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• W2078554706 abstract "Endothelial cells rapidly respond to changes in oxygen homeostasis by regulating gene expression. Regulator of G protein signaling 5 (RGS5) is a negative regulator of G protein-mediated signaling that is strongly expressed in vessels during angiogenesis; however, the role of RGS5 in hypoxia has not been fully understood. Under hypoxic conditions, we found that the expression of RGS5, but not other RGS, was induced in human umbilical vein endothelial cells (HUVEC). RGS5 mRNA was increased when HUVEC were incubated with chemicals that stabilized hypoxia-inducible factor-1α (HIF-1α), whereas hypoxia-stimulated RGS5 promoter activity was absent in HIF-1β−/− cells. Vascular endothelial growth factor (VEGF), which is regulated by HIF-1, did not appear to be involved in hypoxia-induced RGS5 expression; however, VEGF-mediated activation of p38 but not ERK1/2 was increased by RGS5. Overexpression of RGS5 in HUVEC exhibited a reduced growth rate without affecting the cell proliferation. Annexin V assay revealed that RGS5 induced apoptosis with significantly increased activation of caspase-3 and the Bax/Bcl-2 ratio. Small interfering RNA-specific for RGS5, caspase-3 inhibitor, and p38 inhibitor resulted in an attenuation of RGS5-stimulated apoptosis. Matrigel assay proved that RGS5 significantly impaired the angiogenic effect of VEGF and stimulated apoptosis in vivo. We concluded that RGS5 is a novel HIF-1-dependent, hypoxia-induced gene that is involved in the induction of endothelial apoptosis. Moreover, RGS5 antagonizes the angiogenic effect of VEGF by increasing the activation of p38 signaling, suggesting that RGS5 could be an important target for apoptotic therapy. Endothelial cells rapidly respond to changes in oxygen homeostasis by regulating gene expression. Regulator of G protein signaling 5 (RGS5) is a negative regulator of G protein-mediated signaling that is strongly expressed in vessels during angiogenesis; however, the role of RGS5 in hypoxia has not been fully understood. Under hypoxic conditions, we found that the expression of RGS5, but not other RGS, was induced in human umbilical vein endothelial cells (HUVEC). RGS5 mRNA was increased when HUVEC were incubated with chemicals that stabilized hypoxia-inducible factor-1α (HIF-1α), whereas hypoxia-stimulated RGS5 promoter activity was absent in HIF-1β−/− cells. Vascular endothelial growth factor (VEGF), which is regulated by HIF-1, did not appear to be involved in hypoxia-induced RGS5 expression; however, VEGF-mediated activation of p38 but not ERK1/2 was increased by RGS5. Overexpression of RGS5 in HUVEC exhibited a reduced growth rate without affecting the cell proliferation. Annexin V assay revealed that RGS5 induced apoptosis with significantly increased activation of caspase-3 and the Bax/Bcl-2 ratio. Small interfering RNA-specific for RGS5, caspase-3 inhibitor, and p38 inhibitor resulted in an attenuation of RGS5-stimulated apoptosis. Matrigel assay proved that RGS5 significantly impaired the angiogenic effect of VEGF and stimulated apoptosis in vivo. We concluded that RGS5 is a novel HIF-1-dependent, hypoxia-induced gene that is involved in the induction of endothelial apoptosis. Moreover, RGS5 antagonizes the angiogenic effect of VEGF by increasing the activation of p38 signaling, suggesting that RGS5 could be an important target for apoptotic therapy. Hypoxia is a common pathophysiological phenomenon that has a profound impact on endothelial cell properties during many cardiovascular disease processes and tumorigenesis. It has been extensively suggested that cell response to hypoxia is mainly regulated by hypoxia inducible factor-1α (HIF-1α) 2The abbreviations used are: HIF-1αhypoxia-inducible factor-1αsiRNAsmall interfering RNAVEGFvascular endothelial growth factorGPCRG protein-coupled receptorTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labelingHUVEChuman umbilical vein endothelial cellsDHBdihydroxybenzoateRGS5 o/eoverexpressing RGS5ERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinaseMAPKmitogen-activated protein kinaseGFPgreen fluorescent proteinZ-VAD-FMKcabobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone.2The abbreviations used are: HIF-1αhypoxia-inducible factor-1αsiRNAsmall interfering RNAVEGFvascular endothelial growth factorGPCRG protein-coupled receptorTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labelingHUVEChuman umbilical vein endothelial cellsDHBdihydroxybenzoateRGS5 o/eoverexpressing RGS5ERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinaseMAPKmitogen-activated protein kinaseGFPgreen fluorescent proteinZ-VAD-FMKcabobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone., which is rapidly ubiquitinated and degraded in normal conditions but can be stabilized by hypoxia (1.Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3873) Google Scholar, 2.Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2720) Google Scholar). Under hypoxic conditions, HIF-1 activates diverse genes involved in both cell growth and cell death (3.Carmeliet P. Dor Y. Herbert J.M. Fukumura D. Brusselmans K. Dewerchin M. Neeman M. Bono F. Abramovitch R. Maxwell P. Koch C.J. Ratcliffe P. Moons L. Jain R.K. Collen D. Keshert E. Nature. 1998; 394: 485-490Crossref PubMed Scopus (2215) Google Scholar). In endothelial cells, hypoxia stimulates the secretion of vascular endothelial growth factor (VEGF) and other angiogenic factors and receptors through transcriptional regulation by HIF-1, which leads to neovascularization and protection against ischemic injury (4.Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M. Yu A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (2044) Google Scholar, 5.Brunelle J.K. Chandel N.S. Apoptosis. 2002; 7: 475-482Crossref PubMed Scopus (146) Google Scholar, 6.Liu Y. Cox S.R. Morita T. Kourembanas S. Circ. Res. 1995; 77: 638-643Crossref PubMed Scopus (813) Google Scholar, 7.Mandriota S.J. Pyke C. Di Sanza C. Quinodoz P. Pittet B. Pepper M.S. Am. J. Pathol. 2000; 156: 2077-2089Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Conversely, HIF-1 has been shown to be a factor mediating hypoxia-induced apoptosis, growth of tumors with HIF-1α−/− was not retarded but accelerated because of decreased hypoxia-induced apoptosis (3.Carmeliet P. Dor Y. Herbert J.M. Fukumura D. Brusselmans K. Dewerchin M. Neeman M. Bono F. Abramovitch R. Maxwell P. Koch C.J. Ratcliffe P. Moons L. Jain R.K. Collen D. Keshert E. Nature. 1998; 394: 485-490Crossref PubMed Scopus (2215) Google Scholar). hypoxia-inducible factor-1α small interfering RNA vascular endothelial growth factor G protein-coupled receptor terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling human umbilical vein endothelial cells dihydroxybenzoate overexpressing RGS5 extracellular signal-regulated kinase c-Jun NH2-terminal kinase mitogen-activated protein kinase green fluorescent protein cabobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone. hypoxia-inducible factor-1α small interfering RNA vascular endothelial growth factor G protein-coupled receptor terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling human umbilical vein endothelial cells dihydroxybenzoate overexpressing RGS5 extracellular signal-regulated kinase c-Jun NH2-terminal kinase mitogen-activated protein kinase green fluorescent protein cabobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone. Regulator of G protein signaling (RGS) proteins are responsible for the rapid turnoff of G protein-coupled receptor signaling pathways via the GTPase-stimulating protein activity of their RGS domain (8.Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 428-432Crossref PubMed Scopus (335) Google Scholar, 9.Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). More than 25 RGS proteins have been identified, and there are indications that each will specifically regulate a particular G protein-coupled receptor pathway (10.De Vries L. Gist Farquhar M. Trends Cell Biol. 1999; 9: 138-144Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 11.Wieland T. Mittmann C. Pharmacol. Ther. 2003; 97: 95-115Crossref PubMed Scopus (114) Google Scholar). RGS5 belongs to the R4 subfamily of RGS proteins and is enriched in cardiovascular tissues, especially in pericytes and endothelial cells (12.Kirsch T. Wellner M. Luft F.C. Haller H. Lippoldt A. Brain Res. 2001; 910: 106-115Crossref PubMed Scopus (50) Google Scholar, 13.Adams L.D. Geary R.L. McManus B. Schwartz S.M. Circ. Res. 2000; 87: 623-631Crossref PubMed Scopus (93) Google Scholar, 14.Cho H. Kozasa T. Bondjers C. Betsholtz C. Kehrl J.H. FASEB J. 2003; 17: 440-442Crossref PubMed Scopus (148) Google Scholar) but not in cultured vascular smooth muscle cells (15.Grant S.L. Lassègue B. Griendling K.K. Ushio-Fukai M. Lyons P.R. Alexander R.W. Mol. Pharmacol. 