down-regulating their expression can significantly impair cell migration (27Lee E. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 161-166Crossref PubMed Scopus (204) Google Scholar), suggesting the importance of uPA/uPAR in cell migration. In fact, phosphatidylinositol 3-kinase and protein kinase C have been shown to regulate the motility of breast cancer cells by promoting uPA secretion (28Silva D. Rizzo M.T. J. Biol. Chem. 2002; 277: 3150-3157Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 29Silva D. English D. Lyons D. Lloyd Jr., F.P. Biochem. Biophys. Res. Commun. 2002; 290: 552-557Crossref PubMed Scopus (76) Google Scholar). We and others have demonstrated previously that uPA expression is regulated by the p38 MAPK pathway (30Huang S. New L. Pan Z. Han J. Nemerow G.R. J. Biol. Chem. 2000; 275: 12266-12272Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 31Montero L. Nagamine Y. Cancer Res. 1999; 59: 5286-5293PubMed Google Scholar), and this raises the likelihood that p38 MAPK participates in cell migration by regulating uPA expression. The goal of the present study was to define the mechanisms responsible for p38 MAPK-regulated endothelial cell migration. We showed that p38 MAPK activity is required for VEGF-, S-1-P, and soluble VCAM-stimulated human endothelial cell migration. By using dominant negative forms of the signaling components in the p38 MAPK signaling pathway, we identified that only MKK3, p38α/γ MAPK, and MK2 are involved in VEGF-stimulated endothelial cell migration. Furthermore, we found that p38 MAPK-dependent protein expression rather than direct p38 MAPK signaling is essential for VEGF-stimulated cell migration. Because uPA and uPAR are important for endothelial cell migration and their expressions are regulated by the p38 MAPK pathway in various cell types, we examined the involvement of uPA and uPAR in VEGF-stimulated endothelial cell migration. Our results indicate that p38 MAPK regulates uPA expression and that the expression of uPA and the ability of VEGF to stimulate cell migration are closely associated. By using endothelial cells with uPA transgene expression, we demonstrated that forced uPA expression was capable of rescuing VEGF-stimulated cell migration in p38 MAPK-inhibited conditions, which suggests that uPA may be the sole factor responsible for the role of p38 MAPK in VEGF-stimulated endothelial cell migration. Finally, we provide evidence that p38 MAPK-uPA regulated VEGF-stimulated cell migration by facilitating actin reorganization and focal adhesion assembly. Reagents and Cells—VEGF and soluble VCAM were obtained from R&D Systems (Minneapolis, MN). S-1-P was purchased from BioMol (Plymouth Meeting, PA). SB203580 and SB202474 were obtained from Calbiochem. Human umbilical embryonic cells (HUVECs) were purchased from Cascade Biologics (Portland, OR) and were maintained in medium 200 containing low serum growth supplement. Amino-terminal fragment of uPA was obtained from Chemicon (Temecula, CA). The antibodies used in the study are as follows: anti-uPA mAbs 3471 and 394 and anti-uPAR mAb 3936 from American Diagnostica (Greenwich, CT); anti-uPAR, anti-paxillin polyclonal antibodies, and anti-Myc mAb from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-MK2 and anti-phospho-MK2 (Thr-334) polyclonal antibodies from Cell Signaling (MA). Transwell Migration Assay—The migration was performed using Transwells as described previously (30Huang S. New L. Pan Z. Han J. Nemerow G.R. J. Biol. Chem. 2000; 275: 12266-12272Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Briefly, the undersurface of the Transwell was coated with 10 μg/ml collagen I overnight at 4 °C. Cells were added to the upper chamber of the Transwell and allowed to migrate for 4 h. As indicated, various concentrations of VEGF, soluble VCAM, and S-1-P were added into the medium in the under wells to stimulate HUVEC migration. To determine the importance of the p38 MAPK pathway in HUVEC migration, cells were treated with SB203580 or SB202174 for various times prior to the assay. Alternatively, HUVECs were infected with 103 viral particles/cell recombinant adenoviruses encoding dominant MKK3, MKK6, p38α, p38β, p38γ, p38δ, MK2, MK3, PRAK, or control Ad for 36 h prior to the migration assays. Recombinant Adenoviruses Construction—The construction of recombinant adenoviruses (Ads) has been described elsewhere (32Huang S. Jiang Y. Li Z. Nishida E. Mathias P. Lin S. Ulevitch R.J. Nemerow G.R. Han J. Immunity. 