A novel chimeric promoter that is highly responsive to hypoxia and metals

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Abstract

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 × HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS–MRE-3 × HRE (E–M–H) gave a hypoxia induction ratio of 69. The expression induced from E–M–H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E–M–H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1α, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E–M–H chimeric promoter. E–M–H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E–M–H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E–M–H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E–M–H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.

Introduction

Hypoxia induces a wide range of physiological responses and plays a crucial role in the pathogenesis of several human diseases.1 Many hypoxia-responsive genes are regulated by the hypoxia-response element (HRE) and the basic helix–loop–helix/Per-Arnt-Sim (bHLH-PAS) protein family of transcription factors, including hypoxia-inducible factor-1 (HIF-1). The HIF-1/HRE system can be used to achieve selective expression of therapeutic genes under hypoxic conditions. When HREs derived from different genes are placed in plasmids and viral vector systems, they confer hypoxia inducibility upon heterologous promoters in various cell types.2 Recently, the murine phosphoglycerate kinase 1 (PGK-1) HRE has been used successfully to confer hypoxia inducibility upon other promoters in vitro3 and in vivo.4, 5, 6 Moreover, HREs have been incorporated into the minimal simian virus 40 (SV40) promoter7 to achieve an induced expression level comparable to that of the human cytomegalovirus (HCMV) promoter6 in the context of an adenoviral vector.

Hypoxia also induces other transcription factors, such as metal transcription factor-1 (MTF-1) and early growth response factor-1 (Egr-1). Although the roles of MTF-1 and Egr-1 in hypoxia are not fully known, several reports have suggested their possible involvement in hypoxic responses.8, 9, 10 MTF-1 is an evolutionarily conserved zinc-finger transcription factor that regulates target gene expression by binding to the metal-response element (MRE) in response to heavy metals.11 Egr-1 was originally identified as an immediate-early gene that is rapidly induced in response to a variety of stimuli.12 More recently, we and others have focused on the role of Egr-1 in coordinating responses to hypoxia and vascular injury.13, 14, 15, 16

The potential roles of MTF-1 and Egr-1 in hypoxia are significant in that they may represent other types of host response mechanisms in the hypoxic milieu. The target genes regulated by these zinc-finger transcription factors are thought to differ from those under the control of the HIF-1 family. These different yet cooperative mechanisms could contribute to an increased hypoxic response. Moreover, an alternate means of controlling gene expression could be developed through the corresponding DNA motifs. However, efforts to exploit these different aspects of the hypoxic response to boost expression and to diversify induction routes have been limited.

In addition to hypoxia, certain metals such as cobalt, nickel, and manganese and the iron chelator, deferoxamine (DFX), can mimic the hypoxic environment and can be useful for hypoxic induction.17 These hypoxia mimetics are thought to exploit the cellular oxygen-sensing mechanism and the hypoxic signal-transduction pathway that lead to stimulation of several hypoxia-responsive genes.18

In the present study, we hypothesized that combinations of the HRE, MRE and Egr-1-binding site (EBS) might provide novel means by which to enhance the level of hypoxic induction and to control target gene expression with hypoxia mimetics. Once successful, such chimeric enhancer vectors could rigorously restrict therapeutic gene expression to hypoxia- or hypoxia mimetics-enriched tissues, thereby providing greatly enhanced condition-specific expression. Here, we demonstrate that a potent hypoxia-inducible promoter can be generated by the novel chimeric strategy, in which three different enhancers are combined upstream from the minimal promoter. We also present evidence that other stresses such as hydrogen peroxide can finely regulate gene expression from such a chimeric promoter. Gene-based therapies for ischemic diseases and cancer should profit from the novel chimeric enhancer approach, which can achieve hyperinduction of therapeutic genes in hypoxic conditions.

Results

Effect of single enhancer elements on hypoxia responsiveness

To examine the enhancer functions of the putative hypoxia-related cis-acting elements, the EBS from the murine Egr-1 promoter (referred to as ‘E’), the MRE from the murine metallothionein-I (MT-I) promoter (‘M’), and three tandem repeats of the murine PGK-1 HRE (‘H’) were synthesized and placed upstream from the minimal SV40 promoter in the pGL3-promoter plasmid (‘pGL3’). The resulting constructs, E-pGL3, M-pGL3 and H-pGL3, respectively, were then evaluated for the hypoxic induction of a luciferase reporter after transient transfection in HeLa cells (Figure 1a). The H-pGL3 construct served as a positive control for the hypoxia responsiveness of the assay, and the enhancerless pGL3 construct was used as the negative control. Compared with H-pGL3, which displayed a 20.2-fold increase in luciferase expression after 21 h exposure to hypoxia (1% O2), the E-pGL3 and M-pGL3 constructs expressed low levels of luciferase activity and showed an apparent hypoxia inducibility (1.4- and 1.8-fold, respectively) no higher than that of the control (pGL3; 1.8-fold). This indicates that E and M may not be effective hypoxic enhancers by themselves, at least in the context of the minimal SV40 promoter.

