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Hypoxia-specific upregulation of the endogenous human VEGF-A gene by hypoxia-driven expression of artificial transcription factor
Biochemical and Biophysical Research Communications 390 (2009) 845–848
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Hypoxia-specific upregulation of the endogenous human VEGF-A gene by hypoxia-driven expression of artificial transcription factor Tomoaki Mori, Jun Sasaki, Takuya Kanamori, Yasuhiro Aoyama, Takashi Sera * Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
a r t i c l e
i n f o
Article history: Received 24 September 2009 Available online 20 October 2009 Keywords: Artificial transcription factor Hypoxia Hypoxia-response element Ischemic diseases Vascular endothelial growth factor A Zinc-finger protein
a b s t r a c t Activation of vascular endothelial growth factor A (VEGF-A) is an attractive approach to treatment of ischemic diseases. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF-A and constitutive expression of ATFs upregulated the endogenous VEGF-A gene expression, activation of VEGF-A specifically in ischemic tissues is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-specific activation of human VEGF-A gene by hypoxia-driven expression of the ATF. We constructed a hypoxia-driven promoter for the ATF expression and placed it upstream of the ATF-encoding regions. The resulting hypoxia-driven expression plasmid induced the ATF expression in hypoxia but not in normoxia, and the hypoxia-specific expression of the ATF activated the VEGF-A expression specifically in hypoxia. Thus, the engineered expression system of ATFs may enable activation of VEGF-A expression specifically in ischemic tissues without affecting normal, healthy tissues in vivo. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Vascular endothelial growth factor A (VEGF-A) is one of the key gene products in neovascularization. Therefore, activation of the endogenous VEGF-A gene or overexpression of the exogenous transgene may ameliorate ischemic diseases including diabetic angiopathy and arteriosclerosis obliterans [1]. For example, delivery of the cDNA expression plasmid of VEGF-A165 that is one of the major splice variants may improve ischemic diseases [2,3]. Alternatively, methods to activate the endogenous human VEGFA gene using zinc-finger-based artificial transcription factors (ATFs) have been explored [4–7]. One of the advantages is that ATFs are able to upregulate each major splice variant of VEGF-A proportionally [4,5] because ATFs activate the transcription of the endogenous VEGF-A gene. This feature is important because proper isoform balance is crucial for VEGF-A function in vivo [8,9]. Actually, ATFs that were constitutively expressed from plasmids or viral vectors successfully upregulated the endogenous VEGF-A gene in vitro [4,6] and in vivo [5,10–12]. However, in clinical application, it is desirable to express ATFs from transfected expression plasmids or viral vectors specifically in hypoxic ischemic tissues without affecting healthy, normal (normoxic) tissues. To our best knowledge, such a system using ATFs has not yet been developed. In this study, we explored the possibility of hypoxia-specific activation of the human endogenous VEGF-A gene by expressing * Corresponding author. Fax: +81 75 383 2767. E-mail address: [email protected] (T. Sera). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.060
ATFs specifically in hypoxia. Namely, we constructed an artificial hypoxia-driven promoter in which six tandem copies of a hypoxia-response element (HRE) [13] were placed upstream of a constitutive cytomegalovirus (CMV) minimal promoter [14] to yield hypoxia-driven expression plasmids of ATFs. We examined the hypoxia-specific expression of ATFs using the engineered ATF-expression plasmids, and then examined whether the hypoxia-driven expression of the ATFs led to hypoxia-specific activation of VEGFA gene in mammalian cells. Materials and methods Plasmid constructions. The constitutive expression plasmid, pCMV-AZP–VP16, encoding a nuclear localization signal (NLS) from the simian virus 40 large T antigen, a six-finger artificial zinc-finger protein (AZP), a herpes simplex virus VP-16 activation domain (residues 415–490; [15]) and a FLAG epitope tag, was prepared as described previously [6]. The six-finger AZP was designed to recognize the 19-bp target of 50 -GGG GCT GGG GGC GGT GTC T-30 (+516 to +534 in the human VEGF-A gene, where +1 is the transcription start site) [16]. A DNA fragment encoding an AZP mutant in which all recognition amino acids were mutated to alanine [17] was cloned into the BamHI/EcoRI sites of pCMV-AZP–VP16 to generate pCMV-AZPAla–VP16. The hypoxia-driven promoter generated for this study harbored six tandem copies of the HRE (50 -CCACAGTGCATACGTGGGCTCCAACAGGTCCTCTT-30 ; [13]) with arbitrary 5-bp spacers upstream of a CMV minimal promoter (53 to +7 in the CMV promoter, where +1
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is the transcription start site; [14]). The DNA fragment constructed from synthesized DNA oligomers was cloned into the MluI/NheI sites of pCMV-AZP–VP16 to generate pHRE6-AZP–VP16. Assay for endogenous human VEGF-A protein production by transient transfection of plasmids expressing ATF derivatives. An expression plasmid of the ATF derivative described above (0.45 lg) was cotransfected with a pCMV-b-galactosidase plasmid (Clontech) (0.05 lg) into 1 105 HEK293 cells (American Type Culture Collection) by electroporation using a MicroPorator MP-100 (Digital Bio Technology) according to the protocol accompanying the machine. The resulting cell suspension (10 ll) was plated onto a 96-well poly(D-lysine)-coated plate (BD Biosciences) with 90 ll of Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 0.1 mM nonessential amino acids (Invitrogen) and 10% FBS (Invitrogen). After incubation as described in the figure legends, the culture medium was collected, and the supernatant was cleared by centrifugation at 200g and 4 °C for 5 min and frozen at –80 °C until assayed. The VEGF-A protein concentration in the supernatant was assayed by using a human VEGF-A ELISA kit (R&D Systems) according to the