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Development of a high throughput luciferase reporter gene system for screening activators and repressors of human collagen I␣2 gene expression1 Rushita A. Bagchi, Viktoriya Mozolevska, Bernard Abrenica, and Michael P. Czubryt
Abstract: Fibrosis, which is characterized by the excessive production of matrix proteins, occurs in multiple tissues and is associated with increased morbidity and mortality. Despite its significant negative impact on patient outcomes, therapies targeted to treat fibrosis are currently lacking. Screening for inhibitors of the expression of collagen, the primary component of fibrotic lesions, represents an option for the identification of novel lead compounds for therapeutic development with potentially fewer off-target effects compared with the targeting of multifunctional cell signaling pathways. Here we report on the generation of a stable luciferase reporter system using a fibroblast cell line, which can be used for rapidly screening both activators and repressors of human collagen COL1A2 gene transcription in a high throughput setting. This in vitro screening tool was validated using known agonists (scleraxis, TGF-, angiotensin II, CTGF) and antagonists (TNF-␣, pirfenidone) of COL1A2 gene expression. The COL1A2-luc NIH-3T3 fibroblast system provides a useful and effective screen for potential lead compounds with pro- or anti-fibrotic properties. Key words: collagen, luciferase reporter, stable cell line, in vitro screening assay, fibrosis, high-throughput assay, fibroblast. Résumé : La fibrose, caractérisée par une production excessive de protéines de la matrice extracellulaire, survient dans une multitude de tissus, et elle est associée avec un accroissement de morbidité et de mortalité a` l’échelle mondiale. Malgré son impact négatif significatif sur le patient, des thérapies visant a` traiter la fibrose font actuellement défaut. Le criblage d’inhibiteurs de l’expression du collagène, la première composante des lésions fibreuses, constitue une option afin d’identifier de nouveaux médicaments têtes de série en vue d’un développement thérapeutique qui présenteraient potentiellement moins d’effets hors cible, comparativement a` cibler des voies de signalisation cellulaire multifonctionnelles. Les auteurs rapportent ici la production d’un système rapporteur a` la luciférase stable, a` l’aide d’une lignée de fibroblastes qui peut être utilisée pour cribler rapidement tant les activateurs que les répresseurs de la transcription du gène COL1A2 codant le collagène humain, dans un procédé a` haut débit. Cet outil de criblage in vitro a été validé a` l’aide d’agonistes (scleraxis, TGF-, angiotensine II, CTGF) et d’antagonistes (TNF-␣, pirfenidone) connus de l’expression génique de COL1A2. Le système COL1A2-luciférase/fibroblastes NIH-3T3 constitue un outil de criblage utile et efficace afin d’identifier des composés têtes de série potentiels, possédant des propriétés pro- et anti-fibrogènes. [Traduit par la Rédaction] Mots-clés : collagène, rapporteur luciférase, lignée cellulaire stable, test de criblage in vitro, fibrose, test a` haut-débit, fibroblaste.
Introduction The extracellular matrix (ECM) is a complex and dynamic structure that provides cells and organs with mechanical and functional support. This compartment of our body’s architecture is primarily composed of collagens (Karsenty and Park 1995; Di Lullo et al. 2002). Defects in collagen structure or expression are associated with multiple human genetic disorders such as Ehlers Danlos Syndrome and osteogenesis imperfecta (Plopper 2007). Increased synthesis of type I collagen, resulting in ECM build-up, is the hallmark of fibrotic disease (Fan et al. 2012; Roche and Czubryt 2014). Fibrosis can present as a multisystem disorder or as a tissuespecific disease affecting the lungs, heart, kidney, liver, and other tissues (Schnaper and Kopp 2003; Bataller and Brenner 2005; Noble 2006; Krenning et al. 2010). Cardiac fibrosis is quite similar to other forms of tissue fibrosis such as those affecting the lungs,
liver and kidneys, suggesting that common mechanisms may underlie fibrosis in multiple tissues (Weber 1997). Despite being a significant contributor to patient morbidity and mortality, specific therapies directly targeting fibrotic disease are almost completely lacking. Fibroblasts are considered to be the principal determinants of tissue fibrosis. These cells are primarily involved in matrix synthesis and turnover in healthy tissues. However, activation of these cells with pro-fibrotic agonists results in excessive production of fibrotic matrix and impairment of organ function. The commercially available immortalized fibroblast NIH-3T3 cell line provides a useful tool for assessment of fibroblast function in a reproducible manner. These cells synthesize collagens and other ECM components and respond to fibrotic agents such as TGF- and thus serve as a convenient surrogate for primary cells (Bagchi and Czubryt 2012).
Received 12 December 2014. Accepted 21 January 2015. R.A. Bagchi and M.P. Czubryt. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada; Department of Physiology and Pathophysiology, University of Manitoba, St. Boniface Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada. V. Mozolevska and B. Abrenica. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada. Corresponding author: Michael P. Czubryt (e-mail: [email protected]). 1This article is part of a Special Issue entitled “2nd Cardiovascular Forum for Promoting Centers of Excellence and Young Investigators.” Can. J. Physiol. Pharmacol. 93: 1–6 (2015) dx.doi.org/10.1139/cjpp-2014-0521
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In this study, we used the NIH-3T3 cell line to derive a stable luciferase reporter system for detecting the response of the human collagen I␣2 (COL1A2) gene promoter to inducers and repressors of fibrosis in a high-throughput manner. In a 96-well in vitro screening assay format, we confirmed that known activators and repressors of COL1A2 gene transcription significantly increased or decreased, respectively, luciferase expression assayed in an automated reader. Our results demonstrated that this screen can be easily employed to rapidly obtain tightly clustered data with high reproducibility. This study provides a proof-of-principle for the utility of this cell line in first-pass compound library screening for identification of pro- and anti-fibrotic agents.
Can. J. Physiol. Pharmacol. Vol. 93, 2015
Fig. 1. Schematic diagram illustrating the luciferase-based reporter gene assay development and workflow.
