The Gα12/13 Family of Heterotrimeric G Proteins and the Small GTPase RhoA Link the Kaposi Sarcoma-associated Herpes Virus G Protein-coupled Receptor to Heme Oxygenase-1 Expression and Tumorigenesis
2007; Elsevier BV; Volume: 282; Issue: 47 Linguagem: Inglês
10.1074/jbc.m703043200
ISSN1083-351X
AutoresMarÁa José MartÁn, Tamara Tanos, Ana Belén GarcÁa, Daniel Martı́n, J. Silvio Gutkind, Omar A. Coso, María Julia Marinissen,
Tópico(s)Adenosine and Purinergic Signaling
ResumoHeme oxygenase-1 (HO-1), an inducible enzyme that metabolizes the heme group, is highly expressed in human Kaposi sarcoma lesions. Its expression is up-regulated by the G protein-coupled receptor from the Kaposi sarcoma-associated herpes virus (vGPCR). Although recent evidence shows that HO-1 contributes to vGPCR-induced tumorigenesis and vascular endothelial growth factor (VEGF) expression, the molecular steps that link vGPCR to HO-1 remain unknown. Here we show that vGPCR induces HO-1 expression and transformation through the Gα12/13 family of heterotrimeric G proteins and the small GTPase RhoA. Targeted small hairpin RNA knockdown expression of Gα12, Gα13, or RhoA and inhibition of RhoA activity impair vGPCR-induced transformation and ho-1 promoter activity. Knockdown expression of RhoA also reduces vGPCR-induced VEFG-A secretion and blocks tumor growth in a murine allograft tumor model. NIH-3T3 cells expressing constitutively activated Gα13 or RhoA implanted in nude mice develop tumors displaying spindle-shaped cells that express HO-1 and VEGF-A, similarly to vGPCR-derived tumors. RhoAQL-induced tumor growth is reduced 80% by small hairpin RNA-mediated knockdown expression of HO-1 in the implanted cells. Likewise, inhibition of HO-1 activity by chronic administration of the HO-1 inhibitor tin protoporphyrin IX to mice reduces RhoAQL-induced tumor growth by 70%. Our study shows that vGPCR induces HO-1 expression through the Gα12/13/RhoA axes and shows for the first time a potential role for HO-1 as a therapeutic target in tumors where RhoA has oncogenic activity. Heme oxygenase-1 (HO-1), an inducible enzyme that metabolizes the heme group, is highly expressed in human Kaposi sarcoma lesions. Its expression is up-regulated by the G protein-coupled receptor from the Kaposi sarcoma-associated herpes virus (vGPCR). Although recent evidence shows that HO-1 contributes to vGPCR-induced tumorigenesis and vascular endothelial growth factor (VEGF) expression, the molecular steps that link vGPCR to HO-1 remain unknown. Here we show that vGPCR induces HO-1 expression and transformation through the Gα12/13 family of heterotrimeric G proteins and the small GTPase RhoA. Targeted small hairpin RNA knockdown expression of Gα12, Gα13, or RhoA and inhibition of RhoA activity impair vGPCR-induced transformation and ho-1 promoter activity. Knockdown expression of RhoA also reduces vGPCR-induced VEFG-A secretion and blocks tumor growth in a murine allograft tumor model. NIH-3T3 cells expressing constitutively activated Gα13 or RhoA implanted in nude mice develop tumors displaying spindle-shaped cells that express HO-1 and VEGF-A, similarly to vGPCR-derived tumors. RhoAQL-induced tumor growth is reduced 80% by small hairpin RNA-mediated knockdown expression of HO-1 in the implanted cells. Likewise, inhibition of HO-1 activity by chronic administration of the HO-1 inhibitor tin protoporphyrin IX to mice reduces RhoAQL-induced tumor growth by 70%. Our study shows that vGPCR induces HO-1 expression through the Gα12/13/RhoA axes and shows for the first time a potential role for HO-1 as a therapeutic target in tumors where RhoA has oncogenic activity. Heme oxygenase-1 is an inducible and ubiquitous 32-kDa enzyme that controls heme metabolism and iron levels by catalyzing the degradation of the heme group (iron protoporphyrin IX). The products of this enzymatic reaction are the physiological messenger molecule carbon monoxide, free iron, and biliverdin, the last being subsequently reduced to the antioxidant bilirubin (1Maines M.D. Gibbs P.E. Biochem. Biophys. Res. Commun. 