Disease- and cell-type-specific transcriptional targeting of vectors for osteoarthritis gene therapy: further development of a clinical canine model

S. E. Campbell, D. Bennett, L. Nasir, E. A. Gault and D. J. Argyle1

Molecular Therapeutics Research Group, Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK and 1 University of Wisconsin Comparative Oncology Program, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706–1102, USA.

Correspondence to: D. J. Argyle, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706–1102, USA. E-mail: argyled{at}svm.vetmed.wisc.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. The potential for undesirable systemic effects related to constitutive expression of certain therapeutic transgenes may be limited through the development of transcriptionally targeted disease- and cell-type-specific vectors. The objective of this study was to analyse the canine matrix metalloproteinase-9 (MMP-9) promoter and deletion constructs for its ability to drive expression in response to pro-inflammatory cytokines (interleukin-1ß and tumour necrosis factor-{alpha}).

Methods. Initial analysis of MMP-9 deletion constructs was made using a luciferase reporter system. The promoter was subsequently engineered to incorporate multiple NF-{kappa}B sites. In parallel experiments we used the mouse collagen type XI promoter to study cell-type-specific promoter activity in chondrocyte-specific cells (SW1353) and undifferentiated chondroprogenitor cells (ATDC5).

Results. Incorporation of multiple NF-{kappa}B sites into the MMP-9 promoter enhanced activity while maintaining disease specificity. Further, manipulation of the mouse collagen type XI (mColXI) promoter by the incorporation of SOX9 enhancer sites downstream of a reporter gene, increased gene activity while maintaining cell type specificity.

Conclusions. Manipulation of promoter and enhancer regions can improve transcriptionally targeted genes. A combination of these systems, in the context of the canine model, has the potential to improve the safety of osteoarthritis gene therapy vectors.

KEY WORDS: Osteoarthritis, Canine model, Gene therapy, Transcription


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Developing gene-based therapy for osteoarthritis (OA) in both human and veterinary patients represents an exciting challenge. The detailed understanding of disease pathogenesis has already enabled the introduction of ‘structure-modifying’ therapeutic genes into arthritic joints to control disease progression at the molecular level by inhibiting the enzymes responsible for cartilage degradation while enhancing tissue repair. However, despite considerable advances in molecular biology, several technical hurdles must be overcome before gene therapies can be considered acceptable clinical practice. In the development of a gene-based therapy for OA, appropriate therapeutic genes and vector vehicles must be selected and methods devised for efficient delivery and sustained expression. However, it is also necessary to minimize undesirable side-effects by accurately targeting therapeutic gene expression to diseased cells. To this end it is possible that regulatory promoter elements can be modulated to enhance therapeutic levels of gene expression whilst maintaining both disease and cell type specificity.

The construction of vectors enabling tissue-specific gene expression is one of the current challenges in the field of gene therapy. A number of transcriptionally based targeting strategies have been described which target specific cell types or are targeted through specific pathologies within the cell. However, correctly regulated expression may not only require promoter regions but also the distant 5' and 3' elements that influence tissue-specific promoter activity [1]. Tissue-specific regulatory elements have already been used to target gene expression to certain cell types. For example, using the transgenic mouse model for muscular dystrophy, the creatine kinase promoter has been used to restrict dystrophin cDNA expression to skeletal and cardiac muscles to correct the clinical signs of disease without deleterious side-effects [2]. The promoter of the immunoglobulin (Ig) heavy chain has also been used to direct tissue-specific expression of the diphtheria toxin A (DT-A) gene in lymphoid cells [3]. However, vector context is an important parameter when designing tissue-specific targeting systems. Although tissue-specific promoters frequently retain their specificity in the context of retroviral vectors [4] this is not always the case, and the design of the viral vectors may have significant effects on cell type specificity due to promoter interference [5].

Cell-type- or disease-specific targeting is important for systemic and local gene therapy. In OA, it is possible that the efficacy and safety of gene therapy can be improved by the use of cell-type-specific promoters keeping the expression of therapeutic genes in non-target cells to a minimum. In the joint, a number of candidate promoter systems exist which may be developed for transcriptional targeting. For example collagen types II, IX and XI are expressed in chondrocytes [6] and may be used to drive cell-type-specific gene expression in joints. The discovery of synovial-specific promoter elements such as hyaluronan (although the expression of this gene is not entirely tissue specific) may enable the development of targeting systems to accompany the advanced delivery systems already established for this cell type.

In this paper we capitalize on the up-regulation of matrix metalloproteinase 9 (MMP-9) promoter activity in pro-inflammatory environments to develop a transcriptional unit which is active in disease states. Further, we show that the incorporation of multiple NF-{kappa}B sites into the promoter allows for increased levels of expression, while retaining specificity. Subsequent studies, using the collagen type IX promoter, demonstrated that expression could be up-regulated through the manipulation of the downstream enhancer region. The ability of these units to transcriptionally target gene expression in both a disease-specific and cell-specific manner, suggests that a combination of these systems may be employed for optimum vector development.

