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• W2012035062 abstract "The stress-inducible cytoprotective enzyme heme oxygenase-1 (HO-1) may play a critical role in the growth and metastasis of tumors. We demonstrated that overexpressed HO-1 promotes the survival of renal cancer cells by inhibiting cellular apoptosis; we also showed that the proto-oncogene H-Ras becomes activated in these cells under stress following treatment with immunosuppressive agents. However, it is not known if there is an association between Ras activation and HO-1 overexpression. Here, we examined if the activation of H-Ras pathway could induce HO-1, and promote the survival of renal cancer cells (786-0 and Caki-1). In co-transfection assays, using HO-1 promoter-luciferase construct, we found that the activated H-Ras, H-Ras(12V), promoted HO-1 transcriptional activation. The inhibition of endogenous H-Ras by specific dominant-negative mutant/siRNA markedly ablated the HO-1 promoter activity. Active H-Ras increased HO-1 mRNA and protein expression. Moreover, transfection with effector domain mutant constructs of active H-Ras showed that H-Ras-induced HO-1 overexpression was primarily mediated through the Raf signaling pathway. Using pharmacological inhibitor, we observed that ERK is a critical intermediary molecule for Ras-Raf-induced HO-1 expression. Activation of H-Ras and ERK promoted nuclear translocation of the transcription factor Nrf2 for its binding to the specific sequence of HO-1 promoter. The knockdown of Nrf2 significantly inhibited H-Ras-induced HO-1 transcription. Finally, by FACS analysis using Annexin-V staining, we demonstrated that the H-Ras-ERK-induced and HO-1-mediated pathway could protect renal cancer cells from apoptosis. Thus, targeting the Ras-Raf-ERK pathway for HO-1 overexpression may serve as novel therapeutics for the treatment of renal cancer. The stress-inducible cytoprotective enzyme heme oxygenase-1 (HO-1) may play a critical role in the growth and metastasis of tumors. We demonstrated that overexpressed HO-1 promotes the survival of renal cancer cells by inhibiting cellular apoptosis; we also showed that the proto-oncogene H-Ras becomes activated in these cells under stress following treatment with immunosuppressive agents. However, it is not known if there is an association between Ras activation and HO-1 overexpression. Here, we examined if the activation of H-Ras pathway could induce HO-1, and promote the survival of renal cancer cells (786-0 and Caki-1). In co-transfection assays, using HO-1 promoter-luciferase construct, we found that the activated H-Ras, H-Ras(12V), promoted HO-1 transcriptional activation. The inhibition of endogenous H-Ras by specific dominant-negative mutant/siRNA markedly ablated the HO-1 promoter activity. Active H-Ras increased HO-1 mRNA and protein expression. Moreover, transfection with effector domain mutant constructs of active H-Ras showed that H-Ras-induced HO-1 overexpression was primarily mediated through the Raf signaling pathway. Using pharmacological inhibitor, we observed that ERK is a critical intermediary molecule for Ras-Raf-induced HO-1 expression. Activation of H-Ras and ERK promoted nuclear translocation of the transcription factor Nrf2 for its binding to the specific sequence of HO-1 promoter. The knockdown of Nrf2 significantly inhibited H-Ras-induced HO-1 transcription. Finally, by FACS analysis using Annexin-V staining, we demonstrated that the H-Ras-ERK-induced and HO-1-mediated pathway could protect renal cancer cells from apoptosis. Thus, targeting the Ras-Raf-ERK pathway for HO-1 overexpression may serve as novel therapeutics for the treatment of renal cancer. Heme oxygenase-1 (HO-1), 3The abbreviations used are: HO-1heme oxygenase-1AREanti-oxidant response elementCoPPcobalt protoporphyrin. a member of the heat shock protein family, plays a key role as a sensor and regulator of oxidative stress (1Agarwal A. Nick H.S. J. Am Soc. Nephrol. 