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Materials and methods Subcloning of the human COL1A2 proximal promoter The human COL1A2 proximal promoter luciferase parent vector (pGL3-COL1A2) has been previously described (Bagchi and Czubryt 2012). This vector was digested with BsgI to excise the promoterluciferase fragment intact, and the ends were blunted using Klenow DNA polymerase (New England Biolabs). This fragment was further digested using XbaI to yield the COL1A2.luc insert, which was gel purified. pcDNA3.1 vector was subjected to restriction enzyme digestion with EcoRV and XbaI followed by gel purification. The COL1A2.luc insert was ligated to pcDNA3.1 vector at 16 °C overnight using T4 DNA ligase (Life Technologies). The ligation reaction product was used to transform competent bacterial cells (Bioline) and plasmids were isolated and purified using standard methods. The construct nucleotide sequence was verified at the DNA Sequencing Core (Manitoba Institute of Cell Biology). This plasmid was used for generation of a NIH-3T3-derived stable reporter cell line (Fig. 1). Generation of a stable COL1A2 reporter system The pcDNA3.1-COL1A2.luc plasmid construct containing a neomycin selection marker was stably transfected into NIH-3T3 fibroblasts (ATCC CRL-1658) using Lipofectamine 3000 reagent (Life Technologies) for 48 h. Transfected cells were then selected using G418 antibiotic (800 g/mL; Enzo Life Sciences) for 2–3 weeks. Individual antibiotic-resistant clones were isolated, subcultured, and maintained in 10% fetal bovine serum-supplemented Dulbecco’s modified essential medium at 37 °C with 5% CO2 in air. Cells were subjected to G418 selection (400 g/mL) every 2–3 passages to maintain purity. Transient transfections COL1A2-luc NIH-3T3 cells were seeded at 4 × 103 cells per well in 100 L growth medium in 96-well sterile tissue culture plates (Corning Life Sciences). Cells were transfected (50 ng/well) with the following expression vectors after overnight recovery: scleraxis (ScxWT), DNA binding domain scleraxis mutant (Scx⌬BD), or scleraxis mutant lacking both DNA-binding and protein interaction domains (Scx⌬⌬) (Espira et al. 2009; Bagchi and Czubryt 2012). pECE plasmid was used for control transfections, whereas pRL (1.0 ng/well) was used as internal control for all experiments. Lipofectamine 3000 reagent and OptiMEM (Life Technologies) were used for all transfections. After 24 h, cells were rinsed once with phosphate-buffered saline (PBS) and lysed to assay for luciferase activity. Treatment of stable COL1A2-luciferase reporter cells with pro- and anti-fibrotic compounds Cells were seeded at 4 × 103 cells per well in 96-well microtiter plates (Corning Life Sciences) in growth medium. Cells were briefly equilibrated in serum-free media prior to the administration of compounds. TGF-1 (1.0, 5.0, 10.0, and 20.0 ng/mL, Peprotech), angiotensin II (1.0, 5.0, 10.0, and 20.0 nmol/L; Sigma Aldrich) and CTGF (10.0, 50.0, 100.0, and 200.0 ng/mL; Life Technologies) were used as pro-fibrotic agents. Pirfenidone (0.5, 1.0, 5.0, and
Selection of positive clones with G418
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10.0 mmol/L; Sigma–Aldrich) and TNF-␣ (1.0, 5.0, 10.0, and 20.0 ng/mL; R&D Systems) were used to examine their antagonist activity on Col1␣2 gene promoter activation. An equal volume of vehicle (water or DMSO) was added to the control wells. After 24 h of treatment, cells were lysed and luciferase activity was assayed. Analysis of luciferase reporter gene activity Stable reporter-transformed cells, transfected with expression vectors or treated with compounds, were lysed using passive lysis buffer (20 L; Promega). The firefly and Renilla luciferase activities were measured on a GloMax-Multi+ Microplate Multimode Reader (Promega) using a dual luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase for transfections and further normalized to the experimental control (pECE). The firefly luciferase activity was normalized to the vehicle controls for pro- and anti-fibrotic agents to calculate relative fold-change in Col1␣2 promoter activation. Statistical analysis A minimum of 4 independent experiments were performed in quadruplicate for each treatment or transfection (n = 4). Results are reported as the mean ± SEM. Statistical comparison was done using unpaired Student’s t test or one-way ANOVA followed by a Student–Newman–Keuls post-hoc test. Values for P < 0.05 were considered to be statistically significant.
Results Induction of COL1A2-driven luciferase activity in the stable reporter cells is mediated by scleraxis Scleraxis was over-expressed in the transformed cells to confirm responsiveness of the COL1A2-driven luciferase activity in Published by NRC Research Press
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Fig. 2. (A) Luciferase activity in COL1A2-luc NIH-3T3 cells is mediated by scleraxis. Over-expression of intact scleraxis (ScxWT) induced luciferase activity, whereas transfection of its non-functional mutant forms (Scx⌬BD and Scx⌬⌬) failed to alter luciferase expression. pECE was used as empty vector control. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.05 compared with pECE; #, P < 0.05 compared with ScxWT. (B) Renilla raw light units averaged for different transfection conditions across experiments showed no significant variability. Data are the mean ± SEM for n = 4 independent experiments. A
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these cells. In agreement with previously published data from our laboratory (Espira et al. 2009; Bagchi and Czubryt 2012), the reporter cells exhibited a 6-fold induction of luciferase activity following transfection of expression vector encoding intact scleraxis (ScxWT) (Fig. 2A). Conversely, transfection with a DNA binding domain mutant form of scleraxis (Scx⌬BD), which does not transactivate the collagen I␣2 gene promoter, failed to induce luciferase activity in comparison with an empty vector control (pECE), as expected. Both the DNA-binding and proteininteraction domains of scleraxis were deleted, rendering it incapable of mediating promoter activation. Over-expression of this double deletion scleraxis mutant (Scx⌬⌬) in the reporter cells also failed to induce luciferase activity compared with the control. The Renilla luciferase expression exhibited no significant variability between transfection conditions when used as an internal control for normalization (Fig. 2B). Luciferase reporter activity is increased by fibrosis-inducing agents We have previously reported that the pro-fibrotic cytokine TGF-1 induces scleraxis expression and works synergistically with receptor Smad3 to regulate collagen 1␣2 gene expression in cardiac fibroblasts (Bagchi and Czubryt 2012), in agreement with numerous other reports that TGF- induces collagen type I synthesis (Reed et al. 1994; Lijnen and Petrov 2002; Leask and Abraham 2004; Pan et al. 2013). In our study, COL1A2 luciferase activity was induced significantly (>5-fold) in response to TGF-1 treatment in the stable reporter cells, thus demonstrating the capability of this system in a high-throughput setting (Fig. 3A). We also observed a significant increase in luciferase activity (4–5 fold) in response to other fibrotic agonists, including angiotensin II (Lijnen et al. 2000; Tharaux et al. 2000) and connective tissue growth factor (CTGF) (Ponticos et al. 2009; Koshman et al. 2013) using this transformed cell line (Fig. 3A). Dose–response assays for all these established pro-fibrotic agents demonstrated the sensitivity of the reporter system to low concentrations of these compounds (Figs. 3B–3D).Thus, this stable reporter cell line is strongly responsive to stimulators of collagen expression. Anti-fibrotic compounds suppress COL1A2 promoter-driven luciferase activity The pro-apoptotic cytokine TNF-␣ has been shown to play a pleiotropic role in the regulation of collagen genes. However sev-