Ethical approval was not required for this study.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines and reagents
Human chondrosarcoma cells (SW1353 cells) were maintained in Dulbecco's MEM/F-12 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 1x non-essential amino acids, 100 U/ml of penicillin and streptomycin, 20 U/ml nystatin. Undifferentiated chondroprogenitor cells (ATDC5) (a gift from Dr David Thomas, Kings Hospital, London) were cultured in a mix of Dulbecco's MEM/F-12 medium supplemented with 5% FCS 3 x 108 M sodium selenite, 10 mg/ml human apo-transferrin and 10 mg/ml insulin.

Relative semi-quantitative reverse transcription PCR
Reverse transcription PCR (RT-PCR) was used to assess the endogenous levels of MMP-9 and collagen type XI gene transcription in the SW1353 and ATDC5 cells. Densitometric analysis was not performed on RT-PCR samples as these studies were simply included to identify relevant cell lines for receptor gene assays and up-regulation was visually assessed. Briefly, SW1353 cells and ATDC5 cells were seeded onto six-well plates at a concentration of 5.5 x 104 and 5 x 104 cells/ml respectively and allowed to grow to confluence over 24 h. SW1353 cells and ATDC5 cells were serum-starved for 24 h prior to stimulation with interleukin-1ß (IL-1ß) (10 ng/ml) and tumour necrosis factor-{alpha} (TNF{alpha}) (10 ng/ml) (R&D Systems) for a further 24 h prior to harvesting. Total RNA was harvested from both untreated (basal) and treated (IL-1ß and TNF{alpha}) cells using RNAzolTM B solution (AMS Biotechnology) following the manufacturer's protocol and then treated with DNA-freeTM (Ambion) to remove contaminating DNA from the RNA preparation. First strand cDNA synthesis reactions were performed using 2 mg total RNA samples Molony murine leukaemia virus reverse transcriptase (MMLV-RT, GIBCO BRL), and random primers. PCR reactions using Ready-To-GoTM PCR beads (25 ml) (Amersham, Pharmacia), with cDNA samples (1 mg) as template, contained 0.4 mM of both sense (f) and antisense (r) primer pairs based on the human (hMMP-9f and hMMP-9r) and mouse (mMMP-9f and mMMP-9r) MMP-9 gene sequences and also on the human (hColXIf and hColXIr) and mouse (mColXIf and mColXIr) collagen type XI gene sequences. The constitutively expressed cyclophilin gene was used as an internal control for the human (NM_021130) and mouse (XM_125205) species. Species-specific primers were based on the human (hcyclophilinf and hcyclophilin) and mouse (mcyclophilinf and mcyclophilinr) genes to amplify a region of 265 base pairs (bp) from a cDNA template spanning an intron, to enable the identification of contaminating genomic DNA (gDNA) (450 bp fragment). Samples were subjected to an initial denaturation at 95°C for 5 min followed by a variable number of cycles for amplification, each cycle consisting of a denaturation step of 95°C for 1 min, an annealing temperature of 67°C followed by an elongation step of 72°C for 1 min. A final elongation step of 72°C for 10 min completed the reaction. Samples were removed at multiple (three to five) intervals five cycles apart to determine the exponential phase of the reaction where basal and treated samples could be semi-quantified and compared. All PCR products were analysed by TAE agarose gel (1%) electrophoresis.

Cloning of manipulated luciferase reporter constructs
Cloning of the canine MMP-9 promoter deletions 1984, 984, 628, 534 has been described previously [7]. Manipulation of the canine MMP-9, mouse collagen type XI promoters and intronic regions were performed by PCR using Ready-To-GoTM PCR beads (Amersham, Pharmacia) in a total volume of 2.5 ml containing 0.4 mM of sense (5NF-{kappa}Bf, 3NF-{kappa}Bf, 1NF-{kappa}Bf, mColXIpromf, ColXIIntronf and 3SOX9intronf) and antisense (cMMP-9r, mColXIpromr, ColXIIntronr and 3SOX9intronr) primer pairs (Table 1). Using a Perkin Elmer (PE) 2400 samples were subjected to an initial denaturation at 95°C for 5 min followed by 30 cycles of amplification, each consisting of a denaturation step of 95°C for 1 min, an annealing temperature of 66°C for 1 min and an elongation step of 72°C for 2 min. A final extension step of 72°C for 30 min completed the reaction. PCR products were cloned into the pCR®2.1-TOPO plasmid (Invitrogen) using the manufacturer's instructions and sequenced. All manipulated promoters were subcloned from the pCR2.1 vectors using the KpnI and EcoRV restriction sites into the luciferase reporter vector pGL3-Basic (Promega) pre-digested with KpnI and SmaI restriction enzymes. The intronic, fragment-containing flanking SOX9 sites, were subcloned into the downstream enhancer site of the pGL3-Basic vector using the BamHI restriction enzyme site. The NF-{kappa}B site was mutated from the canine MMP-9 promoter deletion (628) vector using the in vitro QuikChangeTM site-directed mutagenesis protocol (Stratgene) following the manufacturer's guidelines with sense (NF-{kappa}Bmutnf) and antisense (NF-{kappa}Bmutnr) primers (Table 1).