2000; 11: 965-973Crossref PubMed Google Scholar, 2Nath K.A. Kidney Int. 2006; 70: 432-443Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). It catalyzes the degradation of heme to form biliverdin, carbon monoxide (CO), and free iron (3Otterbein L.E. Soares M.P. Yamashita K. Bach F.H. Trends Immunol. 2003; 24: 449-455Abstract Full Text Full Text PDF PubMed Scopus (1015) Google Scholar). It plays an important protective role in the tissues by reducing oxidative injury, attenuating inflammatory response, inhibiting apoptosis, and also by regulating angiogenesis and cell proliferation (1Agarwal A. Nick H.S. J. Am Soc. Nephrol. 2000; 11: 965-973Crossref PubMed Google Scholar, 4Akagi R. Takahashi T. Sassa S. Contrib. Nephrol. 2005; 148: 70-85Crossref PubMed Scopus (47) Google Scholar, 5Wagener F.A. Eggert A. Boerman O.C. Oyen W.J. Verhofstad A. Abraham N.G. Adema G. van Kooyk Y. de Witte T. Figdor C.G. Blood. 2001; 98: 1802-1811Crossref PubMed Scopus (354) Google Scholar). Although it is a cytoprotective enzyme, a growing body of evidence clearly suggests that HO-1 may also play a significant role in the induction of tumorigenic pathways (6Jozkowicz A. Was H. Dulak J. Antioxid. Redox. Signal. 2007; 9: 2099-2117Crossref PubMed Scopus (328) Google Scholar, 7Sass G. Leukel P. Schmitz V. Raskopf E. Ocker M. Neureiter D. Meissnitzer M. Tasika E. Tannapfel A. Tiegs G. Int. J. Cancer. 2008; 123: 1269-1277Crossref PubMed Scopus (84) Google Scholar, 8Goodman A.I. Choudhury M. da Silva J.L. Schwartzman M.L. Abraham N.G. Proc. Soc. Exp. Biol. Med. 1997; 214: 54-61Crossref PubMed Scopus (151) Google Scholar, 9Miyake M. Fujimoto K. Anai S. Ohnishi S. Kuwada M. Nakai Y. Inoue T. Matsumura Y. Tomioka A. Ikeda T. Tanaka N. Hirao Y. Oncol. Rep. 2011; 25: 653-660Crossref PubMed Scopus (61) Google Scholar). HO-1 is often highly up-regulated in tumor tissues, and its expression is further increased in response to therapies. The overexpressed HO-1 can inhibit apoptosis of tumor cells and promote tumor growth and metastasis (6Jozkowicz A. Was H. Dulak J. Antioxid. Redox. Signal. 2007; 9: 2099-2117Crossref PubMed Scopus (328) Google Scholar, 10Was H. Dulak J. Jozkowicz A. Curr. Drug Targets. 2010; 11: 1551-1570Crossref PubMed Scopus (219) Google Scholar). It has been suggested that the inhibition of HO-1 expression can be a potential therapeutic approach to sensitize tumors to radiation and chemotherapy (11Fang J. Seki T. Maeda H. Adv. Drug. Deliv. Rev. 2009; 61: 290-302Crossref PubMed Scopus (431) Google Scholar, 12Fang J. Akaike T. Maeda H. Apoptosis. 2004; 9: 27-35Crossref PubMed Scopus (178) Google Scholar, 13Alaoui-Jamali M.A. Bismar T.A. Gupta A. Szarek W.A. Su J. Song W. Xu Y. Xu B. Liu G. Vlahakis J.Z. Roman G. Jiao J. Schipper H.M. Cancer Res. 2009; 69: 8017-8024Crossref PubMed Scopus (103) Google Scholar, 14Rushworth S.A. Bowles K.M. Raninga P. MacEwan D.J. Cancer Res. 2010; 70: 2973-2983Crossref PubMed Scopus (55) Google Scholar). heme oxygenase-1 anti-oxidant response element cobalt protoporphyrin. The mechanisms underlying HO-1 induction are complex, and tightly regulated primarily at the transcriptional level (15Lavrovsky Y. Schwartzman M.L. Levere R.D. Kappas A. Abraham N.G. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 5987-5991Crossref PubMed Scopus (355) Google Scholar, 16Ryter S.W. Choi A.M. Antioxid. Redox Signal. 2002; 4: 625-632Crossref PubMed Scopus (167) Google Scholar). The HO-1 gene has two important distal enhancer regions, E1 and E2, located upstream of the transcription start site (16Ryter S.W. Choi A.M. Antioxid. Redox Signal. 2002; 4: 625-632Crossref PubMed Scopus (167) Google Scholar, 17Srisook K. Kim C. Cha Y.N. Antioxid. Redox Signal. 