2000; 57: 460-467Crossref PubMed Scopus (81) Google Scholar). RGS5 mRNA expression was found markedly decreased in a model of three-dimensional capillary morphogenesis (16.Bell S.E. Mavila A. Salazar R. Bayless K.J. Kanagala S. Maxwell S.A. Davis G.E. J. Cell Sci. 2001; 114: 2755-2773Crossref PubMed Google Scholar). However, it is also reported that RGS5 mRNA is highly expressed in endothelial cells in the tumor vasculature of human renal cell carcinoma (17.Furuya M. Nishiyama M. Kimura S. Suyama T. Naya Y. Ito H. Nikaido T. Ishikura H. J. Pathol. 2004; 203: 551-558Crossref PubMed Scopus (58) Google Scholar) and astrocytomas but not in HIF-1α deficient tumors (18.Berger M. Bergers G. Arnold B. Hämmerling G.J. Ganss R. Blood. 2005; 105: 1094-1101Crossref PubMed Scopus (149) Google Scholar). RGS5 is responsible for the abnormal tumor vascular morphology in mice. Loss of RGS5 results in pericyte maturation, vascular normalization, and consequent marked reductions in tumor hypoxia and vessel leakiness (19.Hamzah J. Jugold M. Kiessling F. Rigby P. Manzur M. Marti H.H. Rabie T. Kaden S. Gröne H.J. Hämmerling G.J. Arnold B. Ganss R. Nature. 2008; 453: 410-414Crossref PubMed Scopus (439) Google Scholar). These findings argue for an important role of RGS5 in endothelial function. G protein-coupled receptor (GPCR) signaling pathways are involved in cellular responses to many extracellular stimuli and are major targets for drug discovery. RGS5 is a negative modulator of the angiotensin AT1a receptor. The inhibition of RGS5 expression enhanced angiotensin-stimulated inositol phosphate release (20.Wang Q. Liu M. Mullah B. Siderovski D.P. Neubig R.R. J. Biol. Chem. 2002; 277: 24949-24958Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In addition, RGS5 attenuated endothelin-1-, sphingosine-1-phosphate-, and platelet-derived growth factor-induced ERK phosphorylation, which indicates that RGS5 exerts control over the platelet-derived growth factor receptor β and GPCR-mediated signaling pathways (14.Cho H. Kozasa T. Bondjers C. Betsholtz C. Kehrl J.H. FASEB J. 2003; 17: 440-442Crossref PubMed Scopus (148) Google Scholar). We conducted the present study to elucidate the molecular mechanisms controlling the regulation of RGS5 in hypoxic endothelium and the function of RGS5 in endothelial cells. The results presented here demonstrated that hypoxia increased RGS5 expression, which was mediated by HIF-1. Increased RGS5 expression induced apoptosis in human endothelial cells via increase of p38 MAPK activation. We report here, for the first time, that RGS5 is a novel hypoxia-inducible molecule involved in the regulation of endothelial cell behavior, indicating that RGS5 plays an important role in homeostasis in hypoxic endothelial cells. Human umbilical vein endothelial cells (HUVEC) were obtained from Clonetics (San Diego, CA) and cultured following the manufacturer's instructions. Human dermal microvascular endothelial cells-1 (HMEC-1; Center of Disease Control) were cultured in MCDB-131 (Invitrogen) containing 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 1 μg/ml hydrocortisone, and 2 mm l-glutamine. C4-B13NBii1 (HIF-1β−/−) and vT2 (HIF-1β+/+) cell lines were obtained from American Type Culture Collection and cultured according to the manufacturer's instructions. HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum. Hypoxic exposure was performed using a molecular incubator chamber (Billups-Rothenberg) flushed with 5% CO2 and 95% N2. The concentration of oxygen (∼1%) was determined before and after incubation by using an oxygen analyzer (Vascular Technology). Other reagents used to mimic hypoxia included dimethyloxalylglycine (DMOG) (BIOMOL), cobalt chloride (CoCl2), and 3,4-dihydroxybenzoate (3,4-DHB) (Sigma). Full-length RGS5 cDNA was obtained using reverse transcription-PCR from HUVEC and confirmed to be correct sequences