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TABLE 1. DNA sequence of primers used throughout the experimental procedures. This table can be viewed in colour as supplementary material at Rheumatology Online

 
Expression of luciferase reporter vectors
White, tissue culture treated ViewPlateTM-96 (Packard) were seeded with SW1353 cells, and ATDC5 cells at concentrations of 5.5 x 104 cells/ml and 5 x 104 cells/ml respectively and incubated overnight at 37°C in 5% CO2. Transient transfections were carried out using TransFastTM Reagent (Promega) at a 1:1 ratio with DNA (50 ng per well) according to the manufacturer's instructions. Cells were serum-starved for 24 h prior to stimulation with IL-1ß (10 ng/ml), and TNF{alpha} (10 ng/ml) (R&D Systems) for a further 24 h prior to harvesting the cells. Dual-Luciferase® Reporter Assays were performed 72 h post-transfection, according to the manufacturer's protocol (Promega).

Statistical analysis
The Mann–Whitney statistical analysis was performed on all luciferase data using Minitab software to determine levels of statistical significance where P<0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Characterization of the canine pGL3/cMMP-9 promoter deletions, NF-{kappa}B mutation and multiple NF-{kappa}B sites in human chondrosarcoma cells (SW1353)
Endogenous transcription of MMP-9 in SW1353 cells
To analyse the basal and cytokine induced IL-1ß and TNF{alpha} levels of MMP-9 gene transcription in the human chondrosarcoma cell line (SW1353) relative semi-quantitative RT-PCR was performed. A portion of the gene was amplified from cDNA samples prepared from untreated (basal) and treated (IL-1ß and TNF{alpha}) cells using species-specific primer pairs. Samples were removed at three intervals, 25, 30 and 35 cycles, to enable the semi-quantification of MMP-9 gene transcription over the exponential phase of the PCR. A portion of the human cyclophilin gene was also amplified (255 bp), using species-specific primer pairs, as an internal control. The primers were designed to span an intron and control for the presence of contaminating genomic DNA (absence of a 474 bp PCR product). Basal transcription of the endogenous MMP-9 gene was evident in the human chondrosarcoma cells and could be up-regulated by cytokines, IL-1ß and TNF{alpha} (Fig. 1). Subsequently this cell line was used to analyse all of the cloned MMP-9 promoter constructs.



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FIG. 1. Endogenous levels of MMP-9 transcription in SW1353 cells using relative semi-quantitative RT-PCR. A portion (364 bp) of the human MMP-9 gene (upper band) was amplified from cDNA samples prepared from total RNA isolated from human chondrosarcoma cells (SW1353). During PCR amplification, samples were removed within the exponential phase of the reaction (25 to 35 cycles) revealing that basal levels of MMP-9 transcription were significantly up-regulated by the pro-inflammatory cytokines IL-1ß and TNF{alpha}.

 
Analysis of canine MMP-9 promoter constructs using Dual-Luciferase® Reporter Assays
All Dual-Luciferase® assays were conducted in triplicate for statistical significance and to ensure reproducibility, and all transfections were carried out three times. To account for differences in transfection efficiency the cells were co-transfected with Renilla luciferase vector (Promega) and the firefly luciferase values were adjusted accordingly. Renilla was expressed and shown to be active in the cell type used. The corrected luciferase activity of each construct represents the mean ± S.E.M. (n = 3). The canine MMP-9 deletion reporter constructs, together with the promoter-less luciferase vector pGL3-Basic vector as the negative control, were transiently transfected into human chondrosarcoma cells (SW1353). Basal luciferase activities for each promoter were compared with treated samples (IL-1ß and TNF{alpha}). The Mann–Whitney statistical analysis was performed on all assays.

Basal activities of all pGL3/cMMP-9 deletion constructs were evident in the SW1353 cell line. All constructs MMP-9(1894), (984), (628) and (534) could be significantly induced by TNF{alpha} to varying degrees (*, P<0.05). Although some induction was also observed with IL-1ß the results were not significant. The general trend of promoter activity showed that the smallest construct (534) had significantly less activity than the three largest constructs MMP-9(1894), (984) and (628) at both basal and induced (IL-1ß and TNF{alpha}) levels (•, P<0.05) (Fig. 2).