2005; 7: 1674-1687Crossref PubMed Scopus (101) Google Scholar). The dominant element in the E1 and E2 regions is the anti-oxidant response element (ARE)/stress-responsive element (StRE), which mediates transcriptional activation through the binding of transcription factors in response to most of the HO-1 inducers (18Kivelä A.M. Kansanen E. Jyrkkänen H.K. Nurmi T. Ylä-Herttuala S. Levonen A.L. J. Nutr. 2008; 138: 1263-1268Crossref PubMed Scopus (24) Google Scholar). Several transcription factors, such as nuclear factor-E2-related factor 2 (Nrf2), cAMP responsive element-binding protein-1, activator protein-1, Maf, and nuclear factor-κB play significant roles in the activation of the HO-1 promoter (19Hock T.D. Liby K. Wright M.M. McConnell S. Schorpp-Kistner M. Ryan T.M. Agarwal A. J. Biol. Chem. 2007; 282: 6875-6886Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 20Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 21Ferrándiz M.L. Devesa I. Curr. Pharm. Des. 2008; 14: 473-486Crossref PubMed Scopus (152) Google Scholar). Complex networks of intracellular signaling events are involved in the activation of transcription factors to induce HO-1 overexpression (21Ferrándiz M.L. Devesa I. Curr. Pharm. Des. 2008; 14: 473-486Crossref PubMed Scopus (152) Google Scholar, 22Rojo 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 (54) Google Scholar). However, the intricate signaling mechanism(s) for the induction of HO-1 transcription, particularly in cancer cells, is not well defined. The ras family of proto-oncogenes encodes small proteins that transduce mitogenic signals from tyrosine kinase receptors (23Schlessinger J. Trends Biochem. Sci. 1993; 18: 273-275Abstract Full Text PDF PubMed Scopus (343) Google Scholar, 24Downward J. Nat. Rev. Cancer. 2003; 3: 11-22Crossref PubMed Scopus (2523) Google Scholar). Ras proteins act as molecular switches that cycle between active GTP-bound and inactive GDP-bound forms (25Settleman J. Albright C.F. Foster L.C. Weinberg R.A. Nature. 1992; 359: 153-154Crossref PubMed Scopus (252) Google Scholar, 26Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1759) Google Scholar, 27Khosravi-Far R. Chrzanowska-Wodnicka M. Solski P.A. Eva A. Burridge K. Der C.J. Mol. Cell. Biol. 1994; 14: 6848-6857Crossref PubMed Scopus (128) Google Scholar). The three isoforms of Ras, H-Ras, K-Ras, and N-Ras, are ubiquitously expressed in mammalian cells (28Omerovic J. Hammond D.E. Clague M.J. Prior I.A. Oncogene. 2008; 27: 2754-2762Crossref PubMed Scopus (76) Google Scholar). Hyperactive Ras can promote the growth and development of cancer cells even without being mutated, where it may be activated by persistent upstream signaling events (29Datta D. Flaxenburg J.A. Laxmanan S. Geehan C. Grimm M. Waaga-Gasser A.M. Briscoe D.M. Pal S. Cancer Res. 2006; 66: 9509-9518Crossref PubMed Scopus (131) Google Scholar, 30Mo L. Zheng X. Huang H.Y. Shapiro E. Lepor H. Cordon-Cardo C. Sun T.T. Wu X.R. J. Clin. Invest. 2007; 117: 314-325Crossref PubMed Scopus (97) Google Scholar, 31Eckert L.B. Repasky G.A. Ulkü A.S. McFall A. Zhou H. Sartor C.I. Der C.J. Cancer Res. 2004; 64: 4585-4592Crossref PubMed Scopus (177) Google Scholar). Upon activation, Ras transmits signals to a cascade of protein kinases that have MAP kinase kinase (MEK) as substrate, such as MEK kinase, c-Raf-1, and B-Raf, culminating in the activation of MAP kinase (MAPK) (32Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (766) Google Scholar). It has been suggested that Ras may function primarily to promote the translocation of Raf-1 from the cytosol to the plasma membrane, where subsequent Ras-independent events trigger Raf-1 kinase activation (33Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (885) Google Scholar). However, despite the evidence that Raf-1 is a critical downstream effector of Ras function, there is increasing evidence that Ras may also mediate its action through Raf-independent pathways, including Rho- and phosphatidylinositol 3-kinase (PI3K) pathways (34Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar, 35Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1726) Google Scholar, 36Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (813) Google Scholar). We have recently demonstrated that H-Ras becomes activated in human renal cancer cells under stress following treatment with immunosuppressive agents, and the activated H-Ras induces tumorigenic pathways (37Datta D. Contreras A.G. Basu A. Dormond O. Flynn E. Briscoe