2005; 338: 568-577Crossref PubMed Scopus (201) Google Scholar). The regulation of HO-1 activity depends primarily on the control of HO-1 expression at the transcriptional level (1Maines M.D. Gibbs P.E. 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Chem. 2004; 279: 8919-8929Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). Because of the antioxidant properties of the heme metabolism products, HO-1 has been considered a cytoprotector molecule involved in several physiological responses against inflammation and oxidative and cellular stress (2Alam J. Igarashi K. Immenschuh S. Shibahara S. Tyrrell R.M. Antioxid. Redox Signal. 2004; 6: 924-933Crossref PubMed Scopus (104) Google Scholar). However, an increasing number of studies have now expanded this notion defining HO-1 as an important regulator of the physiology of the vasculature, endothelial cell cycle control, proliferation, vascular endothelial growth factor (VEGF) 5The abbreviations used are: VEGF, vascular endothelial growth factor; KSHV, Kaposi sarcoma-associated herpesvirus; GPCR, G protein-coupled receptor; vGPCR, G protein-coupled receptor from the Kaposi sarcoma-associated herpes virus; HA, hemagglutinin; shRNA, small hairpin RNA; DMEM, Dulbecco's modified Eagle's medium; SnPP, tin protoporphyrin IX; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole; RT, reverse transcription; GFP, green fluorescent protein; KS, Kaposi sarcoma; SRE, serum response element. secretion, angiogenesis, and tumorigenesis (3Dulak J. Loboda A. Zagorska A. Jozkowicz A. Antioxid. 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Kaposi sarcoma (KS) is the most frequent type of tumor in AIDS patients. The multifocal angioproliferative lesions are formed by spindle cells derived from endothelial cells transformed by the KSHV (16Flore O. Rafii S. Ely S. O'Leary J.J. Hyjek E.M. Cesarman E. Nature. 1998; 394: 588-592Crossref PubMed Scopus (354) Google Scholar). Several experimental strategies using animal models revealed that the product of the orf 74 from the KSHV genome, a G protein-coupled receptor (vGPCR), plays a key role in the development of KSHV-induced oncogenesis (16Flore O. Rafii S. Ely S. O'Leary J.J. Hyjek E.M. Cesarman E. Nature. 1998; 394: 588-592Crossref PubMed Scopus (354) Google Scholar, 17Montaner S. Sodhi A. Molinolo A. Bugge T.H. Sawai E.T. He Y. Li Y. Ray P.E. Gutkind J.S. Cancer Cell. 2003; 3: 23-36Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 18Montaner S. Sodhi A. Ramsdell A.K. Martin D. Hu J. Sawai E.T. Gutkind J.S. 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Cancer Cell. 2007; 11: 245-258Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). vGPCR is homologous to the mammalian interleukin-8 receptor CXCR2 (22Arvanitakis L. Geras-Raaka E. Varma A. Gershengorn M.C. Cesarman E. Nature. 1997; 385: 347-350Crossref PubMed Scopus (577) Google Scholar) but contains a D142V mutation in the highly conserved Asp-Arg-Tyr (DRY) motif in homologue mammalian GPCRs, which enables its constitutive, ligand-independent activity. Expression of vGPCR induces transformation in fibroblasts, angiogenesis in endothelial cells (23Bais C. Santomasso B. Coso O. Arvanitakis L. Raaka E.G. Gutkind J.S. Asch A.S. Cesarman E. Gershengorn M.C. Mesri E.A. Nature. 