1997; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 33Han Q. Leng J. Bian D. Mahanivong C. Carpenter K.A. Pan Z.K. Han J. Huang S. J. Biol. Chem. 2002; 277: 48379-48385Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The recombinant adenoviruses were purified by CsCl gradient centrifugation and dialyzed in Tris-buffered saline (10 mm Tris, 0.9% NaCl, pH 8.0). Purified Ad concentrations were determined using the Bio-Rad protein assay solution, and viral particles were calculated based on the equation that 1 μg of viral protein = 4 × 109 viral particles (34Wickham T.J. Mathias P. Cheresh D.A. Nemerow G.R. Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1955) Google Scholar). Retroviral Vectors Construction—Constitutively active MK2, PRAK, and uPA retroviral vectors were generated by subcloning cDNA encoding constitutively active Myc-tagged MK2, PRAK, and uPA into the pBabePuro plasmid. Retroviruses were prepared by transfecting 15 μg of retroviral constructs into an amphotropic packing cell line, LinX-A, with calcium phosphate. After 2 days of viral production at 32 °C, the supernatants containing recombinant retroviruses were collected, filtered through 0.45-μm filter units, mixed with 20% fresh medium, and used to infect HUVECs in the presence of 8 μg/ml Polybrene. Retroviral infection was facilitated by centrifugation of the plates containing cells and retroviruses at 1,600 rpm for 1 h. We typically achieved 30–50% infection rates in HUVECs. The transduced cells were selected out by culturing cells in medium containing 2 μg/ml puromycin. To verify the expression of constitutively active MK2 and uPA, retrovirus-transduced cells were lysed with radioimmunoprecipitation assay (RIPA) buffer. Lysates were electrophoresed on 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane, and the expression of constitutively active MK2, PRAK, and uPA was detected by their respective antibodies. Analysis of uPA and uPAR Expression—HUVECs were treated with various concentrations of SB203580 for 24 h. The cells were lysed in RIPA, and cell lysates were subjected to immunoblotting to detect cell-associated uPA or uPAR with their respective antibodies. To determine the length of time required for SB203580 to down-regulate cell-associated uPA expression, HUVECs were treated with 10 μm SB203580 and then subjected to immunoblotting to detect uPA expression. To determine the effect of dominant negative forms of p38 MAPK pathway signaling components in uPA expression, HUVECs were infected with 103 viral particles/cell recombinant adenoviruses encoding dominant MKK3, MKK6, p38α, p38β, p38γ, p38δ, MK2, PRAK, or control Ad for 36 h followed by immunoblotting to detect uPA expression. Analysis of MK2 Phosphorylation—HUVECs were either treated with SB203580 or left untreated for various lengths of time prior to lysis with RIPA. The lysates were incubated initially with anti-MK2 polyclonal antibody for 4 h at 4 °C, then Gamma-bind beads (Amersham Biosciences) for another hour, followed by five washes with RIPA. The protein-bound beads were boiled in SDS protein sample buffer and subjected to SDS-PAGE analysis. Phosphorylated MK2 was detected with anti-phospho-MK2 (Thr-334) polyclonal antibody. Immunostaining—Mock control or uPA retrovirally transduced HUVECs were cultured on 10 μg/ml collagen I-coated coverslips overnight and then treated with VEGF for 1 h. Cells were fixed with 3% paraformaldehyde, permeabilized with 1% Triton X-100, and blocked with 5% bovine serum albumin. Anti-paxillin polyclonal antibody (1:50 dilution) and rhodamine-conjugated phalloidin (Sigma) were then added to cells for 1 h followed by an 1-h incubation with fluorescein isothiocyanate-conjugated rabbit anti-mouse secondary antibody (Molecular Probe, Eugene, OR). Paxillin and stress fiber staining were visualized by fluorescence microscopy (Axiovert 200M, Zeiss). Statistical Analysis—All migration experiments were performed two or three times, and the results represent mean values of triplicates. p values were calculated by Student t test using Microsoft Excel software. p38 MAPK Activity Is Required for Stimulated Endothelial Cell Migration—Various agents including VEGF, S-1-P, and soluble VCAM are capable of stimulating endothelial cell migration (3Zachary I. Biochem. Soc. Trans. 