D.M. Pal S. Cancer Res. 2009; 69: 8902-8909Crossref PubMed Scopus (51) Google Scholar). We have also observed that the expression of HO-1 is significantly up-regulated in renal cancer tissues, and the overexpressed HO-1 can inhibit tumor cell apoptosis (38Datta D. Banerjee P. Gasser M. Waaga-Gasser A.M. Pal S. J. Biol. Chem. 2010; 285: 36842-36848Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In the present study, we show that activated H-Ras promotes the transcriptional activation of HO-1 in human renal cancer cells; and H-Ras-induced HO-1 overexpression is mediated primarily through the Raf-MAPK signaling pathway involving the transcription factor Nrf2, which leads to the survival of renal cancer cells. Cobalt protoporphyrin (CoPP) was obtained from Frontier Scientific. The gene-specific small interfering RNA (siRNA) for H-Ras, Raf-1, Nrf2, HO-1, and their respective controls were purchased from Qiagen. The transfection of siRNA was performed using Lipofectamine 2000 (Invitrogen). The MEK inhibitor PD98059 and the Raf-1 kinase inhibitor I {RKI; 5-iodo-3-[(3,5-dibromo-4-hydroxy-phenyl)methylene]-2-indolinone} were purchased from Calbiochem. Recombinant human platelet-derived growth factor (PDGF) was purchased from BioLegend. The human renal cancer cell lines (786–0 and Caki-1) were obtained from American Type Culture Collection. 786-0 cells were grown in RPMI 1640, and Caki-1 cells were grown in McCoy's medium supplemented with 10% fetal bovine serum (Gibco). Human renal proximal tubular epithelial cells (RPTEC) were purchased from Clonetics and cultured in complete epithelial medium (REGM BulletKit). Tissue samples of human renal cell cancer (RCC) were obtained from surgical specimens of patients who underwent surgery at the University Hospital (Wurzburg, Germany). The protocol to obtain tissue samples was approved by the review board of the hospital. Normal renal tissues were obtained from normal parts of the surgical specimens, and the normalcy of these tissues was confirmed by histology. A human HO-1 promoter-luciferase construct was obtained as a gift from J. Alam of Alton Ochsner Medical Foundation, New Orleans, LA (20Alam J. Wicks C. Stewart D. Gong P. Touchard C. Otterbein S. Choi A.M. Burow M.E. Tou J. J. Biol. Chem. 2000; 275: 27694-27702Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). The plasmid phHO4luc was constructed by cloning the promoter fragment from the human HO-1 gene (bp −4067 to +70 relative to transcription start site) into the luciferase reporter gene vector pSKluc. All Ras expression constructs encode mutant versions of the transforming human H-Ras(12V), and were obtained as generous gifts from Roya Khosravi-Far (Beth Israel Deaconess Medical Center, Boston, MA). The pDCR-ras(12V), pDCR-ras(12V, 35S), pDCR-ras(12V,37G), and pDCR-ras(12V,40C) mammalian constructs encode effector domain mutants of H-Ras(12V) in which expression is under the control of the cytomegalovirus promoter (34Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar). The Ras(17N) and RhoA(19N) dominant-negative plasmids inhibit the function of endogenous Ras (27Khosravi-Far R. Chrzanowska-Wodnicka M. Solski P.A. Eva A. Burridge K. Der C.J. Mol. Cell. Biol. 1994; 14: 6848-6857Crossref PubMed Scopus (128) Google Scholar) and RhoA (34Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar), respectively. Total RNA was prepared using the RNeasy isolation kit (Qiagen), and cDNA was synthesized using cloned AMV first-strand synthesis kit (Invitrogen). To analyze mRNA expression, we performed real-time PCR using the Assay-on-Demand Gene Expression product (TaqMan, Mammalian Gene Collection probes) according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was amplified and analyzed under identical conditions. Gene-specific primer-probe sets for human HO-1/GAPDH were obtained from Applied Biosystems. Ct value (the cycle number at which emitted fluorescence exceeded an automatically determined threshold) for gene of interest was corrected by the Ct value for GAPDH and expressed as ΔCt. The fold change of mRNA amount was calculated as follows: (fold changes) = 2X (where X = ΔCt for control group - ΔCt for experimental group). 