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eral lines of evidence point to its role as an antagonist of fibrillar collagen expression (Kahari et al. 1990; Abraham et al. 2000; Greenwel et al. 2000; Verrecchia and Mauviel 2004; Lindner et al. 2012). Treatment of the stable luciferase-expressing cells with TNF-␣ resulted in a significant down-regulation of promoter activity compared with the vehicle-treated cells (Fig. 4A). The dosage for TNF-␣ (10.0 ng/mL) was chosen based on previously published studies involving TNF-␣ treatment on NIH-3T3 cells (Bode et al. 2003; Cavadini et al. 2007). Pirfenidone, an anti-fibrotic agent, is currently in clinical trials for the treatment of patients suffering from idiopathic pulmonary fibrosis and has been approved for use in Canada, Europe, and USA under the trade name Esbriet (InterMune, Brisbane, California, USA). It has been tested in multiple in-vitro and in-vivo models for its role in attenuating collagen expression, although its mechanism of action remains unclear (Di Sario et al. 2002; Hisatomi et al. 2012; Conte et al. 2014). Here we exposed cells to this drug to assess its effect on COL1A2 promoter activation as measured by luciferase activity. Pirfenidone administration significantly suppressed luciferase activity by ⬃33% compared with the vehicle control (Fig. 4A). Dose–response assays performed using these compounds also confirmed the sensitivity and responsiveness of the stable reporter cell line to low doses of these antifibrotic agents (Figs. 4B–4C).
Discussion Fibrotic diseases adversely affect nearly every tissue of the body, yet the available therapeutic arsenal is nearly non-existent. Pirfenidone/Esbriet has recently been approved for mild or moderate idiopathic pulmonary fibrosis, and has shown promise in other tissue cell types (Roche and Czubryt 2013). However, its mechanism of action remains to be elucidated, although it may impact fibrosis indirectly via mediation of the inflammation that frequently accompanies or induces fibrosis (Wang et al. 2013). An emerging strategy is to directly target the mechanisms responsible for the elevated expression of fibrillar collagens, which are typically the major components of fibrotic foci: in particular, type I collagen (Roche and Czubryt 2015). The development of a simple, high-throughput assay for regulators of collagen I␣2 gene expression would be an invaluable tool for the rapid screening of potential inhibitors of fibrotic collagen expression. NIH-3T3 cells are an immortalized fibroblast line derived from embryonic NIH-Swiss mice (Todaro and Green 1963). These cells Published by NRC Research Press
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Can. J. Physiol. Pharmacol. Vol. 93, 2015
Fig. 3. The COL1A2-luc stable reporter cell line is responsive to pro-fibrotic agents. (A) TGF-1 (10.0 ng/mL), angiotensin II (AngII; 10.0 nmol/L), and CTGF (50.0 ng/mL) induced luciferase reporter activity in the COL1A2-luc NIH-3T3 cells. Bioluminescence was analysed 24 h post-treatment. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.05 compared with the vehicle control. (B) COL1A2-luc NIH-3T3 cells exposed to different concentrations of TGF-1 (1.0, 5.0, 10.0, or 20.0 ng/mL) showed strong responsiveness with increase in dosage. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.0001 compared with the vehicle control. (C) Increasing concentrations of angiotensin II (1.0, 5.0, 10.0, or 20.0 nmol/L) yielded an increase in luciferase expression in COL1A2-luc NIH-3T3 cells in a dose-dependent manner. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.0001 compared with the vehicle control. (D) Luciferase activity of COL1A2-luc NIH3T3 cells increased significantly in response to increase in CTGF concentrations (10.0, 50.0, 100.0, or 200.0 ng/mL). Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.0001 compared with the vehicle control.
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endogenously express a variety of extracellular matrix proteins, including collagens, are easily maintained under standard tissue culture conditions (10% FBS in DMEM), and reproduce rapidly, thus providing a convenient cell line capable of responding to both positive and negative regulatory signals for matrix production. We therefore generated a stable reporter mammalian cell line (COL1A2-luc) derived from NIH-3T3 fibroblasts, using the human 0.7 kb COL1A2 proximal promoter to direct luciferase transgene expression. This region contains 3 scleraxis-binding E-boxes and a Smad-binding element (SBE), as well as a host of other cis regulatory elements involved in fibrosis (Bagchi and Czubryt 2012; Roche and Czubryt 2014). The proximal promoter has previously been shown to be transactivated by scleraxis and is responsive to TGF- signaling (Bagchi and Czubryt 2012; Espira et al. 2009). Type I collagen is the primary matrix component expressed in tissue fibrosis, thus the selection of the COL1A2 promoter is a reasonable choice of surrogate for assaying fibrotic gene expression in an
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automated assay (Roche and Czubryt 2014). In our transformed stable cell line, luciferase activity was induced by scleraxis, recapitulating previous findings by our group (Fig. 2) (Bagchi and Czubryt 2012). This cell line exhibited a ⬃4–5 fold increase in reporter gene activity in response to treatment with physiologically relevant doses of inducers (at nanogram levels) that included TGF-1, Ang II, and CTGF. This stable cell line thus provides a good model system that can be used in high-throughput systems to screen compound libraries for detecting agonists of COL1A2 expression. We also examined the capability of our cell line to respond to negative regulators of fibrosis. Both TNF-␣ and pirfenidone significantly repressed basal luciferase activity in this line, ranging from 20%–33% inhibition at the levels employed in this study. The ability of the cells to respond significantly to the dose of pirfenidone employed in this study was noteworthy, given the use of typically higher concentrations in previous in-vitro reports (Lin et al. 2009; Shi et al. 2011; Conte et al. 2014). Published by NRC Research Press
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Fig. 4. Anti-fibrotic agents cause down-regulation of basal luciferase expression. (A) Treatment of COL1A2-luc cells with TNF-␣ (10.0 ng/mL) or pirfenidone (1.0 mmol/L) significantly diminished luciferase activity beyond basal expression levels. Cells were exposed to these antagonists for 24 h followed by assay of reporter activity. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.05 compared with the vehicle control. (B) COL1A2-luc NIH-3T3 cells responded to TNF-␣ administration at varying doses (1.0, 5.0, 10.0, or 20.0 ng/mL) and a decrease in luciferase activity was observed. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.01 compared with the vehicle control. (C) Increasing concentrations of pirfenidone (0.5, 1.0, 5.0, or 10.0 mmol/L) resulted in decreased luciferase expression in COL1A2-luc NIH-3T3 cells. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.01 compared with the vehicle control.