manufacturer’s protocol. The remaining transfected cells were washed with PBS and then lysed with a passive lysis buffer (Promega). The protein concentration of the centrifuged supernatant was determined by using an NI Protein Assay kit (G-Bioscience) to normalize the VEGF-A produced [18]. This lysis sample was also analyzed by immunoblotting of ATF derivatives (see below). For determination of transfection efficiency, the transfected cells were stained with a b-gal staining kit (Invitrogen), and the b-galactosidase activities were measured by using a Luminescent b-Galactosidase Detection kit (BD Biosciences). These experiments were carried out in duplicate and repeated independently at least three times. Immunoblotting analysis of ATF derivatives. Cell lysates prepared as described above were also examined for expression of ATF derivatives. Equal amounts of the protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and probed with an anti-FLAG antibody conjugated with horseradish peroxidase (Sigma) or an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Ambion) by standard methods. ECL Plus (GE Healthcare) was used for chemiluminescence detection. The resulting chemiluminescent signals were recorded on X-ray film. The molecular weight of each ATF derivative is indicated in the figure legends. For determination of a relative amount of ATF produced by transfection of pHRE6-AZP–VP16, band intensities of ATF produced by transfection of pCMV-AZP–VP16 were digitized and quantitated by UN-SCAN-IT (Silk Scientific, Inc.), and a standard curve was then generated. Results and discussion First, we examined whether the ATF AZP–VP16 (Fig. 1A) activated the endogenous VEGF-A gene in hypoxia (1% O2). Although we demonstrated that the ATF activated the endogenous VEGF-A gene in HEK293 cells under the normoxic conditions [6], we have not yet examined the effectiveness under the hypoxic conditions. Cells cultured under our hypoxic conditions increased VEGF-A production by >5-fold compared with those cultured under normoxic conditions (data not shown), similarly to a previous study (6-fold activation of VEGF-A in HEK293 cells incubated in a 1% O2 atmosphere; [19]). At 24 h after transfection of a plasmid encoding the ATF downstream of a strong, constitutive CMV promoter (Fig. 1A), the ATF activated VEGF-A expression by 24 ± 9-fold compared with a control (i.e., transfection with pcDNA3.1) (Fig. 2A). In contrast, a control ATF, AZPAla–VP16, in which all recognition amino acids in the zinc-finger domains were mutated to alanine, did not increase the expression level (Fig. 2A). The control protein AZPAla was unable
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to bind to a target DNA even at a protein concentration of 1 lM in the presence of an excess amount of poly(dA–dT)2 competitor DNA in vitro [17]. This result demonstrates that the six-finger AZP in AZP–VP16 recognized the target 19-bp in the VEGF-A 50 -UTR precisely. Immunoblotting of ATF derivatives expressed transiently in the experiments revealed that AZP–VP16 was not expressed more than AZPAla–VP16 (Fig. 2B), indicating that VEGF-A gene activation by AZP–VP16 (Fig. 2A) was not due to much higher expression of AZP–VP16 than of AZPAla–VP16. Because we confirmed that the ATF constitutively expressed activated the endogenous VEGF-A gene efficiently in hypoxia, we next constructed the hypoxia-driven expression plasmid of the ATF. In actual clinical application, the ideal is ATF expression only in target ischemic tissues without affecting healthy, normal tissues. Thus, to accomplish specific expression of ATF in hypoxic tissues, we constructed a hypoxia-driven expression plasmid of the ATF (Fig. 1B). In the engineered promoter, six copies of an HRE [13] were placed upstream of the CMV minimal promoter (53 to +7 in the CMV promoter, where +1 is the transcription start site; [14]). The HRE is known to be a cis-element that is recognized by hypoxia-inducible factors (HIFs), and is responsible for hypoxiaspecific expression of various genes including VEGF-A [13]. We then examined whether the new expression plasmid for AZP–VP16, pHRE6-AZP–VP16 (Fig. 1B), was expressed in hypoxia but not in normoxia. As shown in Fig. 3A, lane 4, the ATF was expressed effectively in hypoxia (1% O2). As expected, we did not detect the significant expression of the same ATF in normoxia; the expression profile was the same as that of cells transfected with a pcDNA3.1 control plasmid, which does not contain an ATF open reading frame (compare lane 3 with lane 1 in Fig. 3A). Thus, the expression plasmid induced the AZP–VP16 expression specifically in hypoxia. From a standard curve (not shown) obtained from band intensities of ATF from pCMV-AZP–VP16, the ATF production from pHRE6-AZP–VP16 was determined to be 13% of that from pCMVAZP–VP16 (Fig. 3B). Finally, we examined whether the hypoxia-driven expression plasmid of AZP–VP16 activated the endogenous VEGF-A gene specifically in hypoxia. Transfection of the pHRE6-AZP–VP16 resulted in 22 ± 12-fold activation of the endogenous VEGF-A gene
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Fig. 2. Activation of endogenous VEGF-A gene by constitutive expression of ATF in hypoxia. (A) VEGF-A production in hypoxia. The plasmid expressing an ATF derivative under the control of a CMV promoter (or pcDNA3.1 as a control) (0.45 lg) and pCMV-b-galactosidase plasmid (0.05 lg) were cotransfected into 1 105 HEK293 cells by electroporation. After incubation at 37 °C for 24 h in 1% O2, the culture medium and the transfected cells were collected separately. The VEGF-A concentration and the b-galactosidase activities were quantified as described in Materials and methods. Each point represents means ± SD obtained from three independent experiments. The plasmid used for each cotransfection with pCMV-bgalactosidase is indicated below the figure. (B) Immunoblots of ATF derivatives in the cell lysates prepared for panel A. The molecular weights of AZP–VP16 and AZPAla–VP16 are 32.2 and 30.8 kDa, respectively. Lane 1, sample from cells transfected with pcDNA3.1 as a control; lane 2, sample from cells transfected with pCMV-AZP–VP16; lane 3, sample from cells transfected with pCMV-AZPAla–VP16.