786-0 or Caki-1 (2.5 × 105 cells) were transfected with the Ras expression plasmids or the HO-1 promoter-luciferase plasmid using Effectene Transfection Reagent (Qiagen), according to the manufacturer's protocol. The total amount of transfected plasmid DNA was normalized using a control empty expression vector. For luciferase assay, cells were harvested 48 h after transfection, and luciferase activity was measured using a standard assay kit (Promega) in a luminometer. As an internal control for transfection efficiency, the cells were co-transfected with β-galactosidase gene under control of cytomegalovirus immediate early promoter, and β-galactosidase activity was measured using standard assay system (Promega); luciferase activity measurement was corrected for the transfection efficiency by calculating the ratio of luciferase units to β-galactosidase units, represented as the relative luciferase counts. Protein samples were run on SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore Corporation). The membranes were incubated with the following primary antibodies: anti-Ras (BD Transduction Laboratories); anti-HO-1 (R & D systems); anti-H-Ras, anti-Raf-1, anti-Lamin A, and anti-Nrf2 (Santa Cruz Biotechnology); anti-β-actin and anti-GAPDH (Sigma-Aldrich). The membranes were subsequently incubated with peroxidase-linked secondary antibody, and the reactive bands were detected by using chemiluminescent substrate (Pierce). Immunohistochemistry was performed on frozen sections of human renal cell cancer (RCC) tissues and normal renal tissues. Briefly, acetone-fixed sections were incubated first with anti-human Nrf2 (Abcam), and second with a species-specific horseradish peroxidase-conjugated secondary antibody. Specimens were washed thoroughly in between incubations, developed in 3,3′-diaminobenzidine (BioGenex), and counterstained in Meyer's Hemalaun using standard techniques. Cytoplasmic and nuclear extracts were prepared using a Nuclear Extract kit (Active Motif). The purities of cytoplasmic and nuclear fractions were confirmed by checking the expression of a cytoplasmic protein GAPDH and a nuclear protein Lamin A. The binding of the activated transcription factor Nrf2 to the specific DNA sequence was measured by using TransAM Nrf2 ELISA kit (Active Motif). Briefly, the kit contains stripwell plate to which multiple copies of specific double-stranded oligonucleotide for Nrf2 consensus-binding site (ARE) has been immobilized. When nuclear extract is added to each well, the activated Nrf2 binds specifically to this plate-bound oligonucleotide. Nrf2-specific primary antibody is then added followed by subsequent incubation with secondary antibody and developing solution. Quantitative analysis is performed by spectrophotometry at 450 nm. Cellular apoptosis was measured by Annexin-V and propidium iodide (PI) staining using Annexin-V FITC/APC Apoptosis Detection Kit (eBioscience) according to the manufacturer's protocol. Following staining, the cells were analyzed by flow cytometry on a FACSCalibur. Statistical evaluation for data analysis was determined by Student's t test. Differences with p < 0.05 were considered statistically significant. We have recently demonstrated that the activation of H-Ras plays a crucial role in the accelerated growth of human renal tumors under stress following treatment with immunosuppressive agents (37Datta D. Contreras A.G. Basu A. Dormond O. Flynn E. Briscoe D.M. Pal S. Cancer Res. 2009; 69: 8902-8909Crossref PubMed Scopus (51) Google Scholar); we have also observed that HO-1 is markedly overexpressed in renal cancer tissues, and the overexpressed HO-1 can mediate anti-apoptotic signals in renal cancer cells (38Datta D. Banerjee P. Gasser M. Waaga-Gasser A.M. Pal S. J. Biol. Chem. 2010; 285: 36842-36848Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Thus, both H-Ras and HO-1 may induce pro-tumorigenic pathways in human renal