1998; 391: 86-89Crossref PubMed Scopus (752) Google Scholar), and KS-like angioproliferative lesions in mice (17Montaner S. Sodhi A. Molinolo A. Bugge T.H. Sawai E.T. He Y. Li Y. Ray P.E. Gutkind J.S. Cancer Cell. 2003; 3: 23-36Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 23Bais C. Santomasso B. Coso O. Arvanitakis L. Raaka E.G. Gutkind J.S. Asch A.S. Cesarman E. Gershengorn M.C. Mesri E.A. Nature. 1998; 391: 86-89Crossref PubMed Scopus (752) Google Scholar, 24Yang T.Y. Chen S.C. Leach M.W. Manfra D. Homey B. Wiekowski M. Sullivan L. Jenh C.H. Narula S.K. Chensue S.W. Lira S.A. J. Exp. Med. 2000; 191: 445-454Crossref PubMed Scopus (360) Google Scholar). We have recently shown that vGPCR induces HO-1 mRNA and protein levels in fibroblasts and endothelial cells and that these increased levels correlate with increased cell proliferation and survival, as well as increased VEGF-A expression, one of the determinant events in KS development. Additionally, inhibition of HO-1 expression or activity critically impairs vGPCR-induced tumorigenesis in allograft tumor animal models (25Marinissen M.J. Tanos T. Bolos M. de Sagarra M.R. Coso O.A. Cuadrado A. J. Biol. Chem. 2006; 281: 11332-11346Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Despite the implication of HO-1 as a vGPCR downstream target, the nature of the molecular pathways connecting the receptor to HO-1 expression remains unknown. New studies show that vGPCR contributes to KS development by switching on a complex network of signaling pathways, including the direct or autocrine/paracrine activation and expression of receptors, cytokines, signaling molecules, and transcription factors (18Montaner S. Sodhi A. Ramsdell A.K. Martin D. Hu J. Sawai E.T. Gutkind J.S. Cancer Res. 2006; 66: 168-174Crossref PubMed Scopus (89) Google Scholar, 26Polson A.G. Wang D. DeRisi J. Ganem D. Cancer Res. 2002; 62: 4525-4530PubMed Google Scholar). As other GPCRs, vGPCR activates downstream effectors by coupling to distinct α subunits of heterotrimeric G proteins (27Cannon M.L. Cesarman E. Oncogene. 2004; 23: 514-523Crossref PubMed Scopus (67) Google Scholar, 28Dadke D. Fryer B.H. Golemis E.A. Field J. Cancer Res. 2003; 63: 8837-8847PubMed Google Scholar, 29Shepard L.W. Yang M. Xie P. Browning D.D. Voyno-Yasenetskaya T. Kozasa T. Ye R.D. J. Biol. Chem. 2001; 276: 45979-45987Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Among these effectors are the monomeric or small G proteins of the Rho family represented by RhoA, Rac1, and Cdc42 (30Hall A. Biochem. Soc. Trans. 2005; 33: 891-895Crossref PubMed Scopus (628) Google Scholar). Indeed, Rac1 is activated by the receptor and mediates vGPCR-induced transformation in cells and tumorigenesis in animal models (28Dadke D. Fryer B.H. Golemis E.A. Field J. Cancer Res. 2003; 63: 8837-8847PubMed Google Scholar, 31Montaner S. Sodhi A. Servitja J.M. Ramsdell A.K. Barac A. Sawai E.T. Gutkind J.S. Blood. 2004; 104: 2903-2911Crossref PubMed Scopus (87) Google Scholar), whereas RhoA is required for vGPCR-induced NFκB activation and interleukin-8 secretion (29Shepard L.W. Yang M. Xie P. Browning D.D. Voyno-Yasenetskaya T. Kozasa T. Ye R.D. J. Biol. Chem. 2001; 276: 45979-45987Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Although it is known that some GPCRs induce HO-1 expression (5Lam C.W. Getting S.J. Perretti M. J. Immunol. 2005; 174: 2297-2304Crossref PubMed Scopus (43) Google Scholar, 32Ishizaka N. Griendling K.K. Hypertension. 1997; 29: 790-795Crossref PubMed Scopus (57) Google Scholar, 33Matsuoka Y. Kitamura Y. Kakimura J. Taniguchi T. Neuropharmacology. 