compared with that of cells transfected with a pcDNA3.1 control plasmid (Fig. 3C). As shown in Fig. 3D, in normoxia, the ATF did not significantly activate VEGF-A expression (i.e., 1.1 ± 0.4-fold activation compared with a control). Systemic administration of constitutive ATF-expression plasmids for activation of endogenous VEGF-A expression may stimulate excess blood-vessel formation unnecessary for healthy normal tissues. Therefore, regulated systems to express ATFs are necessary for application of ATF technology to clinical treatment. One such approach is to regulate the expression of ATFs by using chemicals (i.e., chemical gene switches). For example, a chemical that noncovalently linked a zinc-finger protein and a transcriptional activation domain upregulated endogenous VEGF-A in a chemicaldependent manner [20]. Such a system also enables adjustment of the timing of ATF expression. However, administration of chemicals specifically to ischemic tissues seems to be difficult. Another approach is local administration or injection of cell-permeable ATF molecules [6] or expression plasmids of ATFs only to diseased tissues. A recent study demonstrated that intramuscular injection of an expression plasmid encoding fibroblast growth factor-1 (FGF-1) resulted in FGF-1 expression only in injected sites [21]. In the pres-
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T. Mori et al. / Biochemical and Biophysical Research Communications 390 (2009) 845–848
Fig. 3. Hypoxia-specific activation of endogenous VEGF-A gene by hypoxia-driven expression of ATF. (A) Hypoxia-specific expression of ATF by using pHRE6-AZP– VP16. The plasmid expressing the ATF under the control of six HRE enhancers (or pcDNA3.1 as a control) (0.45 lg) and pCMV-b-galactosidase plasmid (0.05 lg) were cotransfected into 1 105 HEK293 cells by electroporation. After incubation at 37 °C for 24 h in normoxia or in 1% O2, the culture medium and transfected cells were collected separately. The cell lysates were used for immunoblotting. Lane 1, sample from cells transfected with pcDNA3.1 and then incubated in normoxia; lane 2, sample from cells transfected with pcDNA3.1 and then incubated in hypoxia; lane 3, sample from cells transfected with pHRE6-AZP–VP16 and then incubated in normoxia; lane 4, sample from cells transfected with pHRE6-AZP–VP16 and then incubated in hypoxia. (B) Comparison of ATF production by the hypoxia-driven promoter with that by the constitutive CMV promoter. The relative amount of ATF produced by transfection of pHRE6-AZP–VP16 was determined from a standard curve (not shown) that was generated from band intensities of ATF produced by transfection of pCMV-AZP–VP16. Amounts of total proteins used for immunoblotting are indicated below the figure. (C) Activation of VEGF-A protein production by pHRE6-AZP–VP16 in hypoxia. The VEGF-A concentration of the culture medium prepared for panel A was quantified. Each point represents means ± SD obtained from three independent experiments. (D) Effect of transfection with pHRE6-AZP– VP16 on VEGF-A protein production in normoxia. The VEGF-A concentration of the culture medium prepared for panel A was quantified. Each point represents means ± SD obtained from three independent experiments.
ent study, we explored an alternative approach, the development of a system to express ATFs specifically in hypoxic ischemic tissues.