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FIG. 2. (a) Schematic representation of MMP-9 deletion constructs. (b) Deletion and mutational analysis of the canine MMP-9 promoter. Dual-Luciferase® Reporter Assays were used to determine the activity of the cloned canine MMP-9 promoter deletion MMP-9(1894), (984), (628), (534) and mutation (MMP-9NF-{kappa}Bmutn) constructs in the human chondrosarcoma (SW1353) cells. Basal activities of the canine MMP-9 promoter constructs were significantly enhanced by TNF{alpha} (*, P<0.05 Mann–Whitney test), but not IL-1ß. Basal and induced (TNF{alpha} and IL-1ß) activities of the MMP-9(534) construct were significantly lower than the other MMP-9 promoter deletion constructs (•, P<0.05). Basal and induced (TNF{alpha} and IL-1ß) activities of the MMP-9(NF-{kappa}Bmutn) construct were significantly lower than the other MMP-9(638) promoter ({triangleup}, P<0.05). Basal and induced (TNF{alpha}) activities of the MMP-9(mutn) construct were also significantly lower than the other MMP-9(534) promoter ({square}, P<0.05). This figure can be viewed in colour as supplementary material at Rheumatology Online.

 
Basal activity of pGL3/cMMP-9(628)(NF-{kappa}Bmutn) construct was evident in the SW1353 cell line which could be significantly enhanced by TNF{alpha} (*, P<0.05), and although induction with IL-1ß was evident this again was not significant. Basal and induced (TNF{alpha} and IL-1ß) activity of the mutated construct was significantly lower than the MMP-9(628) promoter construct ({triangleup}, P<0.05). Basal and induced (TNF{alpha}) was also significantly lower than the MMP-9(534) construct ({square}, P<0.05) (Fig. 2).

The canine MMP-9 promoter constructs, manipulated with additional NF-{kappa}B sites, were directly compared with the non-manipulated MMP-9(1894) promoter construct (Fig. 3). The general trend of promoter activity showed that the addition of five NF-{kappa}B sites increased basal and cytokine-induced activity the most, followed by the addition of three NF-{kappa}B sites while the addition of one alone had little effect. More specifically, basal activities of the canine MMP-9 promoter constructs cMMP-9(1894), (5NF-{kappa}B) and (3NF-{kappa}B) were significantly enhanced by TNF{alpha} (*, P<0.05 Mann–Whitney test). In comparison, the MMP-9(1984) and (three NF-{kappa}B) were significantly enhanced by IL-1ß (•, P<0.05). The addition of five NF-{kappa}B sites significantly enhanced basal and induced (TNF{alpha} and IL-1ß) promoter activity ({triangleup}, P<0.05) in comparison with the non-manipulated cMMP-9(1894) promoter construct. The addition of three NF-{kappa}B sites only significantly enhanced TNF{alpha} induced activity ({square}, P<0.05) compared with the non-manipulated MMP-9(1894) promoter construct. The addition of one NF-{kappa}B site had no effect on increasing promoter activity at the basal or induced levels.



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FIG. 3. Manipulation of the canine MMP-9 promoter fragment with multiple NF-{kappa}B sites. Dual-Luciferase® Reporter Assays were used to determine the activity of the cloned canine MMP-9 promoter, MMP-9(1894) and manipulated MMP-9(5NF-{kappa}B) (3NF-{kappa}B) (1NF-{kappa}B) constructs in the human chondrosarcoma (SW1353) cells. Basal activities of the canine MMP-9 promoter constructs MMP-9(1894), (5NF-{kappa}B) and (3NF-{kappa}B) were significantly enhanced by TNF{alpha} (*, P<0.05 Mann–Whitney test). In comparison, the MMP-9(1984) and (3NF-{kappa}B) were significantly enhanced by IL-1ß (•, P<0.05). The addition of five NF-{kappa}B sites significantly enhanced basal and induced (TNF{alpha} and IL-1ß) promoter activity ({triangleup}, P<0.05) over the unmanipulated MMP-9(1894) promoter construct. The addition of three NF-{kappa}B sites only significantly enhanced TNF{alpha}-induced activity ({square}, P<0.05) over the unmanipulated MMP-9(1894) promoter construct. The addition of one NF-{kappa}B site had no effect on increasing promoter activity at the basal or induced levels. This figure can be viewed in colour as supplementary material at Rheumatology Online.