1999; 38: 825-834Crossref PubMed Scopus (18) Google Scholar, 34Sun J. Kim S.J. Park M.K. Kim H.J. Tsoy I. Kang Y.J. Lee Y.S. Seo H.G. Lee J.H. Chang K.C. FEBS Lett. 2005; 579: 5494-5500Crossref PubMed Scopus (16) Google Scholar), the nature of the molecules connecting these receptors to HO-1 expression has not been established. In this study, we show that expression of constitutively activated Gα12 and Gα13 subunits mimicked vGPCR-induced HO-1 expression and transformation and that vGPCR activates HO-1 and transformation through both G α subunits. Our data indicate that vGPCR activates the small GTPase RhoA, a known downstream effector of Gα112/13, and that reduced expression or inhibition of RhoA impairs vGPCR-induced VEGF expression and secretion, cell survival and proliferation, and transformation both in cell culture and in a murine allograft tumor model. Moreover, Gα13- and RhoA-induced tumorigenesis are dramatically impaired when HO-1 expression or activity is inhibited. These data show that the Gα12/13/RhoA signal transduction pathway is an important mediator of vGPCR-induced tumor growth and demonstrate the participation of HO-1 in this process. These findings uncover a more extensive role of HO-1 in tumorigenesis and suggest that the enzyme can be a potential therapeutic target in the treatment not only of KS but also of tumors where RhoA is oncogenic. DNA Constructs—The plasmids pHO1-Luc containing a 5-kb human HO-1 promoter upstream of a luciferase gene and pVEGF-Luc have been previously described (35Goldhar A.S. Vonderhaar B.K. Trott J.F. Hovey R.C. Mol. Cell. Endocrinol. 2005; 232: 9-19Crossref PubMed Scopus (64) Google Scholar, 36Rojo A.I. Salina M. Salazar M. Takahashi S. Suske G. Calvo V. de Sagarra M.R. Cuadrado A. Free Radic. Biol. Med. 2006; 41: 247-261Crossref PubMed Scopus (55) Google Scholar). pCEFL-AU5-vGPCR, pCDNAIII-β-galactosidase, pCEFL-green fluorescent protein (GFP), pCDNAIII-Gα12QL, pCEFL-HA-Gα13QL, pCDNAIII-GαqQL, pCDNAIII-GαiQL, pCDNAIII-GαsQL, pCEFL-HA-m2, pCEFL-HA-G13i5, pCEFL-HA-Gqi5, pCEFL-p115RGS-D, pCEFL-AU5-RhoAQL, pCEFL-HA-HO-1, pCEFL-AU1-PDZRhoGEF, pCEFL-RhoN19, pEF-C3, pCEFL-AU5-Rac1QL, pNFκB-Luc, and pSshRNAHO-1 have been already described (25Marinissen M.J. Tanos T. Bolos M. de Sagarra M.R. Coso O.A. Cuadrado A. J. Biol. Chem. 2006; 281: 11332-11346Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 37Chikumi H. Vazquez-Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 38Marinissen M.J. Chiariello M. Tanos T. Bernard O. Narumiya S. Gutkind J.S. Mol. Cell. 2004; 14: 29-41Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 39Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar, 40Marinissen M.J. Servitja J.M. Offermanns S. Simon M.I. Gutkind J.S. J. Biol. Chem. 2003; 278: 46814-46825Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). pCEP4, a plasmid carrying a hygromycin resistance gene, was commercially purchased (Invitrogen). Plasmids carrying shRNAs sequences were constructed by inserting double-stranded shRNA oligonucleotides in the pSilencer 1.0-U6 plasmid following the manufacturer's instructions (Ambion). The plasmids pSshRNARho1 and pSshRNARho2 carrying shRNA sequences for RhoA were engineered by annealing the following single strand oligonucleotides: 5′-GACATGCTTGCTCATAGTCTTCAAGAGAGACTATGAGCAAGCATGTCTTTTTT and 3′-AATTAAAAAAGACATGCTTGCTCATAGTCTCTCTTGAAGACTATGAGCAAGCATGTCGGCC for pSshRNARho1 (41Pille J.Y. Denoyelle C. Varet J. Bertrand J.R. Soria J. Opolon P. Lu H. Pritchard L.L. Vannier J.P. Malvy C. Soria C. Li H. Mol. Ther. 2005; 11: 267-274Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar) and 5′-GAAACTGGTGATTGTTGGTTTCAAGAGAACCAACAATCACCAGTTTCTTTTTT and 3′-AATTAAAAAAGAAACTGGTGATTGTTGGTTCTCTTGAAACCAACAATCACCAGTTTCGGCC for pSshRNARho2. pSshRNAGα13.1 and pSshRNAGα13.2 were constructed using the following sequences: shRNAGα13.1, 5′-GTCCAAGGAGATCGACAAATTCAAGAGATTTGTCGATCTCCTTGGACTTTTTT and 3′-AATTAAAAAAGTCCAAGGAGATCGACAAATCTCTTGAATTTGTCGATCTCCTTGGACGGCC; shRNAGα13.2, 5′-GCAACGTGATCAAAGGTATTTCAAGAGAATACCTTTGATCACGTTGCTTTTTT