cancer cells to promote rapid growth of the tumors. However, we did not examine if there is any association between H-Ras activation and HO-1 overexpression in human renal cancer. Here, we first wished to evaluate if the activation of H-Ras can regulate HO-1 promoter activity in human renal cancer cell lines (Caki-1 and 786–0). Both the cells express H-Ras, and its expression is higher in cancer cells compared with normal renal epithelial cells (supplemental Fig. S1). The cells were co-transfected with the HO-1 promoter-luciferase construct and either the plasmid expressing activated form of H-Ras, H-Ras(12V), or the empty expression vector (control). The effect of H-Ras on HO-1 promoter activation was assessed by the measurement of luciferase activity in cell lysates. As shown in Fig. 1, A and B, the activation of H-Ras significantly increased HO-1 promoter activity in both the cell lines compared with vector controls. The expression of H-Ras(12V) plasmid in the transfected cells was confirmed by Western blot analysis (Fig. 1, A and B, bottom panels). Next, to check whether the inhibition of endogenous H-Ras can block HO-1 transcription, we first used a dominant-negative plasmid construct of Ras, Ras(17N). 786-0 cells were co-transfected with the HO-1 promoter-luciferase construct and either Ras(17N) or the empty expression vector. We observed that transfection with Ras(17N) significantly inhibited HO-1 promoter activity compared with vector controls (Fig. 1C). The expression of Ras(17N) plasmid in the transfected cells was confirmed by Western blot analysis (Fig. 1C, bottom panel). We also found that the knockdown of endogenous H-Ras in 786-0 cells by siRNA transfection significantly down-regulated HO-1 promoter activity compared with control siRNA-transfected cells (Fig. 1D). The knockdown of H-Ras was confirmed by Western blot analysis (Fig. 1D, bottom panel). It has been reported that the growth factor PDGF can induce Ras pathway through the tyrosine kinase receptor PDGFR (39Ekman S. Thuresson E.R. Heldin C.H. Rönnstrand L. Oncogene. 1999; 18: 2481-2488Crossref PubMed Scopus (53) Google Scholar), known to be expressed in renal cancer cells (40Xu L. Tong R. Cochran D.M. Jain R.K. Cancer Res. 2005; 65: 5711-5719Crossref PubMed Scopus (115) Google Scholar). Thus, we also tested if PDGF treatment can promote HO-1 transcriptional activation in renal cancer cells. We observed that the treatment with PDGF indeed increased HO-1 promoter activity in Caki-1 cells (supplemental Fig. S2A). Together, these observations suggest that activation of H-Ras promotes the transcriptional activation of HO-1 in human renal cancer cells. Here, we examined if the activation of H-Ras could increase mRNA and protein expression of HO-1 in 786-0 and Caki-1 cells. The cells were transfected with either H-Ras(12V) or the empty expression vector. Through real-time PCR, we found that in both the cell lines, transfection of active H-Ras significantly increased the expression of HO-1 mRNA compared with vector controls (Fig. 2, A and B). We next checked the expression level of HO-1 protein following H-Ras activation. Through Western blot analysis, we observed that active H-Ras markedly induced HO-1 protein expression compared with vector controls (Fig. 2, C and D); however, there was no significant change (data not shown) in the expression of HO-2, which is the non-inducible and constitutive isoform of the enzyme. Thus, the activation of H-Ras in renal cancer cells promotes HO-1 overexpression at both mRNA and protein levels. It is established that the activation of Ras can trigger several associated signaling molecules, including Raf, Rho, and PI3K (34Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar, 35Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1726) Google Scholar, 36Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (813) Google Scholar). Here, we wanted to evaluate which effector molecule(s) is involved in channeling H-Ras-induced signals for HO-1 transcriptional activation. To this end, we made use of three effector loop mutant constructs of H-Ras; H-Ras(12V,35S) retains full-length Raf-1 binding activity, H-Ras(12V,37G) retains Rho binding activity, and H-Ras(12V,40C) retains PI3K binding activity (34Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar). 