IL-1β induces VEGF, independently of PGE2 induction, mainly through the PI3-K/mTOR pathway in renal mesangial cells
2006; Elsevier BV; Volume: 70; Issue: 11 Linguagem: Inglês
10.1038/sj.ki.5001948
ISSN1523-1755
AutoresDavid Solà-Villà, Mercedes Camacho, R. Solà, Marta Soler, José María Díaz Fernández, Luı́s Vila,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoVascular endothelial growth factor (VEGF) could play a relevant role in angiogenesis associated with chronic allograft nephropathy. Interleukin-1β (IL-1β) has a key role in inflammatory response. It induces prostaglandin (PG) E2, which is involved in VEGF release by some normal and tumor cells. In the present work, we studied the effect of IL-1β on VEGF release by rat mesangial cells, the transduction signal, and whether or not PGE2 is involved in this effect. IL-1β induced a time-dependent formation of VEGF (analyzed by enzyme-linked immunosorbent assay) and PGE2 (analyzed by enzyme immunoassay). The latter correlated with microsomal-PGE-synthase (mPGES)-1 expression rather than with cyclooxygenase (COX)-2 in terms of protein, determined by Western blotting. No effect of IL-1β on COX-1, cytosolic PGES, or mPGES-2 expression was observed. Indomethacin exerted a nonsignificant effect on IL-1β-induced VEGF, and exogenously added PGE2 exhibited a nonsignificant stimulatory effect on VEGF formation. SB 203580, a p38 mitogen-activated protein kinase inhibitor, weakly inhibited the induction of VEGF by IL-1β in a concentration-dependent manner, whereas LY 294002, a phosphoinoside 3-kinase (PI3-K) inhibitor, and rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, strongly inhibited both IL-1β- and tumor necrosis factor-α-induced VEGF formation in a concentration-dependent manner. Rapamycin also decreased glomerular VEGF levels in the anti-Thy1.1 model of experimental glomerulonephritis. In conclusion, the PI3-K-mTOR pathway seems to be essential in cytokine-induced release of VEGF in mesangial cells. Vascular endothelial growth factor (VEGF) could play a relevant role in angiogenesis associated with chronic allograft nephropathy. Interleukin-1β (IL-1β) has a key role in inflammatory response. It induces prostaglandin (PG) E2, which is involved in VEGF release by some normal and tumor cells. In the present work, we studied the effect of IL-1β on VEGF release by rat mesangial cells, the transduction signal, and whether or not PGE2 is involved in this effect. IL-1β induced a time-dependent formation of VEGF (analyzed by enzyme-linked immunosorbent assay) and PGE2 (analyzed by enzyme immunoassay). The latter correlated with microsomal-PGE-synthase (mPGES)-1 expression rather than with cyclooxygenase (COX)-2 in terms of protein, determined by Western blotting. No effect of IL-1β on COX-1, cytosolic PGES, or mPGES-2 expression was observed. Indomethacin exerted a nonsignificant effect on IL-1β-induced VEGF, and exogenously added PGE2 exhibited a nonsignificant stimulatory effect on VEGF formation. SB 203580, a p38 mitogen-activated protein kinase inhibitor, weakly inhibited the induction of VEGF by IL-1β in a concentration-dependent manner, whereas LY 294002, a phosphoinoside 3-kinase (PI3-K) inhibitor, and rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, strongly inhibited both IL-1β- and tumor necrosis factor-α-induced VEGF formation in a concentration-dependent manner. Rapamycin also decreased glomerular VEGF levels in the anti-Thy1.1 model of experimental glomerulonephritis. In conclusion, the PI3-K-mTOR pathway seems to be essential in cytokine-induced release of VEGF in mesangial cells. Although immunosuppression has improved considerably over the last 10 years, chronic allograft nephropathy (CAN) is still a major cause of late graft failure.1.McLaren A.J. Fuggle S.V. Welsh K.I. et al.Chronic allograft failure in human renal transplantation: a multivariate risk factor analysis.Ann Surg. 2000; 232: 98-113Crossref PubMed Scopus (67) Google Scholar This slowly progressing