 
Characterization of mouse collagen type XI promoter and intronic constructs manipulated with SOX9 sites in SW1353 and ATDC5 cells
Endogenous transcription of the collagen type XI genes in SW1353 cells and ATDC5 cells
To analyse endogenous activity of collagen type XI transcription in the SW1353 and ATDC5 cell lines to be used for the transfection of the mouse collagen type XI promoter fragment, relative semi-quantitative RT-PCR was once again performed. A portion of each gene was amplified from cDNA samples prepared from untreated (basal) and treated (IL-1ß and TNF{alpha}) cells using species-specific primer pairs. Samples were removed at three intervals, three cycles apart, to enable the semi-quantification of collagen type XI gene transcription over the exponential phase of the PCR. A portion of the cyclophilin gene was also amplified (255 bp) using species-specific primers as an internal control. The primers were designed to span an intron and control for the presence of contaminating genomic DNA (absence of a 474 bp PCR product). Basal levels of endogenous transcription were present in both the SW1353 cells (Fig. 4) and the ATDC5 cells (Fig. 5), neither of which could be up-regulated by IL-1 and TNF. These cell lines could therefore be used to analyse the mouse collagen type XI promoter fragments.



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FIG. 4. Endogenous levels of collagen type XI (mColXI) transcription in SW1353 cells using relative semi-quantitative RT-PCR. A portion (395 bp) of the human collagen type XI gene (upper band) was amplified from cDNA samples prepared from total RNA isolated from human chondrosarcoma cells (SW1353). During PCR amplification, samples were removed within the exponential phase of the reaction (30 to 40 cycles) revealing that basal levels of collagen type XI transcription were present which were not up-regulated by the pro-inflammatory cytokines IL-1ß and TNF{alpha}.

 


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FIG. 5. Endogenous levels of collagen type XI (Col XI) transcription in ATDC5 cells using relative semi-quantitative RT-PCR. A portion (395 bp) of the mouse collagen type XI gene (upper band) was amplified from cDNA samples prepared from total RNA isolated from undifferentiated chondrosarcoma cells (ATDC5). During PCR amplification samples were removed within the exponential phase of the reaction (25–35 cycles) revealing that basal levels of collagen type XI transcription were present which were not up-regulated by the pro-inflammatory cytokines IL-1ß and TNF{alpha}.

 
Analysis of mouse collagen type XI promoter and intronic sequences manipulated with SOX9 sites using Dual-Luciferase® Reporter Assays
Dual-Luciferase® Reporter Assays were conducted in triplicate as described for the various MMP-9 promoter constructs using the Renilla luciferase vector as the internal control. Renilla was expressed and shown to be active in the cell types used. Representative Renilla values for the SW1353 and ATDC5 cells were 322 and 225 respectively. The different mouse collagen type XI promoter ± intron luciferase reporter constructs were transiently transfected into human chondrosarcoma cells (SW1353) and undifferentiated chondrosarcoma cells (ATDC5). Basal luciferase activities of these constructs were directly compared along with a promoter-less luciferase vector (pGL3-Basic) as a negative control. The Mann–Whitney statistical test was used to analyse the results.

Activities of the cloned mouse collagen type XI promoter and manipulated (3SOX9) and (5SOX9) promoter constructs were significantly higher than the negative control in both the SW1353 (*, P<0.05) and ATDC5 (•, P<0.05) cell lines. However, the activities of the mouse collagen type XI promoter and the two manipulated promoter constructs mColXI(3SOX9) and (5SOX9) were significantly higher in the SW1353 cells than the ATDC5 cells ({triangleup}, P<0.05). Nevertheless, the addition of these SOX9 sites did not enhance activity in either cell line (Fig. 6).



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FIG. 6. Analysis of the mouse collagen type XI promoter ± 3 and 5 SOX9 sites. Dual-Luciferase® Reporter Assays were used to determine the activity of the cloned mouse collagen type XI promoter with additional three and five SOX9 sites in the human chondrosarcoma cells (SW1353) and undifferentiated chondroprogenitor cells (ATDC5). Activities of cloned mouse collagen type XI promoter and manipulated promoters (3SOX9; 5SOX9) were significantly higher than the negative control in both the SW1353 (*, P<0.05) and ATDC5 (•, P<0.05) cells (P<0.05 using the Mann–Whitney test). Activity of the three promoter constructs was also significantly higher in the SW1353 cells than the ATDC5 cells ({triangleup}, P<0.05); however, the addition of the three and five SOX9 sites did not significantly enhance activity in either cell line. This figure can be viewed in colour as supplementary material at Rheumatology Online.

 
Activities of cloned mouse collagen type XI promoter with and without the additional intronic sequence flanked by three SOX9 sites were significantly higher than the negative control in both the SW1353 (*, P<0.05) and ATDC5 (•, P<0.05) cells. Activity of the promoter ± the intronic sequence was also significantly higher in the SW1353 cells than the ATDC5 cells ({triangleup}, P<0.05). Furthermore, the addition of the intronic sequence containing flanking SOX9 sites significantly enhanced promoter activity in the SW1353 cell line ({square}, P<0.05) (Fig. 7).