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Our results demonstrate that the COL1A2-luc NIH-3T3 cell line provides a responsive and effective screening tool for detecting agents that regulate collagen 1␣2 transcription. The capability of using these cells in a 96-well plate format makes it an efficient and attractive tool for high throughput compound screening assays. Of note, intra-sample variability was small, with the standard error of the mean ranging from ⬃0.6% to ⬃9.1% relative to the mean (i.e., SEM/mean) and averaging 4.4% in this study. This tight clustering of data facilitated analysis of inter-sample statistical differences with high confidence. This high reproducibility is essential for effective high throughput assay development. Future refinements of this assay include the generation of cell lines in which the scleraxis, Smad, or other transcription factor binding sites within the COL1A2 proximal promoter are mutated to prevent binding of those transcription factors. This approach will increase the potential utility of the assay, as it can thus be employed for defining the mechanism of action of the compounds (e.g., scleraxis-mediated or scleraxis-independent) as well as to act as a negative control for compounds hypothesized to act via a specific transcriptional regulator. Generation of alternative cell lines in which luciferase is replaced by fluorescent proteins such
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as eGFP will also increase the flexibility of the system by permitting alternative read-out approaches. Further refinement should also permit assay size reduction to a 384-well format for increased efficiency. It should be noted that the assays requiring transfection of expression vectors (Fig. 2) also required a concomitant Renilla luciferase transgene as a transfection control. This required an additional step in the workflow, which may be considered to be potentially problematic, since transfection efficiency can vary. However, this experiment was performed simply as a proof-ofconcept, as the results shown in Figs. 3 and 4 were performed without the need for additional transfection of Renilla. The use of luciferase as an assay readout is therefore not, in and of itself, limiting in the usefulness of the assay compared with, for example, the use of a fluorescent readout. The data in Fig. 2A demonstrate that, even when additional transfection was required, the data was tightly clustered, suggesting that the potential variability introduced by having to perform a transfection step was minimal. The use of Renilla transgene expression for normalization did not interfere with the responsiveness of the luciferase reporter, owing to negligible variability between transfection conditions across experiments (Fig. 2B). Published by NRC Research Press
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The rapidness and ease-of-use of in-vitro assays such as that employed in our study make them attractive screening tools. Our results demonstrate that our assay works well in a highthroughput 96-well format suitable for fast turnaround (24 h), high efficiency, highly-automated systems, and that the COL1A2luc cell line can be successfully used to quantify gene responsiveness quickly, easily, and accurately. This tool will allow sensitive and precise analysis of fibrosis-inducing and blocking agents and provide proof-of-principle for rationalizing testing of potential lead compounds in pre-clinical and clinical trials.
Acknowledgements The authors would like to thank Dr. Nina Aroutiounova and Ms. Sari Yakubovich for technical assistance. This work was supported by the St. Boniface Hospital Foundation (M.P.C.). R.A.B. is a recipient of a doctoral research award from Canadian Institutes of Health Research and Research Manitoba.
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sion. Int. Rev. Immunol. 12(2–4): 177–185. doi:10.3109/08830189509056711. PMID:7650420. Koshman, Y.E., Patel, N., Chu, M., Iyengar, R., Kim, T., Ersahin, C., et al. 2013. Regulation of connective tissue growth factor gene expression and fibrosis in human heart failure. J. Card. Fail. 19(4): 283–294. doi:10.1016/j.cardfail.2013. 01.013. PMID:23582094. Krenning, G., Zeisberg, E.M., and Kalluri, R. 2010. The origin of fibroblasts and mechanism of cardiac fibrosis. J. Cell. Physiol. 225(3): 631–637. doi:10.1002/ jcp.22322. PMID:20635395. Leask, A., and Abraham, D.J. 2004. TGF-beta signaling and the fibrotic response. FASEB J. 18(7): 816–827. doi:10.1096/fj.03-1273rev. PMID:15117886. Lijnen, P., and Petrov, V. 2002. Transforming growth factor-beta 1-induced collagen production in cultures of cardiac fibroblasts is the result of the appearance of myofibroblasts. Methods Find. Exp. Clin. Pharmacol. 24(6): 333–344. doi:10.1358/mf.2002.24.6.693065. PMID:12224439. Lijnen, P.J., Petrov, V.V., and Fagard, R.H. 2000. Induction of cardiac fibrosis by angiotensin II. Methods Find. Exp. Clin. Pharmacol. 22(10): 709–723. doi:10. 1358/mf.2000.22.10.802287. PMID:11346891. Lin, X., Yu, M., Wu, K., Yuan, H., and Zhong, H. 2009. Effects of pirfenidone on proliferation, migration, and collagen contraction of human Tenon’s fibroblasts in vitro. Invest. Ophthalmol. Vis. Sci. 50(8): 3763–3770. doi:10.1167/iovs. 08-2815. PMID:19264889. Lindner, D., Zietsch, C., Becher, P.M., Schulze, K., Schultheiss, H.P., Tschope, C., et al. 2012. Differential expression of matrix metalloproteases in human fibroblasts with different origins. Biochem. Res. Int. 2012: 875742. doi:10.1155/ 2012/875742. PMID:22500233. Noble, P.W. 2006. Idiopathic pulmonary fibrosis: natural history and prognosis. Clin. Chest Med. 27(1 Suppl. 