2003; 31: 1171-1177Crossref PubMed Google Scholar, 4Prager G.W. Breuss J.M. Steurer S. Mihaly J. Binder B.R. Blood. 2004; 103: 955-962Crossref PubMed Scopus (109) Google Scholar, 5Panetti T.S. Biochim. Biophys. Acta. 2002; 1582: 190-196Crossref PubMed Scopus (94) Google Scholar, 6Nakao S. Kuwano T. Ishibashi T. Kuwano M. Ono M. J. Immunol. 2003; 170: 5704-5711Crossref PubMed Scopus (71) Google Scholar). Although it is known that VEGF-stimulated endothelial cell migration requires p38 MAPK activity (19Rousseau S. Houle F. Kotanides H. Witte L. Waltenberger J. Landry J. Huot J. J. Biol. Chem. 2000; 275: 10661-10672Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), it has not been clearly shown whether p38 MAPK activity is required for endothelial cell migration stimulated by nongrowth factor stimulants such as G-protein-coupled receptors ligand S-1-P and integrin ligand-soluble VCAM. To investigate this, we treated HUVECs with increasing concentrations of each stimulant, and we measured their ability to induce cell migration. All three stimulants enhanced HUVEC migration in dose-dependent manner with optimal concentrations at 10 ng/ml, 75 nm, and 15 ng/ml, respectively (Fig. 1A). Subsequently, we treated HUVECs with increasing concentrations of SB203580, a highly specific inhibitor for p38 MAPK, for 24 h and then examined the ability of VEGF, S-1-P, or soluble VCAM to stimulate cell migration. SB203580 at 10 μm completely blocked VEGF-, S-1-P, or soluble VCAM-stimulated HUVEC migration (Fig. 1B), whereas the basal level of cell migration was not significantly altered by the addition of SB203580 (data not shown). In control experiments, we treated the cells with the same concentration of SB202474 (a nonfunctional SB203580 structural analog) and did not detect any inhibitory effects on HUVEC migration (Fig. 1B). To rule out the possibility that SB203580 was toxic to HUVECs, we examined the cell viability of both untreated and SB203580-treated cells, and we found that SB203580 caused negligible toxicity on cell viability as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (data not shown). These results indicate that the p38 MAPK activity is required for endothelial cell migration induced by various stimulants but is not required for basal cell migration. MKK3-p38α/γ-MK2 Pathway Is Involved in VEGF-stimulated Endothelial Cell Migration—The p38 MAPK pathway consists of a 3-tiered signaling cascade with multiple players at each level, e.g. MKK3 and -6 at the MAPK kinase level, p38α, -β,-γ, and -δ at the MAPK level, and MK2, -3, and PRAK at the level of p38 MAPK downstream substrates (9Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar, 10Martin-Blanco E. BioEssay. 2000; 22: 637-645Crossref PubMed Scopus (172) Google Scholar). To identify the specific kinases involved in VEGF-stimulated endothelial cell migration, we expressed dominant negative forms of these proteins in HUVECs with the aid of recombinant adenovirus. Cells were detached 36 h post-infection and then analyzed for VEGF-stimulated cell migration. The dominant negative forms of the molecules showed no significant effect on cell migration in the unstimulated cells (Fig. 2A). This is consistent with the results that SB203580 did not significantly affect basal HUVEC migration. However, dominant negative forms of MKK3, p38α, p38γ, and MK2, but not dominant negative forms of MKK6, p38β, p38δ, MK3, and PRAK, blocked VEGF-stimulated cell migration (Fig. 2A). Co-expressing dominant negative p38α and p38γ completely abrogated VEGF-stimulated migration (Fig. 2A). These results suggest that a pathway consisting of MKK3-p38α/γ-MK2 is involved in VEGF-stimulated endothelial cell migration. To determine further the importance of MK2 in VEGF-stimulated endothelial cell migration, we retrovirally introduced constitutively active MK2 or PRAK into HUVECs. Mock control cells and HUVECs expressing constitutively active MK2 or PRAK displayed a similar extent of basal and VEGF-stimulated cell migration (Fig. 2B), suggesting that active MK2 or PRAK did not confer cells with greater basal motility. The addition of SB203580 failed to inhibit VEGF-stimulated cell migration in HUVECs expressing constitutively active MK2 (Fig. 2B). In contrast, SB203580 blocked VEGF-stimulated cell migration in both mock control and constitutively active PRAK-expressing HUVECs (Fig. 2B). These results suggest that the activity of MK2 alone is essential and sufficient for mediating VEGF-stimulated endothelial cell migration. p38 MAPK Pathway-dependent Gene Expression Rather Than Direct p38 MAPK Signaling Is Responsible for VEGF-stimulated Endothelial Cell Migration—To define the mechanism responsible for p38 MAPK-regulated HUVEC migration, we initially conducted a time course measurement of SB203580 inhibition of p38 MAPK activity. The p38 activity was determined by measuring the phosphorylation extent of MK2 (a direct p38 MAPK substrate). Because MK2 is activated through phosphorylation at Thr-222 and Thr-334 (35Stokoe D. Campbell D.G. Nakielny S. Hidaka H. Leevers S.J. Marshall C. Cohen P. EMBO J. 1992; 11: 3985-3994Crossref PubMed Scopus (392) Google Scholar), we used an antibody that recognizes phospho-MK2 (Thr-334) to detect MK2 activity. HUVECs were treated with 10 μm SB203580 and then harvested at various time points indicated in Fig. 3A. An 80% reduction of phosphorylated MK2 was observed in the 1st h, and the activity was diminished 1 h later (Fig. 3A). In the next experiments, we attempted to correlate the data by analyzing HUVEC migration after a similar time course of SB203580 treatment. Endothelial cells were treated with 10 μm SB203580, and at various time points cells were collected for VEGF-stimulated migration assays (Fig. 3B). The earliest time we could detect a significant SB203580-induced migratory inhibition occurred at 6 h, and a minimum of 10 h was necessary to completely inhibit VEGF-stimulated cell migration. The time disparity between the rapid inhibition of MK2 phosphorylation (within 2 h) and delayed onset of migration inhibition (after 6 h) would argue that a p38 MAPK-dependent regulation of gene expression is responsible for VEGF-stimulated cell migration rather than a direct p38 signaling event. The p38 MAPK pathway has been shown to regulate the expression of various proteins (9Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1393) Google Scholar). We thus determined whether p38 MAPK-dependent protein expression is required for VEGF-stimulated endothelial cell migration. HUVECs were treated with SB203580 for 24 h, washed thoroughly to remove SB203580, and cultured in complete medium in the absence or presence of 2 μg/ml actinomycin (RNA synthesis inhibitor) or 20 μg/ml cycloheximide (protein synthesis inhibitor) for 4 h. Cells were lysed and lysates immunoprecipitated with anti-MK2 polyclonal antibody. Immunoblotting with the immunoprecipitates using anti-phospho-MK2 (Thr-334) polyclonal antibody showed similar levels of MK2 phosphorylation in cells treated or untreated with either inhibitor (Fig. 4A), demonstrating that the p38 MAPK activity was not compromised in actinomycin- or cycloheximide-treated cells compared with the untreated cells. However, the ability of VEGF to stimulate cell migration was completely lost in actinomycin or cycloheximide-treated cells (Fig. 4B). These results suggest that p38 MAPK pathway-dependent protein expression is required for VEGF-stimulated endothelial cell migration. uPA Expression Is Regulated by p38 MAPK Pathway and Is Required for Cell Migration—Our previous studies (30Huang S. New L. Pan Z. Han J. Nemerow