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FIG. 7. Analysis of the mouse collagen type XI promoter with intronic SOX9 sites. Dual-Luciferase® Reporter Assays were used to determine the activity of the cloned mouse collagen type XI promoter with and without the additional intronic sequence containing three flanking SOX9 sites in the human chondrosarcoma cells (SW1353) and undifferentiated chondroprogenitor cells (ATDC5). Activities of cloned mouse collagen type XI promoter with and without the additional intronic sequence were significantly higher than the negative control in both the SW1353 (*, P<0.05 using the Mann–Whitney test) and ATDC5 (•, P<0.05) cells. Activity of these two constructs was also significantly higher in the SW1353 cells than the ATDC5 cells ({triangleup}, P<0.05). Furthermore, the addition of the intronic sequence containing flanking SOX9 sites significantly enhanced promoter activity in the SW1353 cells line ({square}, P<0.05). This figure can be viewed in colour as supplementary material at Rheumatology Online.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ultimate success of any gene-based therapy strategy will rely on the efficiency and duration of transcription, directed by promoter DNA sequences and associated regulatory elements, to provide adequate levels of transgene expression. Manipulation of these regulatory sequences may enable the rate of transcription initiation, crucial in the achievement of modulated therapeutic levels of transgene expression, to be obtained in vivo. The modular nature of the promoter region allows a considerable degree of flexibility when deciding on the design of the promoter/enhancer elements to be used to control gene expression using artificial transcriptional units. For example, enhancers isolated from one regulatory region can be incorporated into a promoter with which they are not necessarily associated to produce an optimized, manipulated promoter for the regulated control of therapeutic gene expression. Many different methods of targeting gene expression with promoter manipulation have been investigated.

Some eukaryotic promoters are ideally suited for gene therapy since they combine strong transcriptional activity with a high degree of specificity; this is true for the melanocyte-specific tyrosinase promoter [8]. However, often the application of highly specific promoters results in inefficient levels of transcriptional activity. A typical example is the von Willebrand factor (vWF) promoter which is highly specific for endothelial cells but is a poor activator of transcription [9]. Subsequently, different methods for enhancing the transcriptional activity of eukaryotic promoters, while maintaining their specificity, have been developed with the incorporation of transcriptional control elements into promoter regions [10–12]. Furthermore, it may also be desirable to switch gene expression on or off with the use of regulatable promoters [13, 14].

The aim of this current study was to investigate the MMP gene promoter and chondrocyte-specific promoter to target therapeutic gene expression to the canine OA joint. We used a series of reporter experiments to answer the following questions.

Can the canine MMP-9 promoter be manipulated to enhance activity while maintaining disease-specificity?
Basal and cytokine-stimulated endogenous levels of MMP-9 transcription were examined in the human chondrosarcoma cell line (SW1353) using relative semi-quantitative RT-PCR to investigate the potential for analysing the numerous canine MMP-9 promoter/luciferase constructs in this cell line. The SW1353 cell line was selected since it serves as an appropriate model for primary chondrocytes in OA [15]. The levels of MMP-9 transcription were up-regulated by inflammatory cytokines IL-1ß and TNF{alpha} which complements previous studies showing MMP-9 up-regulated by IL-1ß, Phorbol Myristate Acetate (PMA) [16] and TNF{alpha} [17] in human chondrosarcoma cell lines. The TNFR2, RasMAPK, P13K-Akt and PKC cascades are reported to be involved in TNF{alpha}-dependent MMP secretion [18]. TNF{alpha} did not up-regulate the collagen type XI promoter in our system.

The MMP-9 promoter deletion constructs were transiently transfected into the SW1353 cells in an attempt to identify promoter elements involved in both basal and pro-inflammatory (IL-1ß and TNF{alpha}) induced gene transcription. All of the MMP-9 promoter deletion constructs cMMP-9(1894), (984), (628), (534) demonstrated basal activity that could be significantly enhanced by the addition of TNF{alpha}, and some induction was observed with IL-1ß. This suggested that the distal AP-1 sites (–73 to –67; –111 to 105), and other promoter elements adjacent to the TATA box, were sufficient for up-regulating gene expression in response to both cytokines. This result complemented earlier experiments, where it was demonstrated that basal activity of canine MMP-9 deletion constructs could be significantly enhanced by PMA [7]. However, the actual levels of promoter activity were much greater in the three largest constructs pGL3/cMMP-9(1984), (984) (628) in comparison with the smallest vector pGL3/cMMP-9(534), suggesting that additional cytokine-responsive elements, located upstream of position 534 bp, may cooperate with the distal promoter elements. This may be explained by the presence of a conserved NF-{kappa}B DNA binding domain (–554 to –545) that is able to cross-couple with the AP-1 site acting synergistically to increase promoter activity in response to pro-inflammatory cytokines [19]. To investigate this further the NF-{kappa}B binding site was mutated from the pGL3/cMMP-9(628) construct.