Figure 1
figure1

Effect of varying number of enhancers on hypoxia responsiveness. After transient transfection with the indicated vectors for 24 h, HeLa cells were incubated under the hypoxic condition (1% O2) for 21 h and the luciferase activity measured. The H/N ratio refers to the ratio of luciferase activity under hypoxic condition (1% O2) to that under normoxic condition (21% O2). RLU/mg protein, relative light units normalized to milligrams of protein. (a) Effect of a single enhancer element on hypoxia responsiveness. The vector harboring a single enhancer element upstream from the minimal simian virus 40 (SV40) promoter was examined for its ability to express the luciferase reporter gene in response to hypoxia. The data are means±s.e.m. (n=3). (b) Effect of two-enhancer combinations on hypoxia responsiveness. Two-enhancer combinations were tested for induction by hypoxia in HeLa cells. The data are means±s.e.m. (n=3). **P<0.01; ***P<0.001 (Student's t-test). (c) Effect of three-enhancer combination on hypoxia responsiveness. Luciferase expression was also compared with human cytomegalovirus (HCMV) promoter in pHCMV-L3 vector. See text for vector abbreviations. The data are means±s.e.m. (n=3). ***P<0.001 (Student's t-test).

Effect of two-enhancer combinations on hypoxia responsiveness

We next investigated whether E or M is responsive to hypoxia in the presence of H. The E–H and M–H combinations of enhancers were tested for the hypoxic induction of the luciferase reporter and compared with induction from H. As shown in Figure 1b, inclusion of E or M with H led to a significant increase in luciferase expression and an increase in the induction ratio (more than 1.9-fold) relative to that of H. In contrast, the M–M and E–E combinations did not produce significant levels of gene expression and are therefore not induced by hypoxia (data not shown). These data suggest that E and M can function as enhancers in the presence of H.

Effect of three-enhancer combination on hypoxia responsiveness

To extend the successful increase in the induction ratio achieved by chimeric constructs, we examined three different enhancer combinations for their ability to cause further enhancement of induction and expression levels. As shown in Figure 1c, E–M–H-pGL3 increased luciferase expression 69-fold under hypoxia, which is 38- and 3.4-fold higher than the levels achieved with the control (pGL3) and H-pGL3, respectively. In terms of the levels of induced expression, E–M–H-pGL3 expressed 3.5- and 2.4-fold greater luciferase activity than did the HCMV-driven pHCMV-L3 and H-pGL3 constructs, respectively. The basal level of luciferase expression from E–M–H-pGL3 under normoxic conditions did not exceed the expression from the control or from H-pGL3, which accounts in part for the higher induction ratio of E–M–H-pGL3 compared with that of H-pGL3. On the other hand, under normoxic conditions, the promoterless construct containing only the E–M–H chimeric enhancer exhibited an apparent luciferase activity near the limit of detection and well below that of the control (pGL3) plasmid (data not shown). Thus, the E–M–H chimeric enhancer does not appear to work as a promoter. Taken together, these data suggest that the heterologous enhancer elements operate on a single minimal SV40 promoter in a synergistic manner and thereby boost the transcription of downstream genes.

Effect of order

As the three-enhancer combination conferred increased hypoxia inducibility, we next investigated the best or proper arrangement of the three enhancers (Figure 2). Constructs differing in the order of the enhancers were made and the level of luciferase activity measured. Surprisingly, the initial three-enhancer construct, E–M–H, gave the highest induction ratio as well as the greatest absolute expression level. The chimeric designs starting with 3 × HRE (H–M–E and H–E–M) displayed lower levels of induction than the other arrangements, in which 3 × HRE occurred in the middle or at the end of the chimeric enhancer. With 3 × HRE at the same position (either the first or third position), it appeared to be more advantageous to place EBS before MRE. However, differences between the groups were not statistically significant, except those between E–M–H– or M–H–E-pGL3 and H–M–E-pGL3 (P<0.05).

Figure 2
figure2

Optimization of three heterologous enhancers for maximal induction. The order of the three enhancers was changed to achieve the maximum expression and the indicated constructs were examined for induction by hypoxia in HeLa cells. After transfection, the cells were incubated under normoxia or hypoxia (1% O2) for 21 h and the luciferase activity measured. The pGL3-promoter plasmid was used as a negative control. H/N ratio refers to the hypoxia induction ratio. RLU/mg protein, relative light units normalized to milligrams of protein. See text for vector abbreviations. The data are means±s.e.m. (n=3). *P<0.05 (one-way analysis of variance, Turkey's multiple comparison test).

Validation in different cells and with other cDNAs

We tested whether the induction results for E–M–H could be replicated in cells other than HeLa. We examined human primary cells such as skeletal myocytes (SkMC) and vascular smooth muscle cells (VSMC) as well as C2C12 and LLC1 cell lines (Figure 3a). Consistent with the data in HeLa, the E–M–H enhancer construct showed hypoxia-induced luciferase expression that was significantly higher than the H construct in all cells tested. Although we cannot discount the possible limitation that the basal expression from the E–M–H or H was higher than that from the control (pGL3) (data not shown), the consistent pattern of higher induction in five different cell types strongly suggests that the chimeric enhancer is functional in a variety of cells.