corresponding to GenbankTM (NM_003617). RGS5 cDNA was cloned into pBMN-GFP vectors (Orbigen). In 10-cm dishes, 6 × 106 293T cells were transfected with pBMN-GFP-RGS5, pMD-VSVG, pJK3, and pCMV-tat using Polyethylenimine (Polysciences). Forty-eight h post transfection, virus-containing medium was collected, filtered through a 0.45-μm filter, and used to transduce HUVEC. Overexpression of RGS5 was confirmed by Western blotting. siRNA targeting human RGS5 were synthesized by Genpharma, Inc. (ZhangJiang). Two duplexes of siRNA (siRNA-1: 5′-AGAUGGCUGAGAAGGCAAATT-3′, 5′-UUUGCCUUCUCAGCCAUCUTG-3′ and siRNA-2: 5′-GCGUGAUUCCCUGGACAAATT-3′, 5′-UUUGUCCAGGGAAUCACGCCA-3′) were confirmed to have knockdown ability by Northern and Western blotting. Another duplex of RNA that is not targeted to any human genes was used as a control. HUVEC were transfected with siRNA at a final concentration of 50 nm using Lipofectamine 2000 (Invitrogen). Total RNA was extracted from cells by TRIzol (Invitrogen). Fifteen μg of RNA were loaded per lane, separated on a 1.3% formaldehyde-agarose gel, and transferred to a nylon membrane (Millipore). The membrane was then UV-cross-linked. Northern blot was performed by using a digoxin Northern starter kit (Roche Applied Science) as per the manufacturer's instruction. An RNA probe for human RGS5 containing digoxin-labeled dUTP was used for hybridization. Cells were lysed in radioimmune precipitation assay buffer (Boston BioProducts), and proteins were resolved by SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore). After being blocked for 1 h in Tris-buffered saline-Tween 20 with 5% nonfat milk, the polyvinylidene difluoride membrane was then probed with primary antibodies (RGS5 polyclonal antiserum was obtained by immunized rabbits, 1:2,000; RGS2 and RGS4 antibodies were from Santa Cruz Biotechnology, 1:1,000; Bax, Bcl-2, and p53 antibodies were from BD Bioscience, 1:1,000; and total and cleaved caspase-3, total and phosphorylated p38, and ERK antibodies were from Cell Signaling, 1:1,000) overnight at 4 °C and then with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The blots were detected with SuperSignal chemiluminescent substrate (Pierce Biotechnology). A series of fragments encompassing the 5′-flanking region of the human RGS5 gene (GenBankTMNT_004487) were obtained by PCR from human genomic DNA and cloned into PGL-3 vector (Promega). The cultured cells including HMEC-1, HIF-1β−/−, and HIF-1β+/+ were transfected with the constructs using Lipofectamine 2000. Twenty-four h after transfection, cells were exposed to CoCl2 or hypoxia for 5 h, and luciferase activity was determined using the Dual-Luciferase assay system (Promega). Matrigel assays were carried out as described (21.Zeng H. Qin L. Zhao D. Tan X. Manseau E.J. Van Hoang M. Senger D.R. Brown L.F. Nagy J.A. Dvorak H.F. J. Exp. Med. 2006; 203: 719-729Crossref PubMed Scopus (131) Google Scholar). SKMEL/VEGF cells (1 × 107) alone or mixed with 1 × 107 Phoenix cells infected with retrovirus expressing RGS5 were suspended in 0.5 ml of growth factor-reduced matrigel (BD Biosciences) and injected subcutaneously into nu/nu mice. Tissues were harvested, photographed, and fixed with 4% paraformaldehyde for terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and immunostaining. Three individual experiments were performed, n = 4 per group in each experiment. Frozen sections of the matrigel blocks were washed and incubated with anti-CD31 antibody (BD Biosciences) and counterstained by hematoxylin as described previously (22.Pettersson A. Nagy J.A. Brown L.F. Sundberg C. Morgan E. Jungles S. Carter R. Krieger J.E. Manseau E.J. Harvey V.S. Eckelhoefer I.A. Feng D. Dvorak A.M. Mulligan R.C. Dvorak H.F. Lab. Invest. 2000; 80: 99-115Crossref PubMed Scopus (361) Google Scholar). For immunofluorescent CD31 and TUNEL double staining, sections were incubated with anti-CD31 antibody for 1 h at room temperature followed by incubation with a Texas Red-conjugated secondary antibody for 1 h. Subsequent TUNEL staining was based on instruction of an apoptosis detection kit (Roche Applied Science). All slides were imaged on a Leica DM IRB fluorescent microscope. 