1): S11–S16. doi:10.1016/j.ccm.2005.08.003. PMID: 16545628. Pan, X., Chen, Z., Huang, R., Yao, Y., and Ma, G. 2013. Transforming growth factor beta1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts. PLoS One, 8(4): e60335. doi:10.1371/journal.pone.0060335. PMID:23560091. Plopper, G. 2007. The extracellular matrix and cell adhesion. In Cells. Edited by B. Lewin, L. Cassimeris, V. Lingappa, and G. Plopper. Jones and Bartlett Publishers, Sudbury, Mass. pp. 645–702. Ponticos, M., Holmes, A.M., Shi-wen, X., Leoni, P., Khan, K., Rajkumar, V.S., et al. 2009. Pivotal role of connective tissue growth factor in lung fibrosis: MAPKdependent transcriptional activation of type I collagen. Arthritis Rheum. 60(7): 2142–2155. doi:10.1002/art.24620. PMID:19565505. Reed, M.J., Vernon, R.B., Abrass, I.B., and Sage, E.H. 1994. TGF-beta 1 induces the expression of type I collagen and SPARC, and enhances contraction of collagen gels, by fibroblasts from young and aged donors. J. Cell. Physiol. 158(1): 169–179. doi:10.1002/jcp.1041580121. PMID:8263022. Roche, P., and Czubryt, M.P. 2013. Pirfenidone and the inflammasome: getting to the heart of cardiac remodeling. Cardiology, 126(1): 59–61. doi:10.1159/ 000351983. PMID:23867498. Roche, P., and Czubryt, M.P. 2014. Transcriptional control of collagen I gene expression. Cardiovasc. Hematol. Disord. Drug Targets, 14(2): 107–120. doi:10. 2174/1871529X14666140505122510. PMID:24801728. Roche, P.L., and Czubryt, M.P. 2015. Current and future strategies for the diagnosis and treatment of cardiac fibrosis. In press. In Cardiac fibrosis and heart failure. Edited by I.M.C. Dixon and J.T. Wigle. Springer Science and Business Media. Schnaper, H.W., and Kopp, J.B. 2003. Renal fibrosis. Front. Biosci. 8: e68–e86. doi:10.2741/925. PMID:12456333. Shi, Q., Liu, X., Bai, Y., Cui, C., Li, J., Li, Y., et al. 2011. In vitro effects of pirfenidone on cardiac fibroblasts: proliferation, myofibroblast differentiation, migration and cytokine secretion. PLoS One, 6(11): e28134. doi:10.1371/journal.pone. 0028134. PMID:22132230. Tharaux, P.L., Chatziantoniou, C., Fakhouri, F., and Dussaule, J.C. 2000. Angiotensin II activates collagen I gene through a mechanism involving the MAP/ER kinase pathway. Hypertension, 36(3): 330–336. doi:10.1161/01.HYP. 36.3.330. PMID:10988260. Todaro, G.J., and Green, H. 1963. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17(2): 299–313. doi:10.1083/jcb.17.2.299. PMID:13985244. Verrecchia, F., and Mauviel, A. 2004. TGF-beta and TNF-alpha: antagonistic cytokines controlling type I collagen gene expression. Cell Signal. 16(8): 873–880. doi:10.1016/j.cellsig.2004.02.007. PMID:15157666. Wang, Y., Wu, Y., Chen, J., Zhao, S., and Li, H. 2013. Pirfenidone attenuates cardiac fibrosis in a mouse model of TAC-induced left ventricular remodeling by suppressing NLRP3 inflammasome formation. Cardiology, 126(1): 1–11. doi:10.1159/000351179. PMID:23839341. Weber, K.T. 1997. Monitoring tissue repair and fibrosis from a distance. Circulation, 96(8): 2488–2492. PMID:9355880.
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ARTICLE
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Development of a high throughput luciferase reporter gene system for screening activators and repressors of human collagen I␣2 gene expression1 Rushita A. Bagchi, Viktoriya Mozolevska, Bernard Abrenica, and Michael P. Czubryt
Abstract: Fibrosis, which is characterized by the excessive production of matrix proteins, occurs in multiple tissues and is associated with increased morbidity and mortality. Despite its significant negative impact on patient outcomes, therapies targeted to treat fibrosis are currently lacking. Screening for inhibitors of the expression of collagen, the primary component of fibrotic lesions, represents an option for the identification of novel lead compounds for therapeutic development with potentially fewer off-target effects compared with the targeting of multifunctional cell signaling pathways. Here we report on the generation of a stable luciferase reporter system using a fibroblast cell line, which can be used for rapidly screening both activators and repressors of human collagen COL1A2 gene transcription in a high throughput setting. This in vitro screening tool was validated using known agonists (scleraxis, TGF-, angiotensin II, CTGF) and antagonists (TNF-␣, pirfenidone) of COL1A2 gene expression. The COL1A2-luc NIH-3T3 fibroblast system provides a useful and effective screen for potential lead compounds with pro- or anti-fibrotic properties. Key words: collagen, luciferase reporter, stable cell line, in vitro screening assay, fibrosis, high-throughput assay, fibroblast. Résumé : La fibrose, caractérisée par une production excessive de protéines de la matrice extracellulaire, survient dans une multitude de tissus, et elle est associée avec un accroissement de morbidité et de mortalité a` l’échelle mondiale. Malgré son impact négatif significatif sur le patient, des thérapies visant a` traiter la fibrose font actuellement défaut. Le criblage d’inhibiteurs de l’expression du collagène, la première composante des lésions fibreuses, constitue une option afin d’identifier de nouveaux médicaments têtes de série en vue d’un développement thérapeutique qui présenteraient potentiellement moins d’effets hors cible, comparativement a` cibler des voies de signalisation cellulaire multifonctionnelles. Les auteurs rapportent ici la production d’un système rapporteur a` la luciférase stable, a` l’aide d’une lignée de fibroblastes qui peut être utilisée pour cribler rapidement tant les activateurs que les répresseurs de la transcription du gène COL1A2 codant le collagène humain, dans un procédé a` haut débit. Cet outil de criblage in vitro a été validé a` l’aide d’agonistes (scleraxis, TGF-, angiotensine II, CTGF) et d’antagonistes (TNF-␣, pirfenidone) connus de l’expression génique de COL1A2. Le système COL1A2-luciférase/fibroblastes NIH-3T3 constitue un outil de criblage utile et efficace afin d’identifier des composés têtes de série potentiels, possédant des propriétés pro- et anti-fibrogènes. [Traduit par la Rédaction] Mots-clés : collagène, rapporteur luciférase, lignée cellulaire stable, test de criblage in vitro, fibrose, test a` haut-débit, fibroblaste.