allograft nephropathy is characterized by vascular obliteration owing to proliferation and scarring of intima and media in renal vessels, and membrane multilayering in the peritubular capillaries. The interstitium also shows gradual fibrosis and generation of extracellular matrix. Tubules develop atrophic features.2.Racusen L.C. Solez K. Colvin R.B. et al.The Banff 97 working classification of renal allograft pathology.Kidney Int. 1999; 55: 713-723Abstract Full Text Full Text PDF PubMed Scopus (2771) Google Scholar Chronic inflammation is accepted to be the driving process of CAN. Inflammation mediators such as inflammatory cells, cytokines, growth factors, and vasoactive agents including eicosanoids can be found at different stages of progressive CAN. Inflammatory angiogenesis could be involved in chronic endothelial activation leading to arteriosclerosis-like CAN. Infiltration of leukocytes seems to be essential for CAN development.3.Ozdemir B.H. Ozdemir F.N. Gungen Y. et al.Role of macrophages and lymphocytes in the induction of neovascularization in renal allograft rejection.Am J Kidney Dis. 2002; 39: 347-353Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Angiogenesis has been shown to facilitate leukocyte recruitment through its interaction with microvascular endothelium.3.Ozdemir B.H. Ozdemir F.N. Gungen Y. et al.Role of macrophages and lymphocytes in the induction of neovascularization in renal allograft rejection.Am J Kidney Dis. 2002; 39: 347-353Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Leukocyte activation and neovascularization form a vicious circle that results in high levels of cytokines and other inflammatory mediators that potentially promote interstitial fibrosis. Interleukin-1 (IL-1) is a primary inflammatory cytokine produced by a variety of cells such as monocytes/macrophages and endothelial cells, which play a fundamental role in orchestrating the inflammatory response.4.Dinarello C.A. The interleukin-1 family: 10 years of discovery.FASEB J. 1994; 8: 1314-1325Crossref PubMed Google Scholar IL-1 has been found to be a pro-angiogenic factor that promotes tumor progression5.Kitahara T. Hiromura K. Ikeuchi H. et al.Mesangial cells stimulate differentiation of endothelial cells to form capillary-like networks in a three-dimensional culture system.Nephrol Dial Transplant. 2005; 20: 42-49Crossref PubMed Scopus (19) Google Scholar, 6.Voronov E. Shouval D.S. Krelin Y. et al.IL-1 is required for tumor invasiveness and angiogenesis.Proc Natl Acad Sci USA. 2003; 100: 2645-2650Crossref PubMed Scopus (795) Google Scholar, 7.Yano S. Nokihara H. 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Wilder R.L. et al.Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis.FEBS Lett. 1995; 372: 83-87Abstract Full Text PDF PubMed Scopus (495) Google Scholar, 11.Jung Y.D. Liu W. Reinmuth N. et al.Vascular endothelial growth factor is upregulated by interleukin-1 beta in human vascular smooth muscle cells via the p38 mitogen-activated protein kinase pathway.Angiogenesis. 2001; 4: 155-162Crossref PubMed Scopus (100) Google Scholar Although several molecules have been shown to be important in angiogenesis, VEGF stands out because it is a direct specific mitogen for endothelial cells and appears to function as a key regulator of physiologic as well as pathologic angiogenesis.12.Thomas K.A. Vascular endothelial growth factor, a potent and selective angiogenic agent.J Biol Chem. 1996; 271: 603-606Crossref PubMed Scopus (566) Google Scholar In humans, VEGF has been reported to be induced in association with chronic interstitial inflammation in renal allografts with evidence of CAN.13.Pilmore H.L. Eris J.M. Painter D.M. et al.Vascular endothelial growth factor expression in human chronic renal allograft.Transplantation. 1999; 67: 929-933Crossref PubMed Scopus (71) Google Scholar IL-1 can exert its biological effect in part by induction of prostanoid generation.10.Ben-Av P. Crofford L.J. Wilder R.L. et al.Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis.FEBS Lett. 1995; 372: 83-87Abstract Full Text PDF PubMed Scopus (495) Google Scholar, 14.Miura S. Tatsuguchi A. Wada K. et al.Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G444-G451Crossref PubMed Scopus (61) Google Scholar, 15.Stocks J. Bradbury D. Corbett L. et al.Cytokines upregulate vascular endothelial growth factor secretion by human airway smooth muscle cells: role of endogenous prostanoids.FEBS Lett. 2005; 579: 2551-2556Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar Indeed, prostaglandin (PG) E2 is involved in neovascularization16.Form D.M. Auerbach R. PGE2 and angiogenesis.Proc Soc Exp Biol Med. 1983; 172: 214-218Crossref PubMed Scopus (333) Google Scholar,17.Díaz-Flores L. Gutierrez R. Valladares F. et al.Intense vascular sprouting from rat femoral vein induced by prostaglandins E1 and E2.Anat Rec. 1994; 238: 68-76Crossref PubMed Scopus (57) Google Scholar and induces endothelial-targeted growth factors.18.Cheng T. Cao W. Wen R. et al.Prostaglandin E2 induces vascular endothelial growth factor and basic fibroblast growth factor mRNA expression in cultured rat Muller cells.Invest Ophthalmol Vis Sci. 1998; 39: 581-591PubMed Google Scholar,19.Seno H. Oshima M. Ishikawa T.O. et al.Cyclooxygenase 2- and prostaglandin E(2) receptor EP(2)-dependent angiogenesis in Apc(Delta716) mouse intestinal polyps.Cancer Res. 2002; 62: 506-511PubMed Google Scholar VEGF expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1).20.Yamakawa M. Liu L.X. Date T. et al.Hypoxia-inducible factor-1 mediates activation of cultured vascular endothelial cells by inducing multiple angiogenic factors.Circ Res. 2003; 93: 664-673Crossref PubMed Scopus (269) Google Scholar HIF-1 regulates a number of genes, globally leading to cell adaptation to a lack of oxygen and orchestrating the neovascularization process by promoting expression of matrix metalloproteinases (MMPs), adhesion molecules, and chemokines.21.Lee J.-W. Bae S.-H. Jeong J.-W. et al.Hypoxia inducible factor (HIF)-1α: its protein stability and biological functions.Exp Mol Med. 2004; 36: 1-12Crossref PubMed Scopus (786) Google Scholar It has been reported that PGE2 induces not only stabilization of HIF-1α,22.Liu X.H. Kirschenbaum A. Lu M. et al.Prostaglandin E2 induces hypoxia inducible factor-1α stabilization and nuclear localization in a human prostate cancer cell line.J Biol Chem. 2002; 277: 50081-50086Crossref PubMed Scopus (200) Google Scholar the oxygen-regulated subunit of HIF-1, but also HIF-1α protein synthesis.23.Fukuda R. Kelly B. Semenza G.L. Vascular endothelial growth factor gene expression in colon cancer cells exposed to prostaglandin E2 is mediated by hypoxia-inducible factor 1.Cancer Res. 2003; 63: 2330-2334PubMed Google Scholar PGH2, formed from arachidonic acid by the catalytic action of cyclooxygenase (COX), is the common intermediate in the biosynthesis of the two series of prostanoids, including PGE2. Three isoforms of COX have been fully characterized to date. Two of these are encoded by the COX-1-gen (COX-1 and COX-3) and COX-2 is encoded by a separate gene. In general, COX-1 can be viewed as a constitutive enzyme and prostanoids formed through the action of COX-1 (also COX-3) mediate so-called 'housekeeping' functions, such as the regulation of renal function, maintenance of the gastric mucosa integrity, and hemostasis. In contrast, COX-2 is an inducible enzyme, which is normally expressed in few tissues and cell types including renal macula densa, but is expressed transiently in response to hormones, growth factors, pro-inflammatory cytokines, bacterial endotoxins, and tumor promoters. COX-2 is typically induced at the inflammatory sites (reviewed by Vila24.Vila L. Cyclooxygenase and 5-lipoxygenase pathways in the vessel wall: role in atherosclerosis.Med Res Rev. 2004; 24: 399-424Crossref PubMed Scopus (93) Google Scholar). COX-2 induction results in an increase in PGE2 production, and in some cell types, such as endothelial