and 3′-AATTAAAAAAGCAACGTGATCAAAGGTATTCTCTTGAAATACCTTTGATCACGTTGCGGCC. Gα12 shRNA oligonucleotides (shRNAGα12) have been previously described (42Shin K.J. Wall E.A. Zavzavadjian J.R. Santat L.A. Liu J. Hwang J.I. Rebres R. Roach T. Seaman W. Simon M.I. Fraser I.D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13759-13764Crossref PubMed Scopus (267) Google Scholar). A control construct carrying a "scramble" sequence, pSshRNAscramble, was made, annealing the oligonucleotides 5′-GCTAATTCCGAATCGGTCTTTCAAGAGAAGACCGATTCGGAATTAGCTTTTTT and 3′-AATTAAAAAAGCTAATTCCGAATCGGTCTTCTCTTGAAAGACCGATTCGGAATTAGCGGCC. Cell Lines and Transfections—Transient transfections of NIH-3T3 and HEK (human embryonic kidney) 293T were performed using the Lipofectamine Plus reagent (Invitrogen). HEK 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. NIH-3T3 fibroblasts were maintained in DMEM (Invitrogen) supplemented with 10% calf serum. NIH-vGPCR cells were prepared as described (25Marinissen M.J. Tanos T. Bolos M. de Sagarra M.R. Coso O.A. Cuadrado A. J. Biol. Chem. 2006; 281: 11332-11346Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Stable transfection of NIH-3T3 cells was performed using the calcium phosphate technique (40Marinissen M.J. Servitja J.M. Offermanns S. Simon M.I. Gutkind J.S. J. Biol. Chem. 2003; 278: 46814-46825Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Cells were plated at 20% confluence in 10-cm plates and transfected with 10 μg of pCEFL (NIH-3T3c), pCEFL-HA-m2 (NIH-m2), pCEFL-HA-Gα13QL (NIH-Gα13QL), or pCEFL-AU5-RhoAQL (NIH-RhoAQL). Stably transfected cells were selected with 750 μg/ml G418 (Promega). NIH-m2 cells were further transfected with 5 μg of pCEFL-G13i5 (NIH-m2-G13i5) or pCEFL-Gqi5 (NIH-m2-Gqi5) along with 100 ng of pCEP4 to allow selection by hygromycin. NIH-RhoAQL cells were transfected with 5 μg of pSshRNAHO-1 (NIH-RhoAQLshHO-1) and 100 ng of pCEP4. NIH-vGPCR cells were transfected with 100 ng of pCEP4 and 5 μg of pSshRNARho1 or pSshRNARho2 (NIH-vGPCRshRho1, NIH-vGPCRshRho2). NIH-vGPCR cells were also stably transfected with 5 μg of pSshRNAGα13.1, pSshRNAGα13.2, pSshRNAGα12, or pSshRNAscramble (NIH-vGPCRshG13.1, NIH-vGPCRshG13.2, NIH-vGPCRshG12, or NIH-vGPCRshScram, respectively). Transfected cells were selected with 200 μg/ml hygromycin B from Streptomyces (Sigma). Gene-specific Real Time PCR—Total RNA from cells was extracted by homogenization in TRIzol (Invitrogen). Briefly, cells were grown to 80% confluence, serum-starved for 24 h, washed with cold PBS, and lysed in TRIzol according to the manufacturer's indications. Equal amounts of RNA (1 μg) were reverse-transcribed to obtain cDNA with the Enhanced Avian First Strand Synthesis kit (Sigma). Real time PCR was performed using the ABI prism 7700 sequencer detector system and Qiagen's Quantitect SYBR Green PCR kit (Qiagen, Chatsworth, CA), following the manufacturer's protocol. In brief, the reaction mixture (50-μl total volume) contained 500 ng of cDNA, gene-specific forward and reverse primers for each gene at 1 mm final concentration, and 25 μl of Quantitect SYBR Green PCR Master Mix. The real time cycler conditions were as follows: PCR initial activation step at 95 °C for 10 min, 40 cycles each of melting at 95 °C for 15 s, and annealing/extension at 60 °C for 1 min. A negative control without template was included in parallel to assess the overall specificity of the reaction. The PCR products were analyzed on a 1.2% agarose gel to confirm the size of the amplified product. After PCR, a comparative delta cycle threshold method was used to determine the relative amounts of specific gene and actin mRNA. The sequences of the primers used were as follows: HO-1, 5′-CAACAGTGGCAGTGGGAATTT and 