104 cells were plated in each well of 12-well plates. At 5 h after plating (day 1) and at 2, 3, and 4 days after plating, the cells were fixed in 100% ethanol and subsequently stained with 0.1% crystal violet dissolved in 10% ethanol. After staining and thorough washing, the dye was extracted with 10% acetic acid, and absorbance was measured at 590 nm. Caspase inhibitor, cabobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (Z-VAD-FMK) (BIOMOL) was used in the cell growth measurements. To measure cell proliferation, HUVEC transduced with RGS5 or control were seeded in 12-well plates and incubated with 1 μCi/ml 3H[thymidine] at 37 °C for 4 h. Medium was then removed, and the wells were washed three times with phosphate-buffered saline. Radioactivity was extracted with 1 n NaOH and added to a scintillation vial containing 4 ml of ScintiVerse II (Fisher) solution. Thymidine incorporation was measured using a scintillation counter. Wells that contained no cells were labeled and counted to provide background counts. The cell migration rate was measured by wound healing assay. Equal numbers of cells (1 × 105) were plated in 12-well plates. The monolayer of cells were wounded by manual scratching with a pipette tip and then photographed in phase contrast with a Nikon microscope (0 h point) and placed into complete growth medium. Matching wound regions were photographed after 6, 12, and 24 h. The DNA ladder was examined in the infected HUVEC that were cultured in serum-free medium for 24 h. Cells were then removed from tissue culture plates by trypsin and resuspended in a lysis buffer (150 mm NaCl, 10 mm Tris-HCl, pH 8.0, 10 mm EDTA, 0.5% SDS, and 100 ng/ml proteinase K) for 4 h at 50 °C. DNA was extracted using phenol and chloroform followed by ethanol precipitation. The pellet was resuspended in Tris-EDTA buffer (10 mm Tris-HCl and 1 mm EDTA) and treated with RNase for 30 min at 37 °C. Ten micrograms of the DNA were fractionated by electrophoresis on a 2% agarose gel. Using an apoptosis assay kit (Invitrogen), annexin V binding assay was performed in confluent infected HUVEC in serum-free medium under hypoxic or normoxic conditions for 24 h and analyzed by flow cytometry using a FACScan (BD Biosciences). For each treatment, 10,000 cells were counted and then evaluated using Cell Quest software. Cells that were annexin V-positive and propidium iodide-negative were counted for early stages of apoptosis. Statistical significance was assessed by Student's t test, and p < 0.05 was considered statistically significant. HUVEC were exposed to hypoxic conditions (O2<1%) for periods of 0.5, 1, 3, 6, 12, or 24 h. As compared with cultured HUVEC in normoxia (21% O2), RGS5 mRNA expression was induced by hypoxia immediately from 0.5 h, continuously increased up to 12 h, and peaked at 3 h (2.30 ± 0.25-fold). The increased RGS5 expression returned to baseline at 24 h (Fig. 1A). In addition to the increase of RGS5 mRNA, the protein level was also induced from 3 h to 12 h after hypoxia and declined at 24 h. RGS5 protein expression showed a time-dependent pattern, whereas the other two RGS family members that were also expressed in endothelial cells, RGS2 and RGS4, were not induced by hypoxia (Fig. 1B). These results suggest that hypoxia exclusively up-regulate the expression of RGS5 in endothelial cells. We next focused on which mechanism was involved in the regulation of RGS5 by hypoxia. CoCl2 is recognized as a hypoxia-mimicking compound that stabilizes HIF, a key transcriptional regulator activated only in hypoxic conditions. Incubation of HUVEC in the presence of 150 μm CoCl2 resulted in an induction of RGS5 mRNA (Fig. 2A) with the same time-dependent pattern as the hypoxia induction (data not shown). To further investigate whether the induction of RGS5 is HIF-1-regulated in endothelial cells, HUVEC were incubated in the presence of prolyl hydroxylases inhibitor, ethyl 3,4-DHB or DMOG. As shown in Fig. 2B, exposure of HUVEC to 200 μm DHB for 6 h resulted in a strong increase of RGS5 mRNA expression. Similarly, the cells incubated in the presence of 500 μm DMOG for 6 h, in which the HIF-1α degradation would be blocked for the same duration, showed a significant increase of RGS5 mRNA expression (Fig. 2C). In addition to the increase of RGS5 mRNA, the expression