and mesangial cells, it also results in the release of untransformed PGH2,25.Camacho M. López-Belmonte J. Vila L. Rate of vasoconstrictor prostanoids released by endothelial cells depends on cyclooxygenase-2 expression and PGI-synthase activity.Circ Res. 1998; 83: 353-365Crossref PubMed Scopus (123) Google Scholar, 26.Soler M. Camacho M. Escudero J.R. et al.Human vascular smooth muscle cells but not endothelial cells express prostaglandin E synthase.Circ Res. 2000; 87: 504-507Crossref PubMed Scopus (99) Google Scholar, 27.Soler M. Camacho M. Solá R. et al.Mesangial cells release untransformed prostaglandin H2 as a major prostanoid.Kidney Int. 2001; 59: 1283-1289Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar which is a potent vasoconstricting agent. The enzymes that catalyze conversion of PGH2 to PGE2 are known as PGE-synthases (PGES). The first to be identified and characterized was a 16 kDa protein (PIG12, MGST1-L1), which is a member of the MAPEG (Membrane-Associated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily with glutathione-dependent PGES activity.28.Jakobsson P.J. Thorén S. Morgenstern R. et al.Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target.Proc Natl Acad Sci USA. 1999; 96: 7220-7225Crossref PubMed Scopus (893) Google Scholar This enzyme, now called microsomal-PGE-synthase (mPGES)-1, is located in chromosome 9 and is inducible by pro-inflammatory cytokines. mPGES-1 is induced by IL-1β and tumor necrosis factor-α (TNFα).26.Soler M. Camacho M. Escudero J.R. et al.Human vascular smooth muscle cells but not endothelial cells express prostaglandin E synthase.Circ Res. 2000; 87: 504-507Crossref PubMed Scopus (99) Google Scholar,28.Jakobsson P.J. Thorén S. Morgenstern R. et al.Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target.Proc Natl Acad Sci USA. 1999; 96: 7220-7225Crossref PubMed Scopus (893) Google Scholar Moreover, functional coupling of mPGES-1 with COX-2 has been reported.29.Murakami M. Naraba H. Tanioka T. et al.Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2.J Biol Chem. 2000; 275: 32783-32792Crossref PubMed Scopus (854) Google Scholar The tandem COX-2/mPGES-1 appears to be responsible for the inflammatory production of PGE2. Conversely, a cytosolic PGES (cPGES) seems to act coupled with COX-1.30.Tanioka T. Nakatani Y. Semmyo N. et al.Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis.J Biol Chem. 2000; 275: 32775-32782Crossref PubMed Scopus (628) Google Scholar cPGES is ubiquitously expressed and identical to p23, a protein somehow related with steroid hormone receptor-mediated signal transduction. Another type of mPGES, known as mPGES-2, has been considered to use substrate coming from both COX-1 and COX-2 activities.31.Murakami M. Nakashima K. Kamei D. et al.Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2.J Biol Chem. 2003; 278: 37937-37947Crossref PubMed Scopus (295) Google Scholar It has been reported that mesangial cells can release VEGF32.Iijima K. Yoshikawa N. Connolly D.T. et al.Human mesangial cells and peripheral blood mononuclear cells produce vascular permeability factor.Kidney Int. 1993; 44: 959-966Abstract Full Text PDF PubMed Scopus (157) Google Scholar and promote capillary formation in vitro.5.Kitahara T. Hiromura K. Ikeuchi H. et al.Mesangial cells stimulate differentiation of endothelial cells to form capillary-like networks in a three-dimensional culture system.Nephrol Dial Transplant. 2005; 20: 42-49Crossref PubMed Scopus (19) Google Scholar On the other hand, COX-2-derived PGE2 appears to be essential for inflammatory cytokine-induced angiogenesis.14.Miura S. Tatsuguchi A. Wada K. et al.Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G444-G451Crossref PubMed Scopus (61) Google Scholar,33.Kuwano T. Nakao S. Yamamoto H. et al.Cyclooxygenase 2 is a key enzyme for inflammatory cytokine-induced angiogenesis.FASEB J. 2004; 18: 300-310Crossref PubMed Scopus (248) Google Scholar IL-1 induces COX-2-mediated production of PGE2 formation in mesangial cells.27.Soler M. Camacho M. Solá R. et al.Mesangial cells release untransformed prostaglandin H2 as a major prostanoid.Kidney Int. 2001; 59: 1283-1289Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Hence, the present work was conducted to study the effect of IL-1 on the expression of VEGF in mesangial cells, and to determine whether or not PGE2 biosynthetic machinery is involved in this effect. Figure 1a shows the effect of IL-1β on VEGF-release by rat glomeruli mesangial cells (RGMCs) as a function of time. IL-1β significantly induced VEGF in a time-dependent manner. This effect was not only observed in terms of protein but also in terms of mRNA (Figure 1b). IL-1β also significantly induced PGE2 formation with a time-course pattern similar to that of VEGF (Figure 2a). The time course of COX-2 expression (Figure 2b and c) indicated that IL-1β time dependently induced COX-2 with a pattern shifted to the left respect to that of PGE2. Downstream enzymes in the PGE2 biosynthetic pathway were also studied. Figure 2c shows a representative Western-blotting analysis of mPGES-1, mPGES-2, and cPGES. RGMC expressed all three PGES isozymes, although mPGES-1 was the only enzyme induced by IL-1β in a time-dependent manner. The time-course pattern of mPGES-1 was shifted to the right respect to that of COX-2. Moreover, the PGE2 time-course curve matched that of mPGES-1 better than that of COX-2. Of note, despite the fact that RGMC expressed mPGES-2 and cPGES, production of PGE2 was very low in IL-1β-untreated cells and only when COX-2 and mPGES-1 were induced PGE2 was produced in considerable amounts.Figure 2Time course of PGE2 release, and COX-2 and mPGES-1 expression in response to IL-1β. Time course of (a) IL-1β-stimulated release of PGE2 and (b and c) enzyme expression in RGMC. RGMC were incubated with 100 U/ml IL-1β for the indicated periods of time. PGE2 levels were then evaluated. N=4, mean±s.e.m., *P<0.05, **P<0.01 when compared with controls. Protein expression was analyzed by Western blotting. (b) Density was normalized to the most dense band; n=4, mean±s.e.m. (c) Representative gels from four independent analysis are also shown.View Large Image Figure ViewerDownload (PPT) Despite the fact that IL-1β-induced expression of PGE2 biosynthetic machinery, results in Figure 3 show that 10 μmol/l indomethacin, which inhibited PGE2 formation more than 90%, did not modify IL-1β-induced VEGF release by RGMC. In addition, incubation of IL-1β-untreated cells with 0.1 and 1 μmol/l of purified PGE2 did not modify VEGF release (Figure 3). Nevertheless, 1 μmol/l of purified PGE2 significantly induced MMP-2 expression in terms of mRNA (2−ΔCt: 7.3 × 10−3±1.7 × 10−3 and 20.9 × 10−3±4.3 × 10−3; mean±s.e.m., n=4 for control and PGE2-treated cells, respectively). In our cells, we observed EP-1, EP-3, and EP-4 expression in terms of mRNA with an almost undetectable expression of EP-2 (2−ΔCt: 0.37 × 10−4±0.09 × 10−4 for EP-1, 2.43 × 10−4±1.55 × 10−4 for EP-3, and 2.04 × 10−4±0.69 × 10−4 for EP-4; mean±s.e.m., n=6 different cell lines in independent experiments; EP-2 was not amplified except in one cell line, 2−ΔCt: 0.29 × 10−4). To explore the signaling pathways involved in IL-1β-induced VEGF expression, RGMCs were incubated with IL-1β plus a fixed concentration (see Materials and Methods section) of several kinase inhibitors in the presence of indomethacin to guarantee an effect independent of PGE2. Three separate experiments were performed. A high concentration of the general protein kinase C (PKC) inhibitor GF 109203X did not inhibit the effect of IL-1β on VEGF production. Nor did the Ca2+-dependent PKC inhibitor, Gö 6976. Moreover, 10 nmol/l phorbol-12-myristate-13-acetate was unable to induce VEGF, whereas it clearly induced COX-2. The MAPK/Extracellular signal-regulated Kinases 1 and 2 (MEK 1/2) inhibitor U0126 did not significantly modify IL-1β-induced production of VEGF (results not shown). Only three of the signaling inhibitors tested