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To accomplish this, we constructed an artificial promoter that contained six tandem copies of an HRE upstream of a CMV minimal promoter and generated a hypoxia-driven ATF-expression plasmid by using the artificial promoter. As shown in Fig. 3A, the expression plasmid enabled expression of ATF specifically in hypoxia. Although significant leaky expression in normoxia was observed in other hypoxia-inducible systems using HREs [22], it was not observed in the present study. The hypoxia-driven expression system reduced ATF production to 13% of that by a strong, constitutive CMV promoter (Fig. 3B). However, using the new expression system, we activated the endogenous VEGF-A gene by >20-fold in hypoxia, similar to that of the CMV promoter (Fig. 3C). Furthermore, leaky activation of VEGF-A was not observed in normoxia (Fig. 3D), corresponding to the ATF expression profile (Fig. 3A). Thus, the hypoxia-driven ATF system for VEGF-A activation is potentially applicable to treatment of ischemic diseases without affecting healthy normal tissues. As the next step, we need to carefully examine whether the hypoxia-driven expression system will be effective for treatment of ischemic diseases in animal models without causing undesirable side effects. In conclusion, we have constructed the first hypoxia-driven expression system of ATFs, and demonstrated hypoxia-specific activation of endogenous human VEGF-A gene expression in cultured cells. We will evaluate our hypoxia-specific expression system in vivo as the next step. The 50 -UTR of mouse VEGF-A is similar to the human one, and the 19-bp target recognized by our ATFs is also located present in the mouse 50 -UTR [23]. Therefore, our ATFs can be used directly in in vivo experiments using mice. Acknowledgments We thank Tadayuki Imanaka and Haruyuki Atomi for the use of their DNA sequencer. This work was supported in part by a Grantin-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan to T.S. References [1] N. Ferrara, Molecular and biological properties of vascular endothelial growth factor, J. Mol. Med. 77 (1999) 527–543. [2] J.M. Isner, A. Pieczek, R. Schainfeld, R. Blair, L. Haley, T. Asahara, K. Rosenfield, S. Razvi, K. Walsh, J.F. Symes, Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischemic limb, Lancet 348 (1996) 370–374. [3] H. Leong-Poi, M.A. Kuliszewski, M. Leka, M. Sibbald, K. Teichert-Kuliszewska, A.L. Klibanov, D.J. Stewart, J.R. Lindner, Therapeutic angiogenesis by ultrasound-mediated VEGF165 plasmid gene delivery to chronically ischemic skeletal muscle, Circ. Res. 101 (2007) 295–303. [4] P.-P. Liu, E.J. Rebar, L. Zhang, Q. Liu, A.C. Jamieson, Y. Liang, H. Qi, P.-X. Li, B. Chen, M.C. Mendel, X. Zhong, Y.-L. Lee, S.P. Eisenberg, S.K. Spratt, C.C. Case, A.P. Wolffe, Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions, J. Biol. Chem. 276 (2001) 11323–11334.
[5] E.J. Rebar, Y. Huang, R. Hickey, A.K. Nath, D. Meoli, S. Nath, B. Chen, L. Xu, Y. Liang, A.C. Jamieson, L. Zhang, S.K. Spratt, C.C. Case, A. Wolffe, F.J. Giordano, Induction of angiogenesis in a mouse model using engineered transcription factors, Nat. Med. 8 (2002) 1427–1432. [6] K. Tachikawa, O. Schröder, G. Frey, S.P. Briggs, T. Sera, Regulation of the endogenous VEGF-A gene by exogenous designed regulatory proteins, Proc. Natl. Acad. Sci. USA 101 (2004) 15225–15230. [7] T. Sera, Zinc-finger-based artificial transcription factors and their applications, Adv. Drug Deliver Rev. 61 (2009) 513–526. [8] P. Carmeliet, Y.-S. Ng, D. Nuyens, G. Theilmeier, K. Brusselmans, I. Cornelissen, E. Ehler, V.V. Kakkar, I. Stalmans, M. Virginie, J.-C. Perriard, M. Dewerchin, W. Flameng, A. Nagy, F. Lupu, L. Moons, D. Collen, P.A. D’Amore, D.T. Shima, Impaired Myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188, Nat. Med. 5 (1999) 495–502. [9] J. Grunstein, J.J. Masbad, R. Hickey, F. Giordano, R.S. Johnson, Isoforms of vascular endothelial growth factor act in a coordinate fashion to recruit and expand tumor vasculature, Mol. Cell. Biol. 20 (2000) 7282–7291. [10] Q. Dai, J. Huang, B. Klitzman, C. Dong, P.J. Goldschmidt-Clermont, K.L. March, J. Rokovich, B. Johnstone, E.J. Rebar, K. Spratt, C.C. Case, C.D. Kontos, B.H. Annex, Engineered zinc finger-activating vascular endothelial growth factor transcription factor plasmid DNA induces therapeutic angiogenesis in rabbits with hindlimb ischemia, Circulation 110 (2004) 2467–2475. [11] J. Yu, L. Lei, Y. Liang, L. Hinh, R.P. Hickey, Y. Huang, D. Liu, J.L. Yeh, E. Rebar, C. Case, K. Spratt, W.C. Sessa, F.J. Giordano, An engineered VEGF-activating zinc finger protein transcription factor improves blood flow and limb salvage in advanced-age mice, FASEB J. 20 (2006) 479–481. [12] S.A. Price, C. Dent, B. Duran-Jimenez, Y. Liang, L. Zhang, E.J. Rebar, C.C. Case, P.D. Gregory, T.J. Martin, S.K. Spratt, D.R. Tomlinson, Gene