The MMP-9 promoter construct containing the mutated NF-{kappa}B site (cMMP-9/NF-{kappa}Bmutn) was also transfected into SW1353 cells and shown to have similar levels of activity to the pGL3/cMMP-9(534) reporter construct. Again basal levels of activity could be significantly enhanced by TNF{alpha} with some increase in response to IL-1ß, suggesting that the NF-{kappa}B was not essential for cytokine induction and that other elements were also important. However, actual levels of promoter activity were greatly reduced suggesting that the NF-{kappa}B site was probably important for cooperating with the AP-1 and other sites for both TNF and IL-1ß induction. These results are similar to other studies showing the importance of the NF-{kappa}B site in the mouse MMP-9 promoter [20] and that the human and rabbit MMP-9 promoters both require the synergistic action of NF-{kappa}B and AP-1 sites for cytokine induction with TNF and IL-1 and growth factors [21]. Furthermore, site-directed mutagenesis of the NF-{kappa}B site has been shown to decrease the TNF{alpha} induced activity of the human E-selectin promoter [22] and IL-1-induced activity of the human MCP-1 [23] and COX-2 [24] promoters.

Further to suggestions that multiple NF-{kappa}B sites within the inducible nitric oxide synthase gene may cooperate to confer inducibility to both IL-1ß and TNF{alpha} [25] we incorporated multiple (five, three and one) NF-{kappa}B sites into the 5' end of the promoter to enhance the response to cytokines. Activities of the manipulated MMP-9 promoter constructs, pGL3/cMMP-9(5NF-{kappa}B), (3NF-{kappa}B) and (1NF-{kappa}B), were analysed using the SW1353 cells. The addition of five NF-{kappa}B sites significantly increased both basal and cytokine (IL-1ß and TNF{alpha}) induced promoter activity, suggesting that disease specificity had been lost. The addition of one NF-{kappa}B site did not increase promoter activity at the basal or induced levels; in fact promoter activity appeared to have decreased—the reasons for this are not apparent and further studies need to be performed. The addition of three NF-{kappa}B sites appeared to be optimal with a significant increase in promoter activity in response to TNF{alpha} and IL-1ß while basal levels remained unaffected, indicating that disease specificity had been maintained. The differences observed in promoter activity observed after the addition of five, three and one NF-{kappa}B sites into the 5' end of the MMP-9 promoter is difficult to explain but suggests complex transcriptional changes occurring that are closely associated with the number of additional sites. It may also be explained by the presence of the AP-1 site (–1888 to –1877) located 6 bp downstream of the 5' end of the MMP-9 promoter. The binding of the NF-{kappa}B transcription factor to one extra NF-{kappa}B site may interfere with the binding of transcription factors to this proximal AP-1 site required for promoter activity. However, the addition of three and five extra NF-{kappa}B sites may enable the NF-{kappa}B transcription factor to bind further up stream without interfering with the AP-1 binding. In addition, there may be conformational changes in the DNA that are dependent on the number and type of motifs present within the sequence which not only alters the efficiency of the transcription factor binding but may also directly influence other sites present in the adjacent sequence. This may enhance transcription in the case of five and three additional sites or repress activity in the presence of just one site. Finally, there is an obvious discrepancy in the IL-1 response between experiments; this may be explained by the slight variations between batches of IL-1ß bought from the manufacturer and the stability of the recombinant protein after a number of freeze–thaw cycles.

Can the mouse collagen type XI promoter be manipulated to enhance activity while maintaining cell type specificity?
Endogenous levels of collagen type XI gene transcription in the two cell lines, SW1353 and ATDC5, were analysed using relative semi-quantitative RT-PCR. These cell lines were chosen since chondrosarcoma cells had previously been shown to express the collagen type XI gene whilst the undifferentiated ATDC5 cells have not [26].

Basal levels of endogenous transcription were evident in the differentiated SW1353 cells as expected. However, the level of basal transcription could not be enhanced by the addition of TNF{alpha} or IL-1ß. In contrast, studies have shown that TNF{alpha} can up-regulate collagen gene expression such as the collagen type II in chondrosarcoma cells [17], while other groups have reported a TNF-induced decrease in the synthesis of the components of the extracellular matrix (ECM) [27]. More importantly, it was clear that the undifferentiated cells (ATDC5) were also capable of collagen type XI transcription, contradicting reports that collagen type XI was not expressed in this cell line [26]. Since these authors had not analysed the endogenous expression of the gene in the ATDC5 cells but had shown insignificant levels of collagen XI promoter gene expression from a transfected construct it was decided to continue with this cell line using the cloned collagen type XI promoter constructs. It was hoped that there would be a large enough difference in the degree of gene expression between the SW1353 and ATDC5 cell lines to enable the cell-type-specific analysis of the mouse collagen type XI promoter fragments.