significantly inhibited IL-1β-induced release of VEGF. These inhibitors were then assayed again to study their concentration-dependent effect. Figure 4a shows the effect of SB203580 (a p38 mitogen-activated protein kinase (p38-MAPK) inhibitor), rapamycin (a mammalian target of rapamycin (mTOR) inhibitor), and LY-294002 (phosphoinoside 3-kinase (PI3-K) inhibitor), on IL-1β-induced release of VEGF. All three inhibited the IL-1β-induced release of VEGF in a concentration-dependent manner. LY-294002 and rapamycin were able to suppress the effect of IL-1β on VEGF release by more than 80%. The p38-MAPK inhibitor, SB203580, exerted a maximum inhibition of about 40%, at the highest concentration. When 0.1 μmol/l rapamycin and 10 μmol/l SB203580 were used together, a total suppression of IL-1β-induced VEGF was observed. To verify if these pathways were also involved in VEGF expression induced by other cytokines, RGMC were treated with TNFα for 24 h and subjected to the same studies as IL-1β-treated cells. TNFα also significantly induced VEGF formation by RGMC in terms of protein (data not shown). The inhibition pattern of VEGF induction was similar to that of IL-1β, with SB203580, rapamycin, and LY-294002 being the only active inhibitors. Figure 4b shows the concentration-dependent effect of these compounds on TNFα-induced VEGF that was similar to, or even more potent than that on IL-1β-induced VEGF. To observe the effect of mTOR inhibition on VEGF levels in vivo, the anti-Thy1.1 model of experimental glomerulonephritis was used. Figure 5 shows immunoblotting analysis of VEGF in glomeruli of untreated- and rapamycin-treated rats. Results showed a clear reduction on VEGF levels in gomeruli from rapamycin-treated rats. Values of densitometric analysis are shown in the legend of Figure 5. IL-1 appears to be required for both angiogenesis and tumor invasiveness in IL-1-knockout mice.6.Voronov E. Shouval D.S. Krelin Y. et al.IL-1 is required for tumor invasiveness and angiogenesis.Proc Natl Acad Sci USA. 2003; 100: 2645-2650Crossref PubMed Scopus (795) Google Scholar Several studies have reported that IL-1 promotes tumor growth, invasion, and angiogenesis in animal models with concomitantly enhanced production of VEGF, MMPs, chemokines, and adhesion molecules.7.Yano S. Nokihara H. Yamamoto A. et al.Multifunctional interleukin-1beta promotes metastasis of human lung cancer cells in SCID mice via enhanced expression of adhesion-, invasion- and angiogenesis-related molecules.Cancer Sci. 2003; 94: 244-252Crossref PubMed Scopus (66) Google Scholar,8.Saijo Y. Tanaka M. Miki M. et al.Proinflammatory cytokine IL-1β promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction.J Immunol. 2002; 169: 469-475Crossref PubMed Scopus (207) Google Scholar IL-1 also promotes angiogenesis in vivo through the VEGF receptor pathway, possibly by inducing VEGF synthesis.34.Salven P. Hattori K. Heissig B. et al.Interleukin-1α (IL-1α) promotes angiogenesis in vivo via VEGFR-2 pathway by inducing inflammatory cell VEGF synthesis and secretion.FASEB J. 2002; 16: 1471-1473PubMed Google Scholar Mesangial cells are reportedly able to release VEGF32.Iijima K. Yoshikawa N. Connolly D.T. et al.Human mesangial cells and peripheral blood mononuclear cells produce vascular permeability factor.Kidney Int. 1993; 44: 959-966Abstract Full Text PDF PubMed Scopus (157) Google Scholar and this factor is determinant for mesangial cell-induced capillary formation in vitro.5.Kitahara T. Hiromura K. Ikeuchi H. et al.Mesangial cells stimulate differentiation of endothelial cells to form capillary-like networks in a three-dimensional culture system.Nephrol Dial Transplant. 2005; 20: 42-49Crossref PubMed Scopus (19) Google Scholar The present data show that IL-1β substantially increases VEGF expression in terms of both protein and mRNA, by RGMC, indicating an action at the transcriptional level. Based on previous reports concerning various cell types,10.Ben-Av P. Crofford L.J. Wilder R.L. et al.Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis.FEBS Lett. 1995; 372: 83-87Abstract Full Text PDF PubMed Scopus (495) Google Scholar, 14.Miura S. Tatsuguchi A. Wada K. et al.Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G444-G451Crossref PubMed Scopus (61) Google Scholar, 15.Stocks J. Bradbury D. Corbett L. et al.Cytokines upregulate vascular endothelial growth factor secretion by human airway smooth muscle cells: role of endogenous prostanoids.FEBS Lett. 2005; 579: 2551-2556Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar our initial focus was mainly driven by the assumption that PGE2 partially mediates the possible effect of IL-1β. We therefore analyzed the PGE2 production and the biosynthetic pathway involved. In a previous report, we postulated that resting RGMC did not produce PGE2 enzymatically, whereas in IL-1β-treated cells PGE2 was produced by enzymatic catalysis.27.Soler M. Camacho M. Solá R. et al.Mesangial cells release untransformed prostaglandin H2 as a major prostanoid.Kidney Int. 2001; 59: 1283-1289Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Nevertheless, enzymes involved in the transformation on PGH2 to PGE2 were not characterized at that time. In the present study, we show that RGMC constitutively express cPGES and mPGES-2. We also observed that these isozymes were not induced by IL-1β, which is consistent with other reports.30.Tanioka T. Nakatani Y. Semmyo N. et al.Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis.J Biol Chem. 2000; 275: 32775-32782Crossref PubMed Scopus (628) Google Scholar,31.Murakami M. Nakashima K. Kamei D. et al.Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2.J Biol Chem. 2003; 278: 37937-37947Crossref PubMed Scopus (295) Google Scholar Arachidonic acid mobilization is the first step in prostanoid biosynthesis. Hence, phospholipase activation is required for prostanoid synthesis from endogenous substrate. The fact that resting RGMC did not produce substantial PGE2 could be due to low phospholipase activity, which results in a low COX substrate availability. However, we previously observed that PGE2 formation by RGMC from exogenously added substrate was mainly non-enzymatic.27.Soler M. Camacho M. Solá R. et al.Mesangial cells release untransformed prostaglandin H2 as a major prostanoid.Kidney Int. 2001; 59: 1283-1289Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Moreover, specific PGES activity, incubating RGMC with exogenous purified PGH2, was only detected in IL-1β-treated cells (data not shown). Taking our previous data into account, these present results indicate that cPGES and mPGES-2 are not relevant for PGE2 biosynthesis in RGMC. In contrast, mPGES-1 was not detectable in resting RGMC, and production of PGE2 from endogenous substrate was only relevant when IL-1β induced its expression. This indicates that mPGES-1 was the main PGES isozyme responsible for PGH2 conversion to PGE2 in RGMC. Despite the fact that IL-1β significantly increased the ability of RGMC to synthesize PGE2, indomethacin, a strong inhibitor of both COX-1 and COX-2, was unable to alter IL-1β-induced VEGF production. In addition, exogenously added PGE2 was also unable to modify VEGF production in resting cells, whereas consistently with previous reports it increased MMP-2.35.Zahner G. Harendza S. Müller E. et al.Prostaglandin E2 stimulates expression of matrix metalloproteinase 2 in cultured rat mesangial cells.Kidney Int. 1997; 51: 1116-1123Abstract Full Text PDF PubMed Scopus (35) Google Scholar All together, these results allow us to rule out the idea that PGE2 mediates in the effect of IL-1β on VEGF in RGMC. Cell-type-specific regulation of VEGF expression by PGE2 could be related to the presence of the different PGE2 receptor subtypes. In agreement with others,36.Nasrallah R. Landry A. Scholey J.W. Hébert R.L. Characterization of the PGI2/IP system in cultured rat mesangial cells.Prostag Leukot Essent Fatty Acids. 20
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