transfer of an engineered transcription factor promoting expression of VEGF-A protects against experimental diabetic neuropathy, Diabetes 101 (2006) 1847–1854. [13] Y. Liu, S.R. Cox, T. Morita, S. Kourembanas, Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5’ enhancer, Circ. Res. 77 (1995) 638–643. [14] T. Shibata, A.J. Giaccia, J.M. Brown, Development of a hypoxia-responsive vector for tumor-specific gene therapy, Gene Ther. 7 (2000) 493–498. [15] S.J. Triezenberg, R.C. Kingsbury, S.L. McKnight, Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression, Genes Dev. 2 (1988) 718–729. [16] E. Tischer, R. Mitchard, T. Hartman, M. Silva, D. Gospodarowicz, J.C. Fiddes, J.A. Abraham, The human gene for vascular endothelial growth factor, J. Biol. Chem. 266 (1991) 11947–11954. [17] T. Mino, T. Hatono, N. Matsumoto, T. Mori, Y. Mineta, Y. Aoyama, T. Sera, Inhibition of DNA replication of human papillomavirus by artificial zinc finger proteins, J. Virol. 80 (2006) 5405–5412. [18] A.L. Akeson, J.M. Greenberg, J.E. Cameron, F.Y. Thompson, S.K. Brooks, D. Wiginton, J.A. Whitsett, Temporal and spatial regulation of VEGF-A controls vascular patterning in the embryonic lung, Dev. Biol. 264 (2003) 443–455. [19] Y. Liang, X.-Y. Li, E.J. Rebar, P. Li, Y. Zhot, B. Chen, A.P. Wolffe, C.C. Case, Activation of vascular endothelial growth factor A transcription in tumorigenic glioblastoma cell lines by an enhancer with cell type-specific DNase I accessibility, J. Biol. Chem. 277 (2002) 20087–20094. [20] R. Pollock, M. Giel, K. Linher, T. Clackson, Regulation of endogenous gene expression with a small-molecule dimerizer, Nat. Biotechnol. 20 (2002) 729– 733. [21] I. Baumgartner, N. Chronos, A. Comerota, T. Henry, J.-P. Pasquet, F. Finiels, A. Caron, J.-F. Dedieu, R. Pilsudski, P. Delaère, Local gene transfer and expression following intramuscular administration of FGF-1 plasmid DNA in patients with critical limb ischemia, Mol. Ther. 17 (2009) 914–921. [22] H. Su, J. Arakawa-Hoyt, Y.W. Kan, Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model, Proc. Natl. Acad. Sci. USA 99 (2002) 9480–9485. [23] D.T. Shima, M. Kuroki, U. Deutsch, Y.-S. Ng, A.P. Adamis, A.D. D’Amore, The mouse gene for vascular endothelial growth factor, J. Biol. Chem. 271 (1996) 3877–3883.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Hypoxia-specific upregulation of the endogenous human VEGF-A gene by hypoxia-driven expression of artificial transcription factor Tomoaki Mori, Jun Sasaki, Takuya Kanamori, Yasuhiro Aoyama, Takashi Sera * Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
a r t i c l e
i n f o
Article history: Received 24 September 2009 Available online 20 October 2009 Keywords: Artificial transcription factor Hypoxia Hypoxia-response element Ischemic diseases Vascular endothelial growth factor A Zinc-finger protein
a b s t r a c t Activation of vascular endothelial growth factor A (VEGF-A) is an attractive approach to treatment of ischemic diseases. Although zinc-finger-based artificial transcription factors (ATFs) were constructed for human VEGF-A and constitutive expression of ATFs upregulated the endogenous VEGF-A gene expression, activation of VEGF-A specifically in ischemic tissues is desirable for therapeutic application of ATF technology. Here, we describe hypoxia-specific activation of human VEGF-A gene by hypoxia-driven expression of the ATF. We constructed a hypoxia-driven promoter for the ATF expression and placed it upstream of the ATF-encoding regions. The resulting hypoxia-driven expression plasmid induced the ATF expression in hypoxia but not in normoxia, and the hypoxia-specific expression of the ATF activated the VEGF-A expression specifically in hypoxia. Thus, the engineered expression system of ATFs may enable activation of VEGF-A expression specifically in ischemic tissues without affecting normal, healthy tissues in vivo. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Vascular endothelial growth factor A (VEGF-A) is one of the key gene products in neovascularization. Therefore, activation of the endogenous VEGF-A gene or overexpression of the exogenous transgene may ameliorate ischemic diseases including diabetic angiopathy and arteriosclerosis obliterans [1]. For example, delivery of the cDNA expression plasmid of VEGF-A165 that is one of the major splice variants may improve ischemic diseases [2,3]. Alternatively, methods to activate the endogenous human VEGFA gene using zinc-finger-based artificial transcription factors (ATFs) have been explored [4–7]. One of the advantages is that ATFs are able to upregulate each major splice variant of VEGF-A proportionally [4,5] because ATFs activate the transcription of the endogenous VEGF-A gene. This feature is important because proper isoform balance is crucial for VEGF-A function in vivo [8,9]. Actually, ATFs that were constitutively expressed from plasmids or viral vectors successfully upregulated the endogenous VEGF-A gene in vitro [4,6] and in vivo [5,10–12]. However, in clinical application, it is desirable to express ATFs from transfected expression plasmids or viral vectors specifically in hypoxic ischemic tissues without affecting healthy, normal (normoxic) tissues. To our best knowledge, such a system using ATFs has not yet been developed. In this study, we explored the possibility of hypoxia-specific activation of the human endogenous VEGF-A gene by expressing * Corresponding author. Fax: +81 75 383 2767. E-mail address: [email protected] (T. Sera). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.060