3′-CCAGGCAAGATTCTCCCTTAC; GAPDH, 5′-TCCATCACAACTTTGGCATTG and 3′-TCACGCACAAGCTTTCCA; β-actin 5′-AGTACTCCGTGTGGATCGGC and 3-AGGTCGTCTACACCTAGTCG. Analysis of mRNA Levels by Semiquantitative Reverse Transcription-PCR—Total RNA from cells was obtained by extraction in TRIzol (Invitrogen). Total RNA from tumors was obtained by homogenizing the tissue in TRIzol with a Teflon homogenizer followed by sonication. Equal amounts of RNA (1 μg) were reverse-transcribed to obtain cDNA with the Enhanced Avian First Strand Synthesis kit (Sigma). PCRs were performed using the Ready Mix RedTaq PCR reactive mix (Sigma). HO-1 oligonucleotides described above were used to amplify a 106-bp HO-1 fragment. A vGPCR fragment was amplified using the oligonucleotides 5′-GCGAATTCACCATGGCGGCCGAGGATTTCCTAAC and 3′-GCGCGGCCGCCTACGTGGTGGCGCCGGACATGA. The three splice variants of VEGF-A were amplified using 5′-CTGCTCTCTTGGGTGCACTGG and 3′-ACCGCCTTGGCTTGTCACAT primers (40Marinissen M.J. Servitja J.M. Offermanns S. Simon M.I. Gutkind J.S. J. Biol. Chem. 2003; 278: 46814-46825Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Expected product sizes were 431, 563, and 635 bp, corresponding to the VEGF120, VEGF164, and VEGF188 splice variant isoforms (35Goldhar A.S. Vonderhaar B.K. Trott J.F. Hovey R.C. Mol. Cell. Endocrinol. 2005; 232: 9-19Crossref PubMed Scopus (64) Google Scholar). A 982-bp DNA fragment from the coding sequence for AU5-RhoAQL was amplified by the oligonucleotides AU5 (5′-ATGGGATCCACCGACTTCTACC) and RhoA (3′-CTTCCCACGTCTAGCTTGCAGA). HA-Gα13QL was amplified using HA (5′-TACCCATATGACGTACCGGATTACGCA) and Gα13 (3′-GAGATTCTGTAAGGCGATTG). A fragment of 102 bp from the GAPDH housekeeping gene was amplified in parallel to all reactions to ensure that equal amounts of starting cDNA were used in each reaction. After an initial denaturalization step of 2 min at 94 °C, amplification of each cDNA was performed in 22-34 cycles (in increments of 2), to detect the linear amplification phase. Reactions were set at 28 cycles, which also allowed detection of basal HO-1 mRNA levels in control cells. The same amount of cycles was used for VEGF-A, AU5-RhoA, HA-Gα13, and GAPDH, using a thermal profile of 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C. The PCR products were detected by electrophoresis in agarose gels and fluorescence under UV light upon ethidium bromide staining. Luciferase Reporter Assays—NIH-3T3 cells were transfected with different expression plasmids together with 0.1 μg of the indicated reporter plasmid and 100 ng of pRenilla-null (Promega Corp.) per well in 6-well plates. In all cases, the total amount of plasmid DNA was adjusted with pcDNAIII-β-galactosidase or pCEFL-GFP. When indicated, cells were starved and treated for 24 h with 100 μm tin protoporphyrin IX (SnPP) (Frontier Scientific Europe Ltd.) dissolved in 0.1 n NaOH in PBS, pH 7.5. Firefly and Renilla luciferase activities present in cellular lysates were assayed using the Dual Luciferase reporter system (Promega), and light emission was quantified using a BG1 Optocomp I, GEM Biomedical luminometer (Sparks, NV). Western Blot—Cells were lysed, and extracted proteins were resolved in 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Endogenous HO-1 and transfected HA-HO-1 were detected by specific rabbit polyclonal anti-HO-1 (Stressgen Bioreagents) and mouse monoclonal anti-HA antibodies (Covance). RhoA and AU5-RhoA were detected by anti-RhoA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-AU5 (Covance) antibodies, respectively. Gα12 and Gα13 were detected using Gα12 SC-20 and Gα13 A-13 antibodies from Santa Cruz Biotechnology. As a loading control, α-actin was detected using a rabbit polyclonal antibody (Sigma). Membranes were stripped with