The mouse collagen type XI promoter construct was transiently transfected into the SW1353 and ATDC5 cells in an attempt to show cell-type specificity. Basal levels of promoter activity were normalized with the negative control for each cell line and then directly compared. Activity of the collagen type XI promoter construct was evident in both SW1353 and ATDC5 cell types, which is in concordance with the endogenous data; however, the promoter had significantly more activity in the human SW1353 cells. This suggested that the collagen type XI promoter contained cell-type-specific binding elements within its sequence enabling higher levels of promoter activity in the differentiated chondrocytes. These results are in agreement with a series of transfection studies demonstrating that the collagen type XI promoter had comparable activity in similar cell lines [26]. However, DNA sequences containing minimal promoter elements may serve as a promoter if placed upstream of a gene containing all the necessary elements required for transcription. In gDNA, cell type specificity is not only determined by the primary structure of a gene, such as the promoter and enhancer sequences, but also by the secondary and tertiary structural elements in the region of that gene. The three-dimensional folded arrangement of the DNA in combination with the presence of histone acetylation and DNA methylation [28] maintains cell type specificity of a promoter by regulating the binding of transcription factors and thus to the recruitment of RNA polymerase to the transcription initiation site. As such it is very difficult to attain a high level of cell-type-specific regulation when evaluating the level of gene transcription from a promoter positioned in a plasmid vector which contains minimal secondary and tertiary structure. In this situation, the promoter under analysis is easily accessible to the transcriptional machinery of the cell and therefore will not necessarily reflect the action of the promoter in vivo.

The size of the promoter fragment under analysis is also important for cell type specificity. In this study a relatively small portion of the mouse collagen type XI promoter (1.2 kb) was analysed. Although studies have shown this region to contain cartilage-specific elements [29], such elements are often located hundreds of kilobases upstream of the transcription start site. For example an upstream repressor site in the mouse MMP-9 promoter has been identified and shown to regulate basal levels of promoter activity in a tissue-specific manner [30]. However, until vector technology advances, for example with the development of mammalian artificial chromosomes which can deliver the entire genomic fragment containing the promoter and other elements, it is necessary to try and design the best cell-type-targeting vector possible within the constraints of available DNA plasmid vectors. To this end, the collagen type XI promoter sequence was manipulated with chondrocyte-specific enhancers (SOX9 sites), in an attempt to incorporate enhanced promoter activity and maintain cell type specificity.

The first attempt to modify the collagen type XI promoter involved the introduction of three and five SOX9 sites into the 5' end of the promoter sequence as described for the manipulation of the canine MMP-9 promoter with NF-{kappa}B sites. The two constructs pGL3/mColXI(3SOX9) and (5SOX9) were transiently transfected into the SW1353 and ATDC5 cell lines as described before and compared with the non-manipulated pGL3/mCOLXI construct. Although activities of the two manipulated promoter constructs were again significantly higher in the SW1353 cells compared with the ATDC5 cells the addition of SOX9 sites did not enhance activity in either cell line. This contradicted evidence that SOX9 proteins bind to the collagen type XI gene to up-regulate promoter activity in chondrocytes [31]. However, further studies to investigate the role of SOX9 in cartilage specific expression identified enhancer sites in the first intron of the collagen type XI gene [26]. This suggested that SOX9 sites downstream of the transcription start site were important for enhancing chondrocyte-specific expression of the gene. As a result it was decided to clone a 300 bp region of the mouse collagen type XI intron 1, containing two SOX9 sites within its sequence, and flanking it with a further six SOX9 sites. The manipulated intron was then cloned downstream of the luciferase reporter gene into the pGL3/mCOLXI vector luciferase reporter construct. The modified vector, pGL3/ mColXI[3SOX9(intron)3SOX9], was transiently transfected into the SW1353 and ATDC5 cell lines and compared with the non-manipulated mColXI construct. Activity of the modified construct was again significantly higher in the SW1353 cells compared with the ATDC5 cells. However, this time the incorporation of the intronic sequence, containing flanking SOX9 sites, into the promoter sequence significantly enhanced the mouse collagen type XI promoter activity in the SW1353 cell line while maintaining low levels in the ATDC5 cells. This is in agreement with studies showing that SOX9 expression is considerably higher in chondrosarcoma cells than undifferentiated ATDC5 cells [32].

In summary, to enhance gene expression while maintaining disease specificity, the canine MMP-9 promoter was manipulated with multiple NF-{kappa}B sites and the mouse collagen type XI promoter with an intronic region flanked by SOX9 enhancer sites. These systems demonstrate proof of principle for this approach. Work is currently in progress to combine these systems to develop a dual-targeted vector to be specifically used in the canine model. The clinical application of these gene therapy approaches to experimental and natural OA in the dog will have profound implications for similar studies in humans from both a biomechanical and biological perspective [33–36].


    Acknowledgments
 
All in vitro studies were funded by BSAVA Petsavers. The authors thank Professor Mike Steer for help with statistical analysis.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 10 November 2004; revised version accepted 28 January 2005.



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