786-0 and Caki-1 cells were transfected with the HO-1 promoter-luciferase construct, and either one of the H-Ras effector domain mutants or the empty expression vector; and luciferase assay was performed. As shown in Fig. 3, A and B, we observed that in both the cell lines, there was significant increase in HO-1 promoter activity following transfection with H-Ras(12V,35S) compared with vector controls. The mutant H-Ras(12V,37G) also induced HO-1 promoter activity, however, the effect was less compared with H-Ras(12V,35S). In contrast, the mutant H-Ras(12V,40C) failed to induce HO-1 promoter activity in both the cell lines. The protein expression of the H-Ras effector loop mutant constructs in the transfected cells was confirmed by Western blot analysis (Fig. 3, A and B, right panels). Also, we confirmed that only H-Ras(12V,35S) can induce the phosphorylation of ERK (p44), while H-Ras(12V,40C) can induce the phosphorylation of Akt (Fig. 3A, right panels). Together, these observations suggest that the Raf signaling pathway is primarily involved in channeling H-Ras-induced signals for HO-1 transcriptional activation in renal cancer cells; also, the PI3K pathway may not be involved in H-Ras-induced activation of the HO-1 promoter. Our previous experiment suggested a role of the Raf signaling pathway in H-Ras-induced HO-1 transcription. To confirm the role of Raf-1 kinase in this process, we co-transfected our cells (786-0 and Caki-1) with the HO-1 promoter-luciferase construct and the H-Ras(12V) plasmid in absence or presence of Raf-1 kinase inhibitor (RKI); and luciferase assay was performed. As shown in Fig. 4, A and B, the activation of H-Ras significantly increased HO-1 promoter activity compared with empty vector controls; and the inhibition of Raf-1 with pharmacological inhibitor markedly decreased H-Ras-induced HO-1 transcriptional activation compared with vehicle-treated controls. We also confirmed that the knockdown of Raf-1 in 786-0 cells using siRNA significantly down-regulated H-Ras-induced HO-1 transcriptional activation (Fig. 4C). In addition, the knockdown of Raf-1 was associated with a significant decrease in HO-1 protein expression as observed by Western blot analysis (Fig. 4C, right panel). We also found that the knockdown of Raf-1 inhibited PDGF-induced (known to activate the Ras pathway) HO-1 transcriptional activation (supplemental Fig. S2B). Our earlier experiments suggested that although the Raf signaling pathway is primarily involved in channeling H-Ras-induced signals for HO-1 transcription, the Rho pathway may also play some role. Here, we used a dominant-negative plasmid of RhoA [RhoA(19N)] to check if it can downregulate H-Ras-induced HO-1 transcriptional activation. We observed that with the transfection of RhoA(19N), there was a partial, but not significant inhibition of H-Ras-induced HO-1 promoter activity (supplemental Fig. S3). It is established that the extracellular signal-regulated kinase (ERK) is a critical downstream effector molecule of the Ras-Raf signaling cascade (32Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (766) Google Scholar, 34Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar). Thus, we next examined if ERK plays a role in H-Ras-induced HO-1 transcription. 786–0 cells were co-transfected with the HO-1 promoter-luciferase construct and H-Ras(12V) in the absence or presence of MEK inhibitor PD98059 (known to inhibit ERK), and luciferase assay was performed. We found that in presence of PD98059, there was a significant decrease in H-Ras-induced HO-1 promoter activity compared with vehicle-treated controls (Fig. 4D). Together, our findings suggest that Raf and ERK are critical intermediary signaling molecules i" @default.
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