ATFs specifically in hypoxia. Namely, we constructed an artificial hypoxia-driven promoter in which six tandem copies of a hypoxia-response element (HRE) [13] were placed upstream of a constitutive cytomegalovirus (CMV) minimal promoter [14] to yield hypoxia-driven expression plasmids of ATFs. We examined the hypoxia-specific expression of ATFs using the engineered ATF-expression plasmids, and then examined whether the hypoxia-driven expression of the ATFs led to hypoxia-specific activation of VEGFA gene in mammalian cells. Materials and methods Plasmid constructions. The constitutive expression plasmid, pCMV-AZP–VP16, encoding a nuclear localization signal (NLS) from the simian virus 40 large T antigen, a six-finger artificial zinc-finger protein (AZP), a herpes simplex virus VP-16 activation domain (residues 415–490; [15]) and a FLAG epitope tag, was prepared as described previously [6]. The six-finger AZP was designed to recognize the 19-bp target of 50 -GGG GCT GGG GGC GGT GTC T-30 (+516 to +534 in the human VEGF-A gene, where +1 is the transcription start site) [16]. A DNA fragment encoding an AZP mutant in which all recognition amino acids were mutated to alanine [17] was cloned into the BamHI/EcoRI sites of pCMV-AZP–VP16 to generate pCMV-AZPAla–VP16. The hypoxia-driven promoter generated for this study harbored six tandem copies of the HRE (50 -CCACAGTGCATACGTGGGCTCCAACAGGTCCTCTT-30 ; [13]) with arbitrary 5-bp spacers upstream of a CMV minimal promoter (53 to +7 in the CMV promoter, where +1
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is the transcription start site; [14]). The DNA fragment constructed from synthesized DNA oligomers was cloned into the MluI/NheI sites of pCMV-AZP–VP16 to generate pHRE6-AZP–VP16. Assay for endogenous human VEGF-A protein production by transient transfection of plasmids expressing ATF derivatives. An expression plasmid of the ATF derivative described above (0.45 lg) was cotransfected with a pCMV-b-galactosidase plasmid (Clontech) (0.05 lg) into 1 105 HEK293 cells (American Type Culture Collection) by electroporation using a MicroPorator MP-100 (Digital Bio Technology) according to the protocol accompanying the machine. The resulting cell suspension (10 ll) was plated onto a 96-well poly(D-lysine)-coated plate (BD Biosciences) with 90 ll of Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 0.1 mM nonessential amino acids (Invitrogen) and 10% FBS (Invitrogen). After incubation as described in the figure legends, the culture medium was collected, and the supernatant was cleared by centrifugation at 200g and 4 °C for 5 min and frozen at –80 °C until assayed. The VEGF-A protein concentration in the supernatant was assayed by using a human VEGF-A ELISA kit (R&D Systems) according to the manufacturer’s protocol. The remaining transfected cells were washed with PBS and then lysed with a passive lysis buffer (Promega). The protein concentration of the centrifuged supernatant was determined by using an NI Protein Assay kit (G-Bioscience) to normalize the VEGF-A produced [18]. This lysis sample was also analyzed by immunoblotting of ATF derivatives (see below). For determination of transfection efficiency, the transfected cells were stained with a b-gal staining kit (Invitrogen), and the b-galactosidase activities were measured by using a Luminescent b-Galactosidase Detection kit (BD Biosciences). These experiments were carried out in duplicate and repeated independently at least three times. Immunoblotting analysis of ATF derivatives. Cell lysates prepared as described above were also examined for expression of ATF derivatives. Equal amounts of the protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and probed with an anti-FLAG antibody conjugated with horseradish peroxidase (Sigma) or an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Ambion) by standard methods. ECL Plus (GE Healthcare) was used for chemiluminescence detection. The resulting chemiluminescent signals were recorded on X-ray film. The molecular weight of each ATF derivative is indicated in the figure legends. For determination of a relative amount of ATF produced