Figure 3
figure3

Validation of high inducibility of EBS–MRE-3 × HRE (E–M–H) chimeric promoter. (a) Validation in skeletal myocyte (SkMC), vascular smooth muscle cell (VSMC), C2C12 and LLC1 cells. The indicated constructs were examined for the hypoxic induction of luciferase activity. At 1 day after transfection, the cells were exposed to normoxia or hypoxia (1% O2) for 21 h and the luciferase activity measured. The H/N ratio refers to the hypoxia induction ratio. See text for abbreviations. The data are means ±s.e.m. (n=3). *P<0.05 (Student's t-test). (b) and (c) Validation with different cDNAs. The indicated constructs were evaluated for hypoxic induction of bFGF (b) and LacZ (c) expression in HeLa cells. At 1 day after transfection, the cells were exposed to normoxia or hypoxia (1% O2) for 21 and the expression of bFGF and LacZ was measured using an ELISA kit (R&D and Roche, respectively). See text for abbreviations. The data are means±s.e.m. (n=3). **P<0.01; *P<0.05 (Student's t-test).

The hypoxia responsiveness of the E–M–H chimeric enhancer was also validated using other cDNAs, such as bFGF and LacZ, in HeLa cells. As shown in Figures 3b and c, the induction fold with basic fibroblast growth factor (bFGF) and LacZ was 1.6- and 2.3-fold higher from E–M–H than from the H construct, respectively. These results indicate that the greater induction seen with the E–M–H construct also occurs successfully with other cDNAs.

Increased levels of HIF-1α, Egr-1 and MTF-1 are associated with the high performance of the E–M–H chimeric promoter

To examine potential roles of HIF-1α, Egr-1 and MTF-1 in the hypoxic response of E–M–H promoter, we measured the RNA level of each transcription factor by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) (Table 1). In hypoxic conditions (1% O2, 1–6 h), the mRNA level of HIF-1α, Egr-1 and MTF-1 increased significantly in HeLa and C2C12 cells (Table 2), which is consistent with the data published by others.8, 10, 18 We next tested whether the increased expression of each transcription factor translates into increased binding to its target DNA motif. Gel shift assays with the nuclear extracts prepared in both normoxic and hypoxic conditions indicated that the levels of specific DNA–protein complexes were significantly higher in hypoxia than in normoxia (Figures 4a and b). Competition with a 50-fold molar excess of the cold probes as well as supershift experiments using a polyclonal HIF-1α or Egr-1 antibody indicated that the interaction was specific. These results suggest that hypoxia upregulates the expression of endogenous HIF-1α, Egr-1 and MTF-1, which in turn leads to their increased binding to the corresponding DNA motifs.

Table 1 Primer pairs used in quantitative real-time RT-PCR experiments
Table 2 Increase of HIF-1α, MTF-1 and Egr-1 mRNAs under hypoxic condition (1% O2) as measured by quantitative real-time RT-PCR
Figure 4
figure4

Increased levels of hypoxia-inducible factor-1 (HIF-1α), early growth response factor-1 (Egr-1) and metal transcription factor-1 (MTF-1) are associated with high induction profile of EBS–MRE-3 × HRE (E–M–H) chimeric promoter. (a) and (b) DNA binding activity of endogenous HIF-1α, MTF-1 and Egr-1 is increased in response to hypoxia in HeLa (a) and C2C12 (b) cells. Nuclear extracts (5–10 μg of protein) from the cells grown in normoxia (N) or hypoxia (H, 1%O2) for 21 h were prepared and then used in the gel shift assay with a [32P]ATP-labeled consensus probe for HIF-1α, MTF-1 or Egr-1. Competition experiments were performed using cold oligonucleotides (50-fold molar excess) and the polyclonal antibody raised against HIF-1α or Egr-1 was used for the supershifts assay. (c) and (d) Overexpression of HIF-1α, MTF-1 and Egr-1 induces luciferase expression from E–M–H-pGL3 in normoxia. HeLa (c) and C2C12 (d) cells were cotransfected with the reporter construct and expression vectors for HIF-1α, MTF-1 and/or Egr-1. After transfection, the cells were incubated at 37°C for 24 h, and the luciferase activity was measured as described in ‘Materials and methods.’ The fold induction was calculated as the ratio of luciferase activity observed in cotransfection with the specific expression vector(s) to that observed in cotransfection with the empty vector. The data are means±s.e.m. (n=3); *P<0.05 (Student's t-test).