G.R. J. Biol. Chem. 2000; 275: 12266-12272Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 33Han Q. Leng J. Bian D. Mahanivong C. Carpenter K.A. Pan Z.K. Han J. Huang S. J. Biol. Chem. 2002; 277: 48379-48385Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) have shown that p38 MAPK regulates uPA and uPAR expression in invasive breast cancer cells. Other studies further demonstrated that uPA and uPAR are involved in migration of various cell types including endothelial cells (26Koolwijk P. Kapiteijn K. Molenaar B. van Spronsen E. van der Vecht B. Helmerhorst F.M. van Hinsbergh V.W.M. J. Clin. Endocrinol. Metab. 2001; 86: 3359-3367Crossref PubMed Scopus (21) Google Scholar, 36Sandberg T. Ehinger A. Casslén B. J. Clin. Endocrinol. Metab. 2000; 86: 1724-1730Crossref Scopus (18) Google Scholar). We thus hypothesized that the p38 MAPK pathway may participate in endothelial cell migration by regulating uPA/uPAR expression. To test this hypothesis, we first examined the effect of inhibiting p38 MAPK activity on uPA/uPAR expression in endothelial cells. HUVECs were treated with an increasing dose of SB203580 for 24 h and then lysed, and the cell lysates were subjected to immunoblotting to detect uPA and uPAR expression. Although both uPA and uPAR were readily detected, the expression of uPA was significantly down-regulated by SB203580 in a dose-dependent manner (Fig. 5A). Conversely, the level of uPAR expression was unaltered by the presence of SB203580 (Fig. 5A). To identify the specific signaling components in the p38 MAPK pathway controlling uPA expression, we introduced dominant negative forms of these proteins in HUVECs using recombinant adenovirus. Cells were lysed 36 h post-infection and then analyzed for cell-associated uPA expression. The dominant negative forms of MKK3, p38α, p38γ, and MK2, but not dominant negative forms of MKK6, p38β, p38δ, and PRAK, significantly down-regulated uPA expression (Fig. 5B). These results suggest that the same signaling molecules including MKK3, p38α/γ, and MK2 are involved in both endogenous uPA expression and VEGF-stimulated endothelial cell migration. As 10 μm SB203580 could sufficiently inhibit uPA expression, we used this concentration to examine a time course of uPA inhibition. The earliest apparent reduction in uPA expression was observed 6 h after the addition of SB203580 (Fig. 5C). A significant reduction in uPA expression (>90%) was detected after 8–10 h SB203580 treatment (Fig. 5C). This pattern of uPA expression reduction coincided with the timing observed in the SB203580-mediated inhibition of VEGF-stimulated cell migration (Fig. 3B), suggesting a possible association between uPA expression and VEGF-stimulated cell migration. In order to examine the role of uPA expression in VEGF-stimulated cell migration, we introduced a panel of mAbs targeting uPA and uPAR, and we analyzed their effects on HUVEC migration. Addition of either anti-uPA mAb 3471 or anti-uPAR mAb 3936, which both function to prevent uPA binding to uPAR, caused significant inhibition of VEGF-stimulated HUVEC migration (Fig. 6); anti-uPA mAb 394, which neutralizes uPA protease activity but does" @default.
• W2023356119 created "2016-06-24" @default.
• W2023356119 creator A5015126635 @default.
• W2023356119 creator A5063631664 @default.
• W2023356119 creator A5069238061 @default.
• W2023356119 creator A5081288331 @default.
• W2023356119 creator A5082958853 @default.
• W2023356119 creator A5091597260 @default.
• W2023356119 date "2004-11-01" @default.
• W2023356119 modified "2023-10-17" @default.
• W2023356119 title "p38 Mitogen-activated Protein Kinase Regulation of Endothelial Cell Migration Depends on Urokinase Plasminogen Activator Expression" @default.
• W2023356119 cites W1485224770 @default.
• W2023356119 cites W1505825810 @default.
• W2023356119 cites W1566502635 @default.
• W2023356119 cites W1576290062 @default.
• W2023356119 cites W1774966351 @default.
• W2023356119 cites W1795514804 @default.
• W2023356119 cites W1856672768 @default.
• W2023356119 cites W1867782147 @default.
• W2023356119 cites W1933982817 @default.