Restore Western blot stripping buffer (Pierce), following the manufacturer's indications. Proteins were visualized by enhanced chemiluminescence detection (Amersham Biosciences) using goat anti-mouse and anti-rabbit IgGs coupled to horseradish peroxidase as the secondary antibody (Amersham Biosciences). Focus-forming Assays—NIH-3T3 cells were transfected by the calcium phosphate precipitation technique with 1 μg of each indicated expression plasmid as previously described (40Marinissen M.J. Servitja J.M. Offermanns S. Simon M.I. Gutkind J.S. J. Biol. Chem. 2003; 278: 46814-46825Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The day after transfection, cells were washed three times with DMEM and kept in DMEM supplemented with 5% calf serum alone or with 100 μm SnPP for 2-3 weeks until foci were scored. Alternatively, 5 × 104 NIH-3T3c, NIH-vGPCR, NIH-RhoAQL, NIH-vGPCRshRho2, or NIH-RhoAQLshHO-1 cells were seeded on a 70% confluent monolayer of NIH-3T3 cells and cultured as above until foci were detected. Cells were fixed with methanol for 30 min, washed with water, dried, and stained with Giemsa (Sigma). Rho Activation Assay—Rho activity in cultured cells was assessed by a modified Rho binding domain assay as already described (43Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Crossref PubMed Scopus (212) Google Scholar). Briefly, NIH-3T3 cells were transfected with the indicated plasmids, and after serum starvation for 24 h, cells were lysed at 4 °C in a buffer containing 20 mm HEPES, pH 7.4, 0.1 m NaCl, 1% Triton X-100, 10 mm EGTA, 40 mm glycero-phosphate, 20 mm MgCl2, 1 mm Na3VO4, 1 mm dithiothreitol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. Lysates were incubated with glutathione S-transferase-rhotekin-Rho binding domain previously bound to glutathione-Sepharose beads and washed four times with lysis buffer. Associated GTP-bound forms of Rho were released with protein loading buffer and revealed by Western blot analysis using a monoclonal antibody against RhoA (Santa Cruz Biotechnology). Indirect Immunofluorescence—NIH-3T3 cells were seeded on glass coverslips and transfected with Lipofectamine Plus reagents (Invitrogen). Cells were serum-starved for 24 h, washed twice with 1× PBS, and then fixed and permeabilized with 4% formaldehyde and 0.05% Triton X-100 in 1× PBS for 10 min. After washing with PBS, cells were blocked with 1% bovine serum albumin and incubated with anti HO-1 (Stressgen Bioreagents) or anti-AU5 antibodies (Covance) as primary antibodies for 1 h. Following incubation, cells were washed three times with 1× PBS and then incubated for an additional 1 h with the corresponding secondary antibodies (1:200) conjugated with tetramethylrhodamine B isothiocyanate and fluorescein isothiocyanate (Molecular Probes). When indicated, cells were incubated with AlexaFluor 594 phalloidin (1:40) for 30 s (Invitrogen) to stain actin filaments. Cells were washed three times with 1× PBS and stained with DAPI (1 μg/ml) (Molecular Probes) in the last wash. Coverslips were mounted in Fluorosafe mounting medium (Calbiochem) and viewed using a Nikon Eclipse TE2000-S photomicroscope equipped with epifluorescence. Immunohistochemistry—Tumor tissues were removed and fixed in 4% paraformaldehyde in 1× PBS, transferred to 70% ethanol, and embedded in paraffin. Sections were deparaf-finized in SafeClear and hydrated through graded alcohols and distilled water. Antigens were retrieved by heat in 10 mm citrate buffer, and sections were incubated with the anti-HO-1 (Stressgen Bioreagents) and the anti-VEGF (Santa Cruz Biotechnology) antibodies (1:500). Slides were incubated with biotinylated anti-mouse antibody for 1 h and then
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