Introduction The extracellular matrix (ECM) is a complex and dynamic structure that provides cells and organs with mechanical and functional support. This compartment of our body’s architecture is primarily composed of collagens (Karsenty and Park 1995; Di Lullo et al. 2002). Defects in collagen structure or expression are associated with multiple human genetic disorders such as Ehlers Danlos Syndrome and osteogenesis imperfecta (Plopper 2007). Increased synthesis of type I collagen, resulting in ECM build-up, is the hallmark of fibrotic disease (Fan et al. 2012; Roche and Czubryt 2014). Fibrosis can present as a multisystem disorder or as a tissuespecific disease affecting the lungs, heart, kidney, liver, and other tissues (Schnaper and Kopp 2003; Bataller and Brenner 2005; Noble 2006; Krenning et al. 2010). Cardiac fibrosis is quite similar to other forms of tissue fibrosis such as those affecting the lungs,
liver and kidneys, suggesting that common mechanisms may underlie fibrosis in multiple tissues (Weber 1997). Despite being a significant contributor to patient morbidity and mortality, specific therapies directly targeting fibrotic disease are almost completely lacking. Fibroblasts are considered to be the principal determinants of tissue fibrosis. These cells are primarily involved in matrix synthesis and turnover in healthy tissues. However, activation of these cells with pro-fibrotic agonists results in excessive production of fibrotic matrix and impairment of organ function. The commercially available immortalized fibroblast NIH-3T3 cell line provides a useful tool for assessment of fibroblast function in a reproducible manner. These cells synthesize collagens and other ECM components and respond to fibrotic agents such as TGF- and thus serve as a convenient surrogate for primary cells (Bagchi and Czubryt 2012).
Received 12 December 2014. Accepted 21 January 2015. R.A. Bagchi and M.P. Czubryt. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada; Department of Physiology and Pathophysiology, University of Manitoba, St. Boniface Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada. V. Mozolevska and B. Abrenica. Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB R2H 2A6, Canada. Corresponding author: Michael P. Czubryt (e-mail: [email protected]). 1This article is part of a Special Issue entitled “2nd Cardiovascular Forum for Promoting Centers of Excellence and Young Investigators.” Can. J. Physiol. Pharmacol. 93: 1–6 (2015) dx.doi.org/10.1139/cjpp-2014-0521
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In this study, we used the NIH-3T3 cell line to derive a stable luciferase reporter system for detecting the response of the human collagen I␣2 (COL1A2) gene promoter to inducers and repressors of fibrosis in a high-throughput manner. In a 96-well in vitro screening assay format, we confirmed that known activators and repressors of COL1A2 gene transcription significantly increased or decreased, respectively, luciferase expression assayed in an automated reader. Our results demonstrated that this screen can be easily employed to rapidly obtain tightly clustered data with high reproducibility. This study provides a proof-of-principle for the utility of this cell line in first-pass compound library screening for identification of pro- and anti-fibrotic agents.
Can. J. Physiol. Pharmacol. Vol. 93, 2015
Fig. 1. Schematic diagram illustrating the luciferase-based reporter gene assay development and workflow.
COL1A2 promoter
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Materials and methods Subcloning of the human COL1A2 proximal promoter The human COL1A2 proximal promoter luciferase parent vector (pGL3-COL1A2) has been previously described (Bagchi and Czubryt 2012). This vector was digested with BsgI to excise the promoterluciferase fragment intact, and the ends were blunted using Klenow DNA polymerase (New England Biolabs). This fragment was further digested using XbaI to yield the COL1A2.luc insert, which was gel purified. pcDNA3.1 vector was subjected to restriction enzyme digestion with EcoRV and XbaI followed by gel purification. The COL1A2.luc insert was ligated to pcDNA3.1 vector at 16 °C overnight using T4 DNA ligase (Life Technologies). The ligation reaction product was used to transform competent bacterial cells (Bioline) and plasmids were isolated and purified using standard methods. The construct nucleotide sequence was verified at the DNA Sequencing Core (Manitoba Institute of Cell Biology). This plasmid was used for generation of a NIH-3T3-derived stable reporter cell line (Fig. 1). Generation of a stable COL1A2 reporter system The pcDNA3.1-COL1A2.luc plasmid construct containing a neomycin selection marker was stably transfected into NIH-3T3 fibroblasts (ATCC CRL-1658) using Lipofectamine 3000 reagent (Life Technologies) for 48 h. Transfected cells were then selected using G418 antibiotic (800 g/mL; Enzo Life Sciences) for 2–3 weeks. Individual antibiotic-resistant clones were isolated, subcultured, and maintained in 10% fetal bovine serum-supplemented Dulbecco’s modified essential medium at 37 °C with 5% CO2 in air. Cells were subjected to G418 selection (400 g/mL) every 2–3 passages to maintain purity. Transient transfections COL1A2-luc NIH-3T3 cells were seeded at 4 × 103 cells per well in 100 L growth medium in 96-well sterile tissue culture plates (Corning Life Sciences). Cells were transfected (50 ng/well) with the following expression vectors after overnight recovery: scleraxis (ScxWT), DNA binding domain scleraxis mutant (Scx⌬BD), or scleraxis mutant lacking both DNA-binding and protein interaction domains (Scx⌬⌬) (Espira et al. 2009; Bagchi and Czubryt 2012). pECE plasmid was used for control transfections, whereas pRL (1.0 ng/well) was used as internal control for all experiments. Lipofectamine 3000 reagent and OptiMEM (Life Technologies) were used for all transfections. After 24 h, cells were rinsed once with phosphate-buffered saline (PBS) and lysed to assay for luciferase activity. Treatment of stable COL1A2-luciferase reporter cells with pro- and anti-fibrotic compounds Cells were seeded at 4 × 103 cells per well in 96-well microtiter plates (Corning Life Sciences) in growth medium. Cells were briefly equilibrated in serum-free media prior to the administration of compounds. TGF-1 (1.0, 5.0, 10.0, and 20.0 ng/mL, Peprotech), angiotensin II (1.0, 5.0, 10.0, and 20.0 nmol/L; Sigma Aldrich) and CTGF (10.0, 50.0, 100.0, and 200.0 ng/mL; Life Technologies) were used as pro-fibrotic agents. Pirfenidone (0.5, 1.0, 5.0, and
Selection of positive clones with G418
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10.0 mmol/L; Sigma–Aldrich) and TNF-␣ (1.0, 5.0, 10.0, and 20.0 ng/mL; R&D Systems) were used to examine their antagonist activity on Col1␣2 gene promoter activation. An equal volume of vehicle (water or DMSO) was added to the control wells. After 24 h of treatment, cells were lysed and luciferase activity was assayed. Analysis of luciferase reporter gene activity Stable reporter-transformed cells, transfected with expression vectors or treated with compounds, were lysed using passive lysis buffer (20 L; Promega). The firefly and Renilla luciferase activities were measured on a GloMax-Multi+ Microplate Multimode Reader (Promega) using a dual luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase for transfections and further normalized to the experimental control (pECE). The firefly luciferase activity was normalized to the vehicle controls for pro- and anti-fibrotic agents to calculate relative fold-change in Col1␣2 promoter activation. Statistical analysis A minimum of 4 independent experiments were performed in quadruplicate for each treatment or transfection (n = 4). Results are reported as the mean ± SEM. Statistical comparison was done using unpaired Student’s t test or one-way ANOVA followed by a Student–Newman–Keuls post-hoc test. Values for P < 0.05 were considered to be statistically significant.