• W2023356119 cites W1966931906 @default.
• W2023356119 cites W1967265143 @default.
• W2023356119 cites W1967638202 @default.
• W2023356119 cites W1969732387 @default.
• W2023356119 cites W1970546329 @default.
• W2023356119 cites W1977013517 @default.
• W2023356119 cites W1981705099 @default.
• W2023356119 cites W1991070447 @default.
• W2023356119 cites W1991608171 @default.
• W2023356119 cites W2000000541 @default.
• W2023356119 cites W2001587733 @default.
• W2023356119 cites W2008635369 @default.
• W2023356119 cites W2009812837 @default.
• W2023356119 cites W2011234336 @default.
• W2023356119 cites W2011491512 @default.
• W2023356119 cites W2011549770 @default.
• W2023356119 cites W2013041233 @default.
• W2023356119 cites W2020472510 @default.
• W2023356119 cites W2023189063 @default.
• W2023356119 cites W2025198517 @default.
• W2023356119 cites W2026803058 @default.
• W2023356119 cites W2027263866 @default.
• W2023356119 cites W2029119139 @default.
• W2023356119 cites W2030140400 @default.
• W2023356119 cites W2034184306 @default.
• W2023356119 cites W2040421937 @default.
• W2023356119 cites W2040911522 @default.
• W2023356119 cites W2044175451 @default.
• W2023356119 cites W2056142105 @default.
• W2023356119 cites W2061216192 @default.
• W2023356119 cites W2063627953 @default.
• W2023356119 cites W2068872058 @default.
• W2023356119 cites W2072440487 @default.
• W2023356119 cites W2074254585 @default.
• W2023356119 cites W2075457034 @default.
• W2023356119 cites W2082211144 @default.
• W2023356119 cites W2086655427 @default.
• W2023356119 cites W2095130777 @default.
• W2023356119 cites W2097642975 @default.
• W2023356119 cites W2099667017 @default.
• W2023356119 cites W2115341926 @default.
• W2023356119 cites W2116071714 @default.
• W2023356119 cites W2133003864 @default.
• W2023356119 cites W2144210999 @default.
• W2023356119 cites W2147506398 @default.
• W2023356119 cites W2148687850 @default.
• W2023356119 cites W2159413929 @default.
• W2023356119 doi "https://doi.org/10.1074/jbc.m409221200" @default.
• W2023356119 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15371454" @default.
• W2023356119 hasPublicationYear "2004" @default.
• W2023356119 type Work @default.
• W2023356119 sameAs 2023356119 @default.
• W2023356119 citedByCount "56" @default.
• W2023356119 countsByYear W20233561192012 @default.
• W2023356119 countsByYear W20233561192013 @default.
• W2023356119 countsByYear W20233561192014 @default.
• W2023356119 countsByYear W20233561192015 @default.
• W2023356119 countsByYear W20233561192016 @default.
• W2023356119 countsByYear W20233561192017 @default.
• W2023356119 countsByYear W20233561192018 @default.
• W2023356119 countsByYear W20233561192019 @default.
• W2023356119 countsByYear W20233561192020 @default.
• W2023356119 countsByYear W20233561192021 @default.
• W2023356119 countsByYear W20233561192022 @default.
• W2023356119 crossrefType "journal-article" @default.
• W2023356119 hasAuthorship W2023356119A5015126635 @default.
• W2023356119 hasAuthorship W2023356119A5063631664 @default.
• W2023356119 hasAuthorship W2023356119A5069238061 @default.
• W2023356119 hasAuthorship W2023356119A5081288331 @default.
• W2023356119 hasAuthorship W2023356119A5082958853 @default.
• W2023356119 hasAuthorship W2023356119A5091597260 @default.
• W2023356119 hasBestOaLocation W20233561191 @default.
• W2023356119 hasConcept C104317684 @default.
• W2023356119 hasConcept C132149769 @default.
• W2023356119 hasConcept C134018914 @default.
• W2023356119 hasConcept C184235292 @default.
• W2023356119 hasConcept C185592680 @default.
• W2023356119 hasConcept C2776892390 @default.

• next