Next, we investigated whether the high induction profile of the E–M–H chimeric enhancer can be reproduced by overexpression of HIF-1α, Egr-1 or MTF-1. We performed co-transfection experiments in HeLa (Figure 4c) and C2C12 (Figure 4d) cells with the reporter construct and expression vectors for HIF-1α, Egr-1 and MTF-1. In co-transfection with the HIF-1α vector, the E–M–H-pGL3 showed a significantly higher luciferase reporter expression (2.9-fold in HeLa cells and 4.7-fold in C2C12 cells) than co-transfection with the empty pcDNA3.1 vector. The H-pGL3 also showed a similar pattern of induction in both cell lines (6.3- and 7.9-fold, respectively). In contrast, co-transfection with either the MTF-1 or Egr-1 expression vector caused little boosting of expression, implying their minimal contribution by themselves. However, co-transfecting the MTF-1 or Egr-1 vector with the HIF-1α vector significantly increased luciferase expression from the E–M–H in HeLa (7.5- and 6.9-fold, respectively) and C2C12 cells (7.0- and 7.1-fold, respectively), suggesting that MTF-1 and Egr-1 might synergize with HIF-1α to enable potent induction from the E–M–H chimeric enhancer. The MTF-1/Egr-1 combination increased luciferase expression only marginally. The HIF-1α/MTF-1/Egr-1 triple combination produced a significantly lower level of luciferase expression in both cell lines, which can be explained in part by the squelching effect that results from the use of multiple HCMV promoters.19, 20, 21 On the other hand, induction was lower with the H-pGL3 transfected with the MTF-1/HIF-1α (2.9-fold) or the Egr-1/HIF-1α (3.2-fold) combination than the HIF-1α alone in HeLa (6.3-fold) cells. A similar pattern was observed in C2C12 cells (5.1-, 4.7- and 7.9-fold, respectively). This suggests that MTF-1 and Egr-1, which have little effect on the induction from HRE, may interfere with HIF-1α binding to HRE.

Modulation of gene expression from the E–M–H promoter

As certain transition metals and iron chelators can mimic hypoxia, we investigated whether the chimeric promoter could also be modulated by hypoxia mimetics. If so, this would provide an alternate means by which to control target gene expression and extend the utility of the novel chimeric promoter. As shown in Figure 5a, treatment with metal ions such as cobalt and nickel dramatically upregulated (73.2- and 25.4-fold, respectively) luciferase reporter expression from the E–M–H vector. Deferoxamine also greatly increased expression (43.1-fold). In all cases, the induced expression was significantly higher from E–M–H-pGL3 than from H-pGL3. We also tested whether this chimeric promoter can be regulated by other stress conditions such as heat, low glucose concentration, low pH and hydrogen peroxide. As shown in Figure 5b, hydrogen peroxide was a weak inducer of E–M–H in HeLa cells (2.6-fold). However, heat, low glucose concentration and low pH had little effect on the inducibility. Hydrogen peroxide also worked as an inducer of H-pGL3, although the induction ratio was less than that of E–M–H-pGL3 (P<0.05).

Figure 5
figure5

Regulation of gene expression from EBS–MRE-3 × HRE (E–M–H). (a) Induction by hypoxia mimetics. The indicated constructs were examined for hypoxic induction of luciferase activity in transiently transfected HeLa cells. At 1 day after transfection, the cells were incubated in the presence of MnCl2 (500 μ M), NiCl2 (1 mM), deferoxamine (DFX, 260 μ M), or CoCl2 (300 μ M) for 21 h. The cells were then collected and the luciferase activity measured. The fold induction is defined as the luciferase activity relative to control (N; no treatment). The data are means±s.e.m. (n=3); *P<0.05 (Student's t-test). (b) Modulation by a variety of stress conditions. HeLa cells were transfected with the indicated constructs for 24 h and then subjected to heat shock (43°C, 0.5 h followed by 2 h of recovery at 37°C), low glucose (1 mg/ml, 4 h), low pH (6.0, 4 h) or H2O2 (500 μ M, 4 h) under the normoxic condition. The treated cells were collected and the reporter gene activities were determined by luciferase assay. The fold induction refers to the luciferase activity relative to control (no treatment). The data are means±s.e.m. (n=3); *P<0.05 (Student's t-test). (c) and (d) Reversibility of the induced gene expression. In HeLa cells, the indicated constructs were evaluated first for induction by hypoxia (c) or DFX (d) and then examined for the decline in expression after reoxygenation (hypoxia → normoxia) or removal of DFX (DFX (+) → DFX (−)). The data are means±s.e.m. (n=3).

Next, we examined the reversibility of induced gene expression from the E–M–H promoter. We subjected the HeLa cells transfected with the E–M–H-bFGF construct to the cycle of normoxia–hypoxia–normoxia and measured the amount of bFGF secreted into the medium in each condition. In a similar way, we also examined the effects of adding or removing DFX. As shown in Figures 5c and d, the hypoxia-induced secretion of bFGF decreased significantly upon reoxygenation and removal of DFX, suggesting that the induced expression from the E–M–H is reversible. Taken together, these findings suggest that our chimeric promoter can be modulated by various means that can alter or are related to oxygen tension.