Results Induction of COL1A2-driven luciferase activity in the stable reporter cells is mediated by scleraxis Scleraxis was over-expressed in the transformed cells to confirm responsiveness of the COL1A2-driven luciferase activity in Published by NRC Research Press
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Fig. 2. (A) Luciferase activity in COL1A2-luc NIH-3T3 cells is mediated by scleraxis. Over-expression of intact scleraxis (ScxWT) induced luciferase activity, whereas transfection of its non-functional mutant forms (Scx⌬BD and Scx⌬⌬) failed to alter luciferase expression. pECE was used as empty vector control. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.05 compared with pECE; #, P < 0.05 compared with ScxWT. (B) Renilla raw light units averaged for different transfection conditions across experiments showed no significant variability. Data are the mean ± SEM for n = 4 independent experiments. A
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these cells. In agreement with previously published data from our laboratory (Espira et al. 2009; Bagchi and Czubryt 2012), the reporter cells exhibited a 6-fold induction of luciferase activity following transfection of expression vector encoding intact scleraxis (ScxWT) (Fig. 2A). Conversely, transfection with a DNA binding domain mutant form of scleraxis (Scx⌬BD), which does not transactivate the collagen I␣2 gene promoter, failed to induce luciferase activity in comparison with an empty vector control (pECE), as expected. Both the DNA-binding and proteininteraction domains of scleraxis were deleted, rendering it incapable of mediating promoter activation. Over-expression of this double deletion scleraxis mutant (Scx⌬⌬) in the reporter cells also failed to induce luciferase activity compared with the control. The Renilla luciferase expression exhibited no significant variability between transfection conditions when used as an internal control for normalization (Fig. 2B). Luciferase reporter activity is increased by fibrosis-inducing agents We have previously reported that the pro-fibrotic cytokine TGF-1 induces scleraxis expression and works synergistically with receptor Smad3 to regulate collagen 1␣2 gene expression in cardiac fibroblasts (Bagchi and Czubryt 2012), in agreement with numerous other reports that TGF- induces collagen type I synthesis (Reed et al. 1994; Lijnen and Petrov 2002; Leask and Abraham 2004; Pan et al. 2013). In our study, COL1A2 luciferase activity was induced significantly (>5-fold) in response to TGF-1 treatment in the stable reporter cells, thus demonstrating the capability of this system in a high-throughput setting (Fig. 3A). We also observed a significant increase in luciferase activity (4–5 fold) in response to other fibrotic agonists, including angiotensin II (Lijnen et al. 2000; Tharaux et al. 2000) and connective tissue growth factor (CTGF) (Ponticos et al. 2009; Koshman et al. 2013) using this transformed cell line (Fig. 3A). Dose–response assays for all these established pro-fibrotic agents demonstrated the sensitivity of the reporter system to low concentrations of these compounds (Figs. 3B–3D).Thus, this stable reporter cell line is strongly responsive to stimulators of collagen expression. Anti-fibrotic compounds suppress COL1A2 promoter-driven luciferase activity The pro-apoptotic cytokine TNF-␣ has been shown to play a pleiotropic role in the regulation of collagen genes. However sev-
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eral lines of evidence point to its role as an antagonist of fibrillar collagen expression (Kahari et al. 1990; Abraham et al. 2000; Greenwel et al. 2000; Verrecchia and Mauviel 2004; Lindner et al. 2012). Treatment of the stable luciferase-expressing cells with TNF-␣ resulted in a significant down-regulation of promoter activity compared with the vehicle-treated cells (Fig. 4A). The dosage for TNF-␣ (10.0 ng/mL) was chosen based on previously published studies involving TNF-␣ treatment on NIH-3T3 cells (Bode et al. 2003; Cavadini et al. 2007). Pirfenidone, an anti-fibrotic agent, is currently in clinical trials for the treatment of patients suffering from idiopathic pulmonary fibrosis and has been approved for use in Canada, Europe, and USA under the trade name Esbriet (InterMune, Brisbane, California, USA). It has been tested in multiple in-vitro and in-vivo models for its role in attenuating collagen expression, although its mechanism of action remains unclear (Di Sario et al. 2002; Hisatomi et al. 2012; Conte et al. 2014). Here we exposed cells to this drug to assess its effect on COL1A2 promoter activation as measured by luciferase activity. Pirfenidone administration significantly suppressed luciferase activity by ⬃33% compared with the vehicle control (Fig. 4A). Dose–response assays performed using these compounds also confirmed the sensitivity and responsiveness of the stable reporter cell line to low doses of these antifibrotic agents (Figs. 4B–4C).