by transfection of pHRE6-AZP–VP16, band intensities of ATF produced by transfection of pCMV-AZP–VP16 were digitized and quantitated by UN-SCAN-IT (Silk Scientific, Inc.), and a standard curve was then generated. Results and discussion First, we examined whether the ATF AZP–VP16 (Fig. 1A) activated the endogenous VEGF-A gene in hypoxia (1% O2). Although we demonstrated that the ATF activated the endogenous VEGF-A gene in HEK293 cells under the normoxic conditions [6], we have not yet examined the effectiveness under the hypoxic conditions. Cells cultured under our hypoxic conditions increased VEGF-A production by >5-fold compared with those cultured under normoxic conditions (data not shown), similarly to a previous study (6-fold activation of VEGF-A in HEK293 cells incubated in a 1% O2 atmosphere; [19]). At 24 h after transfection of a plasmid encoding the ATF downstream of a strong, constitutive CMV promoter (Fig. 1A), the ATF activated VEGF-A expression by 24 ± 9-fold compared with a control (i.e., transfection with pcDNA3.1) (Fig. 2A). In contrast, a control ATF, AZPAla–VP16, in which all recognition amino acids in the zinc-finger domains were mutated to alanine, did not increase the expression level (Fig. 2A). The control protein AZPAla was unable
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to bind to a target DNA even at a protein concentration of 1 lM in the presence of an excess amount of poly(dA–dT)2 competitor DNA in vitro [17]. This result demonstrates that the six-finger AZP in AZP–VP16 recognized the target 19-bp in the VEGF-A 50 -UTR precisely. Immunoblotting of ATF derivatives expressed transiently in the experiments revealed that AZP–VP16 was not expressed more than AZPAla–VP16 (Fig. 2B), indicating that VEGF-A gene activation by AZP–VP16 (Fig. 2A) was not due to much higher expression of AZP–VP16 than of AZPAla–VP16. Because we confirmed that the ATF constitutively expressed activated the endogenous VEGF-A gene efficiently in hypoxia, we next constructed the hypoxia-driven expression plasmid of the ATF. In actual clinical application, the ideal is ATF expression only in target ischemic tissues without affecting healthy, normal tissues. Thus, to accomplish specific expression of ATF in hypoxic tissues, we constructed a hypoxia-driven expression plasmid of the ATF (Fig. 1B). In the engineered promoter, six copies of an HRE [13] were placed upstream of the CMV minimal promoter (53 to +7 in the CMV promoter, where +1 is the transcription start site; [14]). The HRE is known to be a cis-element that is recognized by hypoxia-inducible factors (HIFs), and is responsible for hypoxiaspecific expression of various genes including VEGF-A [13]. We then examined whether the new expression plasmid for AZP–VP16, pHRE6-AZP–VP16 (Fig. 1B), was expressed in hypoxia but not in normoxia. As shown in Fig. 3A, lane 4, the ATF was expressed effectively in hypoxia (1% O2). As expected, we did not detect the significant expression of the same ATF in normoxia; the expression profile was the same as that of cells transfected with a pcDNA3.1 control plasmid, which does not contain an ATF open reading frame (compare lane 3 with lane 1 in Fig. 3A). Thus, the expression plasmid induced the AZP–VP16 expression specifically in hypoxia. From a standard curve (not shown) obtained from band intensities of ATF from pCMV-AZP–VP16, the ATF production from pHRE6-AZP–VP16 was determined to be 13% of that from pCMVAZP–VP16 (Fig. 3B). Finally, we examined whether the hypoxia-driven expression plasmid of AZP–VP16 activated the endogenous VEGF-A gene specifically in hypoxia. Transfection of the pHRE6-AZP–VP16 resulted in 22 ± 12-fold activation of the endogenous VEGF-A gene
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Fig. 2. Activation of endogenous VEGF-A gene by constitutive expression of ATF in hypoxia. (A) VEGF-A production in hypoxia. The plasmid expressing an ATF derivative under the control of a CMV promoter (or pcDNA3.1 as a control) (0.45 lg) and pCMV-b-galactosidase plasmid (0.05 lg) were cotransfected into 1 105 HEK293 cells by electroporation. After incubation at 37 °C for 24 h in 1% O2, the culture medium and the transfected cells were collected separately. The VEGF-A concentration and the b-galactosidase activities were quantified as described in Materials and methods. Each point represents means ± SD obtained from three independent experiments. The plasmid used for each cotransfection with pCMV-bgalactosidase is indicated below the figure. (B) Immunoblots of ATF derivatives in the cell lysates prepared for panel A. The molecular weights of AZP–VP16 and AZPAla–VP16 are 32.2 and 30.8 kDa, respectively. Lane 1, sample from cells transfected with pcDNA3.1 as a control; lane 2, sample from cells transfected with pCMV-AZP–VP16; lane 3, sample from cells transfected with pCMV-AZPAla–VP16.