of RGS5 protein was also induced in HUVEC incubated with CoCl2, DHB, and DMOG respectively (Fig. 2D). Together, these results indicate that RGS5 mRNA and protein expression are controlled by changes in oxygen level and that HIF-1α is involved in the regulation of RGS5 in hypoxic endothelial cells. To evaluate whether RGS5 is a target gene for HIF-1α, promoter regions of RGS5 were cloned and constructed. In HMEC-1, the activities of the luciferase construct of RGS5 promoter were measured. As shown in Fig. 3A, exposure of HMEC-1 transfected with RGS5 promoter of various length sequences including 2.4 kb (down to −2214); 1.1 kb (to −772); 0.64 kb (to −413); and 0.46 kb (to −236), but not 0.32 kb (to −93) to CoCl2 (150 μm, 6 h) led to an increase in luciferase activity when compared with HMEC-1 not incubated with CoCl2. In addition, when the construct of RGS5 promoter (2.4 kb) was transfected into the cells lacking HIF-1β, with which HIF-1α forms obligate heterodimers (23.Wood S.M. Gleadle J.M. Pugh C.W. Hankinson O. Ratcliffe P.J. J. Biol. Chem. 1996; 271: 15117-15123Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), and then exposed to hypoxia, no induction of RGS5 promoter activity was detected, whereas induction was observed (1.6 ± 0.11-fold) in the control cells normally expressing HIF-1β (Fig. 3B). These results imply that the HIF pathway is required for hypoxia-regulated RGS5 expression. VEGF is an important gene regulated by HIF-1α under hypoxic conditions. VEGF signaling has broad effects on the growth of endothelial cells. To examine the relationship between these two HIF-dependent genes, RGS5 and VEGF, HUVEC were treated with VEGF by different doses and duration. The results showed that VEGF did not regulate RGS5 expression (Fig. 4A). However, in HUVEC with overexpressing RGS5 (RGS5 o/e) (Fig. 4B), VEGF-induced p38 activation was significantly increased, but ERK1/2 activation was not changed (Fig. 4C). A decreased phosphorylation level of p38 was observed in the HUVEC transfected with RGS5 siRNA (Fig. 4, D and E). These results suggest that HIF-1 independently regulates VEGF and RGS5, but RGS5 can affect the VEGF pathway. Hypoxia-induced genes could lead to endothelial cells to undergo opposing processes, either angiogenesis or apoptosis; however, little is known regarding the functional properties of RGS5 in endothelial cells under hypoxia. To test the consequence of up-regulation of RGS5 in hypoxia, functional properties of RGS5 in endothelial cells were examined in HUVEC with RGS5 o/e. As shown in Fig. 5A, a growth curve demonstrated that the HUVEC with RGS5 o/e grew significantly slower than the control cells that were infected with empty vectors. However, this growth defect did not present in parallel to the proliferative rates that were measured by thymidine incorporation (Fig. 5B), which indicated that the ability of RGS5 might not be correlated to hypoxia-induced angiogenesis. Wound healing scratch assays were performed to determine whether stable RGS5 expression would also affect the endothelial migration (Fig. 5C). Both the control and the RGS5 o/e cells completed the wound healing within 24 h. No significant difference was observed during the process. Because RGS5 attenuated the growth rate of endothelial cells but did not impact its proliferation, it has been considered that RGS5 may affect the apoptosis of endothelial cells. To test this possibility, apoptosis was induced by withdrawing serum and growth factors from HUVEC culture medium for 24 h. In Fig. 6A, a DNA laddering analysis illustrated a significant increase of apoptosis in HUVEC with RGS5 o/e compared with controls. In addition, an increase of activated caspase-3 was observed in HUVEC with RGS5 o/e (Fig. 6B), which could be blocked by application of Z-VAD-FMK (Fig. 6C), and consequently resulted in preventing the RGS5-attenuated endothelial cell growth (Fig. 6D). Moreover, in HUVEC with RGS5 o/e, pro-apoptotic protein Bax was significantly increased by nutrient deprivation whereas there was no difference in Bcl-2 expression between RGS5 o/e and control cells, indicating that the apoptosis occurred because of the change in the Bcl-2/Bax ratio (24.Yang E. Korsmeyer S.J. Blood. 