In vivo evaluation of the E–M–H chimeric promoter in the ischemic hindlimb

We next evaluated the efficacy of the chimeric promoter in vivo in a murine model of hindlimb ischemia. The naked DNA vector encoding the luciferase or LacZ gene was injected into the tibialis anterior muscle of C57BL/6 mice, followed by electroporation procedures to increase the gene transfer efficiency.22 After 2 days, unilateral hindlimb ischemia was introduced into the left leg (day 0) and monitored for blood flow until day 3 (Figure 6a). The reduced blood flow in the left hindlimb, which was represented by the low tissue perfusion ratio (ischemic to non-ischemic limb (I/N) ratio), indicated that tissue hypoxia persisted in the left calf muscle. On day 3, the left tibialis anterior muscle was harvested and the luciferase reporter activity measured. The induction was 304-fold higher in the E–M–H-pGL3 than in the control (pGL3) and 3.5-fold higher than in the HRE vector (H-pGL3) (Figure 6b). In the contralateral, non-ischemic calf muscle, all groups displayed negligible levels of induction ratio. Similar experiments with the LacZ reporter also showed that the E–M–H promoter caused the highest level of LacZ expression in the ischemic hindlimb (Figure 6c), which was consistent with the X-gal staining data of the muscle tissue (data not shown). To provide a further proof of the in vivo utility of our chimeric promoter, we investigated whether E–M–H can increase the serum concentration of a secretable protein such as erythropoietin (EPO). Erythropoietin cDNA was cloned into E–M–H vector, transfected into the ischemic hindlimb, and the EPO concentration was measured in the serum at the indicated time points. As shown in Figure 6d, the EPO level was significantly higher in the E–M–H group than in the control group (P<0.001 on days 3 and 7), HCMV group (P<0.05 on day 3) and H group (P<0.05 on days 3 and 7). Taken together, these results demonstrate that our chimeric promoter works efficiently in vivo in a relevant animal model and that it achieves better hypoxic induction than the HRE-based promoter does.

Figure 6
figure6

Validation of the high inducibility of the EBS–MRE-3 × HRE (E–M–H) chimeric promoter in vivo in a murine model of hindlimb ischemia. (a) Representative laser Doppler imaging (LDI) data of C57BL/6 mice with unilateral hindlimb ischemia on days 0 and 3. The perfusion signal is displayed in color codes ranging from dark blue (0) through red to white (1000). CTRL indicates sham operation. The I/N ratio refers to the perfusion ratio of the ischemic to non-ischemic limb. Less than 25% of normal blood flow occurred in the ischemic leg, indicating successful induction of in vivo hypoxia. (b) Comparisons of luciferase activities in tibialis anterior muscles three days after femoral artery excision. The level of luciferase expression was normalized to that from pHCMV-L3. The data are means±s.e.m. (n=6). ***P<0.001; P>0.05 (Student's t-test). (c) Quantitative evaluation of LacZ expression in the ischemic tibialis anterior muscle. Three days after induction of hypoxia, the muscle was homogenized and β-galactosidase was measured using an ELISA kit (Roche). The data are means±s.e.m. (n=8). ***P<0.001 (Student's t-test). (d) Serum concentration of murine erythropoietin (mEPO) at days 3 and 7 after induction of hindlimb ischemia. Blood was obtained from the orbital venous plexus, and the amount of secreted mEPO was measured by an ELISA kit (R&D). The data are means±s.e.m. (n=5). ***P<0.001; **P<0.01; *P<0.05 (Student's t-test).

Discussion

In the present study, we have demonstrated the usefulness and versatility of the chimeric enhancer approach to achieve potent inducible expression. Using cis-acting enhancer elements that differed in size, sequence and responsiveness to stimuli, we constructed a novel expression cassette with a greatly enhanced response to hypoxia and transition metals. The hypoxia-induced expression of the luciferase reporter from the E–M–H chimeric promoter surpassed that achieved using the HRE system7 or the HCMV promoter (Figure 1c). This superior inducibility was also confirmed in experiments with other cDNAs (Figures 3b and c). The E–M–H chimeric promoter was also upregulated by hypoxia mimetics (Figure 5a) and the induced expression was reversible upon removal of inducers (Figures 5c and d). This novel chimeric promoter will be an invaluable tool for targeting gene expression to specific areas of hypoxia.

Ischemic diseases may profit from the use of this novel chimeric promoter. As an extension of therapeutic angiogenesis strategy widely used to increase collateral vessel growth into the ischemic region,23, 24, 25 our chimeric promoter can effectively increase the hypoxic production of angiogenic growth factors and chemokines such as stromal cell-derived factor-1. Indeed, the hypoxic induction from the E–M–H vector in C2C12 cells and in an animal model of hindlimb ischemia has implications for angiogenic gene therapy of ischemic limb diseases. Moreover, the potent chimeric promoter can provide a means to dramatically increase the expression of therapeutic genes in the center of a tumor where a state of cellular hypoxia exists. Tight control of gene expression by such an approach would also help reduce any damage to normal surrounding tissue and improve the therapeutic outcome. Moreover, targeting gene expression to the hypoxic regions of solid tumors will allow the use of lower doses of the vector and decrease the risk of side effects.26, 27 In this respect, the successful performance of the E–M–H vector in LLC1 carcinoma cells should be noted.

The absolute level of gene expression and the induction ratio achieved by the chimeric promoter can be altered by many factors, including the number and arrangement of the enhancers. The number of different DNA motifs used for the chimeric construct had a significant effect on the performance of the final construct (Figures 1b and c). This reflects, in part, the potential threshold number of heterologous cis-acting elements that might be needed for hyperactivation to occur. The proper order also seemed to be important for additional and maximal levels of enhancement within a given set of enhancers (Figure 2). This may be particularly relevant when the strengths of the enhancers differ, as in our study. Furthermore, the orientation and spacing of different enhancers and the distance from the transcription start site28, 29 might be important because of the interactions and potential steric hindrance between trans-acting factors that bind to the enhancers. It is also unclear whether any of these elements will function bidirectionally. Moreover, the degree of hypoxic stress can have a positive effect on the level of induced gene expression.30, 31 Although we did not examine the induction ratio under near-anoxic conditions (0.1% O2), we presume that expression from the E–M–H vector would increase further, based on the previous observation of Binley et al.32

The hyperinduction observed for the E–M–H promoter (Figure 1c) cannot be explained simply by the presence and independent contribution of EBS and MRE because they failed to confer hypoxia responsiveness by themselves (Figures 4c and d and data not shown). By an unknown mechanism, the protein factors recruited to EBS and/or MRE may activate the HRE-bound HIF-1, or conversely, HIF-1-recruited protein factors may help to activate MTF-1 and Egr-1. Another possibility is that protein factors, in addition to HIF-1, Egr-1 and MTF-1, might be required to account for the degree of synergism seen in E–M–H. It is currently unknown whether the roles of the three transcription factors are mutually exclusive, although Egr-1 and MTF-1 may act independently of HIF-1 and the bHLH/PAS family.33 In addition to hypoxia and its mimetics, stimuli that can activate Egr-1, MTF-1 and HIF-1 might reveal a novel route for the induction from E–M–H. As the expression of genes containing MRE or EBS can be induced by a variety of physiological and environmental stresses,34, 35 such as oxidizing agents, phorbol esters,36 ultraviolet and ionizing radiation, and glucocorticoid hormones, it is necessary to test whether such stimuli can also increase the induction ratio and act as different modulators. However, our preliminary experiments showed that combining hypoxia with different cell stresses were less successful because most cells could not survive the combined stress conditions (data not shown). More optimized systematic studies will be able to determine whether the MRE or EBS confer an advantage to the HRE via different mechanisms in the presence of hypoxia.

One limitation of our study is that we have not tested multiple copies of MRE or EBS. As up to five tandem copies of MRE have been used previously for optimal hypoxic induction,8, 37, 38 we have yet to evaluate the consequences of three to five copies of MRE or EBS. Therefore, there is still room for further improvement of the E–M–H construct. Currently, the effects of multiple copies of EBS and MRE are under investigation. Another limitation is that we have not fully explored the impact of Egr-1 and MTF-1 on the performance of the E–M–H vector. Although overexpression of Egr-1 and MTF-1 in the presence of HIF-1α led to synergism in the E–M–H enhancer, functional binding of Egr-1 and MTF-1 to the HRE needs to be examined. In addition, we do not know whether knockdown of Egr-1 and MTF-1 expression can block hypoxic induction from the E–M–H enhancer. Our current and future work will help evaluate the contribution of MRE and EBS in detail. The usefulness of our chimeric promoter must also be validated in a cancer setting via gene transfer.39, 40, 41 One feasible approach would be to introduce the expression cassette into a stably expressing viral vector (lentivirus or adeno-associated virus) and to assess the expression of the target gene in the hypoxic regions of solid tumors implanted into mice. Despite these limitations, our novel chimeric enhancer promises to be a feasible strategy to control target gene expression on demand.

In conclusion, we have developed a potent chimeric promoter that is highly inducible by hypoxia and metals. This novel chimeric enhancer strategy should be widely applicable for the selective expression of therapeutic genes in the treatment of cancer and ischemic diseases.

Materials and methods

Chemicals

NiCl2, CoCl2, DFX and MnCl2 were purchased from Sigma (Sigma-Aldrich Co., St Louis, MO, USA). All reagents were dissolved in distilled water and then filtered through a sterile Millex-GV filter (pore size, 0.22 μm; Millipore Co., Billerica, MA, USA). A freshly prepared stock solution was diluted with serum-free medium at various concentrations.

Cell culture

Human epitheloid carcinoma (HeLa), mouse Lewis lung carcinoma (LLC1) and mouse myoblast (C2C12) cells were cultured in Dulbecco's modified Eagle's medium (Bio-Whittaker, Walkersville, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Primary human SkMCs were cultured in skeletal muscle cell growth medium (SkGM; Bio-Whittaker, Walkersville, MD, USA) and primary human VSMCs were grown in smooth muscle cell growth medium (Bio-Whittaker, Walkersville, MD, USA). All cells were maintained at 37°C as a monolayer in a humidified atmosphere containing 5% CO2.

Cells were subjected to hypoxia (1% O2, 5% CO2, 94% N2) for 21 h using an environmental chamber (Anaerobic System, Thermo Forma, Germany). Hypoxia was also mimicked by treatment with NiCl2 (1 mM), CoCl2 (300 μ M), DFX (260 μ M) or MnCl2 (500 μ M) for 21 h. Cell viability was not adversely affected by exposure to metal ions. For heat treatments, cells were exposed to heat shock at 43°C for 30 min followed by incubation at 37°C for another 2 h to allow for their recovery.