compared with that of cells transfected with a pcDNA3.1 control plasmid (Fig. 3C). As shown in Fig. 3D, in normoxia, the ATF did not significantly activate VEGF-A expression (i.e., 1.1 ± 0.4-fold activation compared with a control). Systemic administration of constitutive ATF-expression plasmids for activation of endogenous VEGF-A expression may stimulate excess blood-vessel formation unnecessary for healthy normal tissues. Therefore, regulated systems to express ATFs are necessary for application of ATF technology to clinical treatment. One such approach is to regulate the expression of ATFs by using chemicals (i.e., chemical gene switches). For example, a chemical that noncovalently linked a zinc-finger protein and a transcriptional activation domain upregulated endogenous VEGF-A in a chemicaldependent manner [20]. Such a system also enables adjustment of the timing of ATF expression. However, administration of chemicals specifically to ischemic tissues seems to be difficult. Another approach is local administration or injection of cell-permeable ATF molecules [6] or expression plasmids of ATFs only to diseased tissues. A recent study demonstrated that intramuscular injection of an expression plasmid encoding fibroblast growth factor-1 (FGF-1) resulted in FGF-1 expression only in injected sites [21]. In the pres-
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T. Mori et al. / Biochemical and Biophysical Research Communications 390 (2009) 845–848
Fig. 3. Hypoxia-specific activation of endogenous VEGF-A gene by hypoxia-driven expression of ATF. (A) Hypoxia-specific expression of ATF by using pHRE6-AZP– VP16. The plasmid expressing the ATF under the control of six HRE enhancers (or pcDNA3.1 as a control) (0.45 lg) and pCMV-b-galactosidase plasmid (0.05 lg) were cotransfected into 1 105 HEK293 cells by electroporation. After incubation at 37 °C for 24 h in normoxia or in 1% O2, the culture medium and transfected cells were collected separately. The cell lysates were used for immunoblotting. Lane 1, sample from cells transfected with pcDNA3.1 and then incubated in normoxia; lane 2, sample from cells transfected with pcDNA3.1 and then incubated in hypoxia; lane 3, sample from cells transfected with pHRE6-AZP–VP16 and then incubated in normoxia; lane 4, sample from cells transfected with pHRE6-AZP–VP16 and then incubated in hypoxia. (B) Comparison of ATF production by the hypoxia-driven promoter with that by the constitutive CMV promoter. The relative amount of ATF produced by transfection of pHRE6-AZP–VP16 was determined from a standard curve (not shown) that was generated from band intensities of ATF produced by transfection of pCMV-AZP–VP16. Amounts of total proteins used for immunoblotting are indicated below the figure. (C) Activation of VEGF-A protein production by pHRE6-AZP–VP16 in hypoxia. The VEGF-A concentration of the culture medium prepared for panel A was quantified. Each point represents means ± SD obtained from three independent experiments. (D) Effect of transfection with pHRE6-AZP– VP16 on VEGF-A protein production in normoxia. The VEGF-A concentration of the culture medium prepared for panel A was quantified. Each point represents means ± SD obtained from three independent experiments.
ent study, we explored an alternative approach, the development of a system to express ATFs specifically in hypoxic ischemic tissues.
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To accomplish this, we constructed an artificial promoter that contained six tandem copies of an HRE upstream of a CMV minimal promoter and generated a hypoxia-driven ATF-expression plasmid by using the artificial promoter. As shown in Fig. 3A, the expression plasmid enabled expression of ATF specifically in hypoxia. Although significant leaky expression in normoxia was observed in other hypoxia-inducible systems using HREs [22], it was not observed in the present study. The hypoxia-driven expression system reduced ATF production to 13% of that by a strong, constitutive CMV promoter (Fig. 3B). However, using the new expression system, we activated the endogenous VEGF-A gene by >20-fold in hypoxia, similar to that of the CMV promoter (Fig. 3C). Furthermore, leaky activation of VEGF-A was not observed in normoxia (Fig. 3D), corresponding to the ATF expression profile (Fig. 3A). Thus, the hypoxia-driven ATF system for VEGF-A activation is potentially applicable to treatment of ischemic diseases without affecting healthy normal tissues. As the next step, we need to carefully examine whether the hypoxia-driven expression system will be effective for treatment of ischemic diseases in animal models without causing undesirable side effects. In conclusion, we have constructed the first hypoxia-driven expression system of ATFs, and demonstrated hypoxia-specific activation of endogenous human VEGF-A gene expression in cultured cells. We will evaluate our hypoxia-specific expression system in vivo as the next step. The 50 -UTR of mouse VEGF-A is similar to the human one, and the 19-bp target recognized by our ATFs is also located present in the mouse 50 -UTR [23]. Therefore, our ATFs can be used directly in in vivo experiments using mice. Acknowledgments We thank Tadayuki Imanaka and Haruyuki Atomi for the use of their DNA sequencer. This work was supported in part by a Grantin-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan to T.S. References [1] N. Ferrara, Molecular and biological properties of vascular endothelial growth factor, J. Mol. Med. 77 (1999) 527–543. [2] J.M. Isner, A. Pieczek, R. Schainfeld, R. Blair, L. Haley, T. Asahara, K. Rosenfield, S. Razvi, K. Walsh, J.F. Symes, Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischemic limb, Lancet 348 (1996) 370–374. [3] H. Leong-Poi, M.A. Kuliszewski, M. Leka, M. Sibbald, K. Teichert-Kuliszewska, A.L. Klibanov, D.J. Stewart, J.R. Lindner, Therapeutic angiogenesis by ultrasound-mediated VEGF165 plasmid gene delivery to chronically ischemic skeletal muscle, Circ. Res. 101 (2007) 295–303. [4] P.-P. Liu, E.J. Rebar, L. Zhang, Q. Liu, A.C. Jamieson, Y. Liang, H. Qi, P.-X. Li, B. Chen, M.C. Mendel, X. Zhong, Y.-L. Lee, S.P. Eisenberg, S.K. Spratt, C.C. Case, A.P. Wolffe, Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions, J. Biol. Chem. 276 (2001) 11323–11334.
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