1996; 88: 386-401Crossref PubMed Google Scholar) (Fig. 6B). In addition, p53 was not regulated in RGS5 o/e endothelial cells (Fig. 6B) although it was correlated with the induction of apoptosis in hypoxic endothelial cells (25.Stempien-Otero A. Karsan A. Cornejo C.J. Xiang H. Eunson T. Morrison R.S. Kay M. Winn R. Harlan J. J. Biol. Chem. 1999; 274: 8039-8045Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Other than changes of apoptotic factors, RGS5 o/e endothelial cells undergoing apoptosis was also confirmed by FACS analysis. Fig. 6E demonstrated an increase in the numbers of annexin V-positive and propidium iodide-negative cells in HUVEC with RGS5 o/e in comparison to controls after exposure to serum free medium for 24 h (18.35% versus 2.73%), whereas application of p38 inhibitor SB203580 (20 μm) to RGS5 o/e HUVEC resulted in a significant decrease in the number of apoptotic cells. Furthermore, with silencing of the RGS5 gene, HUVEC were rescued to a certain extent from hypoxia-induced apoptosis (Fig. 6F). These findings suggest that RGS5 was involved in the enhancement of apoptosis in association with p38 MAPK activation in endothelial cells. To further elucidate the function of RGS5 in the in vivo setting, matrigel assay was performed in nude mice. On day 3 after implantation of matrigel plugs that were incorporated with SK-MEL-2 melanoma cells expressing VEGF-A165 (SK-MEL/VEGF cells) and/or Phoenix cells packaging retroviruses expressing RGS5, expression of RGS5 in the vasculature of matrigel was confirmed by Western blotting and immunostaining (Fig. 7A). The angiogenic responses were evaluated by histology and immunohistochemistry for the endothelial cell marker CD31 (Fig. 7B). Strong angiogenesis was induced in plugs containing SK-MEL/VEGF cells (Fig. 7B, b). However, the matrigel plug that contained RGS5 significantly impaired the VEGF-induced angiogenesis (Fig. 7B, d) evidenced by the decreased vessel density accounted by CD31 positive cells (Fig. 7C). TUNEL and CD31 labeling were performed to evaluate endothelial cell apoptosis. As Fig. 7D illustrates, the concurrent TUNEL/CD31-positive cells were only observed in the matrigel plug that contained expressing RGS5. Hypoxia and ischemia trigger a multitude of responses designed to compensate for reduced oxygen availability. In endothelial cells, these responses include increased expression of growth factors and their receptors to induce angiogenesis (5.Brunelle J.K. Chandel N.S. Apoptosis. 2002; 7: 475-482Crossref PubMed Scopus (146) Google Scholar, 7.Mandriota S.J. Pyke C. Di Sanza C. Quinodoz P. Pittet B. Pepper M.S. Am. J. Pathol. 2000; 156: 2077-2089Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). On the other hand, hypoxia-regulated genes can lead cells to apoptosis to maintain the homeostasis (3.Carmeliet P. Dor Y. Herbert J.M. Fukumura D. Brusselmans K. Dewerchin M. Neeman M. Bono F. Abramovitch R. Maxwell P. Koch C.J. Ratcliffe P. Moons L. Jain R.K. Collen D. Keshert E. Nature. 1998; 394: 485-490Crossref PubMed Scopus (2215) Google Scholar). Thus, a thorough understanding of the mechanisms that hypoxia-related genes use to regulate endothelial cell behavior may aid in the comprehension of vascular homeostasis. In this report, we demonstrated that regulator of G protein signaling 5 is a novel hypoxia-induced gene in endothelial cells. Expression of RGS5 can be significantly induced by hypoxia at both mRNA and protein levels. Hypoxia induces expression of RGS5 but not RGS2 and RGS4, although these three RGS members have been reported to express in endothelial cells (12.Kirsch T. Wellner M. Luft F.C. Haller H. Lippoldt A. Brain Res. 2001; 910: 106-115Crossref PubMed" @default.
• W2078554706 created "2016-06-24" @default.
• W2078554706 creator A5000353838 @default.
• W2078554706 creator A5052883326 @default.
• W2078554706 creator A5063726925 @default.
• W2078554706 creator A5065562998 @default.
• W2078554706 creator A5084319538 @default.
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• W2078554706 date "2009-08-01" @default.
• W2078554706 modified "2023-10-15" @default.
• W2078554706 title "RGS5, a Hypoxia-inducible Apoptotic Stimulator in Endothelial Cells" @default.
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