Plasmids

The oligonucleotides for three copies of the HRE derived from the 5′ flanking region of murine PGK-1, the MRE derived from the murine MT-I proximal promoter and the EBS corresponding to positions −603 to −595 of the murine Egr-1 promoter were synthesized by Cosmo (Seoul, Korea). These oligonucleotides were used to produce double-stranded synthetic enhancers containing NheI and XbaI sites at each end (3 × HRE, 5′-IndexTermCTAGCGTCGTGCAGGACGTGACATCTAGTGTCGTGCAGGACG TGACATCTAGTGTCGTGCAGGACGTGACAT-3′ and 3′-IndexTermGCAGCACGTCCTGCACTGTAGATCACAGCACGTCCTGCAC TGTAGATCACAGCACGTCCTGCACTGTAGATC-5′; MRE, 5′-IndexTermCTAGCAGGGAGCTCTGCACTCCGCCCGAAAAGT-3′ and 3′-IndexTermGTCCCTCGAGACGTGAGGCGGGCTTTTCAGATC-5′; and EBS, 5′-IndexTermCTAGCCGCCCTCGCT-3′ and 3′-IndexTermGGCGGGAGCGAGATC-5′). All oligonucleotides were designed in such a way that each enhancer could be cloned into the NheI site upstream from the minimal SV40 promoter in the pGL3-promoter vector (Promega, Madison, WI, USA). The NheI (GCTAGC) and XbaI (TCTAGA) sticky ends can be ligated (G/CTAGA) but the product is not cleavable with either enzyme. This approach allowed the enhancers to be added without losing the initial NheI site. The pHCMV-L3 vector was generated by inserting luciferase cDNA (derived from the pGL3-promoter vector) into pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA). To make bFGF-expressing vectors pGL3-bFGF, 3 × HRE-pGL3-bFGF and EBS–MRE-3 × HRE-pGL3-bFGF, the HindIII/XbaI fragment from the pcDNA3.1(+)-bFGF vector (kindly provided by Drs Ji Hyung Chung and Yang Soo Jang) was inserted into the HindIII/XbaI sites of the pGL3, 3 × HRE-pGL3 and EBS-MRE-3 × HRE-pGL3 vectors, respectively. The same cloning scheme was used for the LacZ constructs (pG-LacZ, H-pG-LacZ, E–M–H-pG-LacZ). The pG-murine erythropoietin (mEPO), H-mEPO, and E–M–H-mEPO were generated by subcloning the PCR-amplified HindIII/XbaI fragment from the ACP-mEPO plasmid into the HindIII/XbaI site of the pGL3 H-pGL3, and E–M–H-pGL3 vectors, respectively.

Expression vectors for HIF-1α, Egr-1 and MTF-1

The pcDNA3.1-MTF-1 was created by inserting the mMTF-1 cDNA, which was isolated by RT-PCR,42 into the HindIII/XbaI sites of a pcDNA3.1(+) expression vector. A cDNA for mouse Egr-1 was generated by using RT-PCR (mEgr-1 sense primer: 5′-IndexTermTGGTCCGGGATGCGAGC-3′, mEgr-1 antisense primer: 5′-IndexTermATTCCCTTTAGCAAATTTCAAT-3′) from total RNA prepared from mouse proximal tubular epithelial (MCT) cells. The mEgr-1 cDNA was inserted between the HindIII/XbaI sites of a pcDNA3.1(+) vector. A cDNA for human HIF-1α was amplified by Pfu polymerase from a pBOS-HIF-1α construct (kindly provided by Dr Yoshiaki Fujii-Kuriyama). The sense and antisense primers contained the translation start and stop codons, respectively. The amplified product was cloned into the BstXI sites of a pcDNA3.1(+) expression vector. All vectors were verified by DNA sequencing.

Transient transfection and reporter gene assay

The cells were seeded in 12-well plates (50 000 cells/well) and co-transfected with 30–300 ng of test construct and the control pRL-TK (Promega) plasmid using Effectene Reagent (Qiagen, Valencia, CA, USA). After incubation for 24 h, the transfected cells were washed twice with phosphate-buffered saline (PBS) and filled with fresh growth medium. Cells were then exposed to normoxia or hypoxia for 21 h. Cell lysates were prepared and luciferase activities measured by Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions. Replicate transfections were performed with three independent DNA preparations and luciferase activities were normalized to the protein concentration in the cell lysate.

Real-time RT-PCR

Total RNA was prepared from HeLa or C2C12 cells exposed to normoxia or hypoxia for 1–6 h using RNeasy® mini kit (Qiagen). First-strand cDNA was synthesized from 2 μg total RNA using the ABI High Capacity cDNA Achieve kit (Applied Biosystems, Foster City, CA, USA). Standard curves were made using serial dilutions from pooled cDNA samples. Real-time PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol and amplified on the ABI 7900 HT sequence detection system (Applied Biosystems) with primers set as shown in Table 1. For each sample, the gene copy number was normalized to β-actin on the same sample. Real-time RT-PCR reactions were performed in triplicate and each experiment was repeated three times.

Electrophoretic mobility shift assay

HeLa or C2C12 cells were grown to subconfluence and then exposed to normoxia or hypoxia for 21 h before cell lysis. For hypoxic cell extracts, cell lysis was performed under hypoxia to avoid reoxygenation effects. Nuclear extracts were prepared as described by Khachigian et al.43 and the electrophoretic mobility shift assay performed as described previously.44 Synthetic oligonucleotides consisting of consensus sequences from HIF-1α, MTF-1 and Egr-1 were used as probes after annealing the sense and antisense fragments. The probes were 5′-end labeled with [32P]ATP by using T4 polynucleotide kinase and standard procedures. The extracts (5–10 μg of protein in 25 μl) were incubated for 10 min at 37°C in a total volume of 10 μl binding buffer (12 mM HEPES (pH 7.9) containing 60 mM KCl, 0.5 mM dithiothreitol, 12% glycerol, 5 mM MgCl2, 0.2 μg of dI–dC/μg of protein and 2–4 fmol of end-labeled double-stranded oligonucleotide (5000 c.p.m./fmol) for HIF-1α, MTF-1 and Egr-1). For the supershift assay, polyclonal antibody against HIF-1α (R&D Systems, Minneapolis, MN, USA) or Egr-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the completed reaction mixtures and further incubated for 2 h at 4°C before loading. Protein–DNA complexes were loaded directly into non-denaturing polyacrylamide/bisacrylamide (8%) gels. Gels were prerun for 20 min before samples were loaded, and electrophoresis was performed at room temperature for 2–2.5 h at 15 V/cm. In the competition experiments, a 50-fold molar excess of the indicated unlabeled oligonucleotides was added to the binding buffer. After electrophoresis, the gel was dried, and labeled complexes were detected by autoradiography.

Enzyme-linked immunosorbent assay

The amount of human bFGF, murine EPO and bacterial β-galactosidase (β-Gal) was measured with the corresponding bFGF and EPO enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) and β-Gal ELISA kit (Roche Molecular Biochemicals, MN, USA) according to the manufacturer's instructions. The conditioned media, cell lysates or serum were stored at −70°C in 1.5 ml tubes until used.

Electroporation and hindlimb ischemia model

All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). After DNA injection and electroporation procedures, unilateral hindlimb ischemia was induced in 12-week-old male C57BL/6 mice by excision of the femoral artery. Electroporation was performed as described previously.22 Briefly, the animals were anesthetized by intraperitoneal injection of 100 μl of a solution containing 2.215 mg ketamine (Ketalar 50 mg/ml; Yuhan Co., Korea) and 0.175 mg xylazine (Rompun 23.32 mg/ml; Bayer, Korea) for the surgical procedure. The leg was shaved, depilated and 10 μg of DNA in 30 μl of half-saline solution (0.4% NaCl or 75 mM) was injected into the tibialis anterior muscle of each mouse with a 30-G insulin syringe (Becton-Dickinson, Franklin Lakes, NJ, USA). At 30 s after DNA injection, transcutaneous electric pulses were applied to the surface of the injection site using an ECM 830 electroporator (BTX Division of Genetronics, San Diego, CA, USA). Eight electric pulses of 125 V/cm for 50 ms at 1 Hz were applied through two electrodes (BTX Division of Genetronics, San Diego, CA, USA). At 2 days after electroporation, the middle portion of the left hindlimb of the mouse was incised and ligated at both the proximal end of the femoral artery and the distal portion of the saphenous artery. The artery and all side branches were then dissected and excised. After 3 days, the mice were killed, and the tibialis anterior muscle was resected and assayed for luciferase and LacZ. A blood sample was obtained from the orbital venous plexus under anesthesia on days 3 and 7 after ischemia and serum concentration of mEPO was measured using an ELISA kit (R&D Systems).

In vivo blood flow and laser Doppler imaging

Blood flow in both the ischemic and non-ischemic hindlimbs was measured using an LDI system (Moor Instruments, Axminster, Devon, England) on days 0 and 3 after ischemia. The perfusion signal was displayed in color codes ranging from dark blue (0) through red to white (1000). Blood flow in the ischemic limb was normalized to that of the contralateral non-ischemic limb.

Histological analysis

X-gal staining was performed by incubating frozen sections of muscle tissues in X-gal staining solution (1 mM X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2, 0.02% Nonidet P-40, 0.01% sodium deoxycholate in PBS) at 37°C for 4 h. The sections were counterstained in hematoxylin and eosin, dehydrated through graded alcohols, cleared in xylene and mounted in Permount® SP15-100 (Fisher Scientific, USA).

Statistical analysis

All data are expressed as means±s.e.m. Results were analyzed with Prism software ver. 3.0 (GraphPad Software Inc., San Diego, CA, USA). Student's t-test or one-way analysis of variance was used to evaluate the differences between groups. A P-value <0.05 was considered statistically significant.

Accession codes

Accessions

GenBank/EMBL/DDBJ

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Acknowledgements

This work was supported by the National Research Laboratory Grant from the Korea Institute of Science and Technology Evaluation and Planning (M1-0412-00-0048) and the grants from the Korean Ministry of Health and Welfare (01-PJ1-PG1-01CH06-0003) and the Korea Science and Engineering Foundation (SRC, Molecular Therapy Research Center) to DK Kim.

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Correspondence to J Byun or D-K Kim.

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Lee, J., Lee, Y., Kim, J. et al. A novel chimeric promoter that is highly responsive to hypoxia and metals. Gene Ther 13, 857–868 (2006) doi:10.1038/sj.gt.3302728

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Keywords

  • hypoxia
  • inducibility
  • promoter
  • HRE
  • MRE
  • EBS

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