Discussion Fibrotic diseases adversely affect nearly every tissue of the body, yet the available therapeutic arsenal is nearly non-existent. Pirfenidone/Esbriet has recently been approved for mild or moderate idiopathic pulmonary fibrosis, and has shown promise in other tissue cell types (Roche and Czubryt 2013). However, its mechanism of action remains to be elucidated, although it may impact fibrosis indirectly via mediation of the inflammation that frequently accompanies or induces fibrosis (Wang et al. 2013). An emerging strategy is to directly target the mechanisms responsible for the elevated expression of fibrillar collagens, which are typically the major components of fibrotic foci: in particular, type I collagen (Roche and Czubryt 2015). The development of a simple, high-throughput assay for regulators of collagen I␣2 gene expression would be an invaluable tool for the rapid screening of potential inhibitors of fibrotic collagen expression. NIH-3T3 cells are an immortalized fibroblast line derived from embryonic NIH-Swiss mice (Todaro and Green 1963). These cells Published by NRC Research Press
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Can. J. Physiol. Pharmacol. Vol. 93, 2015
Fig. 3. The COL1A2-luc stable reporter cell line is responsive to pro-fibrotic agents. (A) TGF-1 (10.0 ng/mL), angiotensin II (AngII; 10.0 nmol/L), and CTGF (50.0 ng/mL) induced luciferase reporter activity in the COL1A2-luc NIH-3T3 cells. Bioluminescence was analysed 24 h post-treatment. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.05 compared with the vehicle control. (B) COL1A2-luc NIH-3T3 cells exposed to different concentrations of TGF-1 (1.0, 5.0, 10.0, or 20.0 ng/mL) showed strong responsiveness with increase in dosage. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.0001 compared with the vehicle control. (C) Increasing concentrations of angiotensin II (1.0, 5.0, 10.0, or 20.0 nmol/L) yielded an increase in luciferase expression in COL1A2-luc NIH-3T3 cells in a dose-dependent manner. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.0001 compared with the vehicle control. (D) Luciferase activity of COL1A2-luc NIH3T3 cells increased significantly in response to increase in CTGF concentrations (10.0, 50.0, 100.0, or 200.0 ng/mL). Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.0001 compared with the vehicle control.
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endogenously express a variety of extracellular matrix proteins, including collagens, are easily maintained under standard tissue culture conditions (10% FBS in DMEM), and reproduce rapidly, thus providing a convenient cell line capable of responding to both positive and negative regulatory signals for matrix production. We therefore generated a stable reporter mammalian cell line (COL1A2-luc) derived from NIH-3T3 fibroblasts, using the human 0.7 kb COL1A2 proximal promoter to direct luciferase transgene expression. This region contains 3 scleraxis-binding E-boxes and a Smad-binding element (SBE), as well as a host of other cis regulatory elements involved in fibrosis (Bagchi and Czubryt 2012; Roche and Czubryt 2014). The proximal promoter has previously been shown to be transactivated by scleraxis and is responsive to TGF- signaling (Bagchi and Czubryt 2012; Espira et al. 2009). Type I collagen is the primary matrix component expressed in tissue fibrosis, thus the selection of the COL1A2 promoter is a reasonable choice of surrogate for assaying fibrotic gene expression in an
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automated assay (Roche and Czubryt 2014). In our transformed stable cell line, luciferase activity was induced by scleraxis, recapitulating previous findings by our group (Fig. 2) (Bagchi and Czubryt 2012). This cell line exhibited a ⬃4–5 fold increase in reporter gene activity in response to treatment with physiologically relevant doses of inducers (at nanogram levels) that included TGF-1, Ang II, and CTGF. This stable cell line thus provides a good model system that can be used in high-throughput systems to screen compound libraries for detecting agonists of COL1A2 expression. We also examined the capability of our cell line to respond to negative regulators of fibrosis. Both TNF-␣ and pirfenidone significantly repressed basal luciferase activity in this line, ranging from 20%–33% inhibition at the levels employed in this study. The ability of the cells to respond significantly to the dose of pirfenidone employed in this study was noteworthy, given the use of typically higher concentrations in previous in-vitro reports (Lin et al. 2009; Shi et al. 2011; Conte et al. 2014). Published by NRC Research Press
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Fig. 4. Anti-fibrotic agents cause down-regulation of basal luciferase expression. (A) Treatment of COL1A2-luc cells with TNF-␣ (10.0 ng/mL) or pirfenidone (1.0 mmol/L) significantly diminished luciferase activity beyond basal expression levels. Cells were exposed to these antagonists for 24 h followed by assay of reporter activity. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.05 compared with the vehicle control. (B) COL1A2-luc NIH-3T3 cells responded to TNF-␣ administration at varying doses (1.0, 5.0, 10.0, or 20.0 ng/mL) and a decrease in luciferase activity was observed. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.01 compared with the vehicle control. (C) Increasing concentrations of pirfenidone (0.5, 1.0, 5.0, or 10.0 mmol/L) resulted in decreased luciferase expression in COL1A2-luc NIH-3T3 cells. Data are the mean ± SEM for n = 4 independent experiments; *, P < 0.01 compared with the vehicle control.
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Our results demonstrate that the COL1A2-luc NIH-3T3 cell line provides a responsive and effective screening tool for detecting agents that regulate collagen 1␣2 transcription. The capability of using these cells in a 96-well plate format makes it an efficient and attractive tool for high throughput compound screening assays. Of note, intra-sample variability was small, with the standard error of the mean ranging from ⬃0.6% to ⬃9.1% relative to the mean (i.e., SEM/mean) and averaging 4.4% in this study. This tight clustering of data facilitated analysis of inter-sample statistical differences with high confidence. This high reproducibility is essential for effective high throughput assay development. Future refinements of this assay include the generation of cell lines in which the scleraxis, Smad, or other transcription factor binding sites within the COL1A2 proximal promoter are mutated to prevent binding of those transcription factors. This approach will increase the potential utility of the assay, as it can thus be employed for defining the mechanism of action of the compounds (e.g., scleraxis-mediated or scleraxis-independent) as well as to act as a negative control for compounds hypothesized to act via a specific transcriptional regulator. Generation of alternative cell lines in which luciferase is replaced by fluorescent proteins such
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as eGFP will also increase the flexibility of the system by permitting alternative read-out approaches. Further refinement should also permit assay size reduction to a 384-well format for increased efficiency. It should be noted that the assays requiring transfection of expression vectors (Fig. 2) also required a concomitant Renilla luciferase transgene as a transfection control. This required an additional step in the workflow, which may be considered to be potentially problematic, since transfection efficiency can vary. However, this experiment was performed simply as a proof-ofconcept, as the results shown in Figs. 3 and 4 were performed without the need for additional transfection of Renilla. The use of luciferase as an assay readout is therefore not, in and of itself, limiting in the usefulness of the assay compared with, for example, the use of a fluorescent readout. The data in Fig. 2A demonstrate that, even when additional transfection was required, the data was tightly clustered, suggesting that the potential variability introduced by having to perform a transfection step was minimal. The use of Renilla transgene expression for normalization did not interfere with the responsiveness of the luciferase reporter, owing to negligible variability between transfection conditions across experiments (Fig. 2B). Published by NRC Research Press
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The rapidness and ease-of-use of in-vitro assays such as that employed in our study make them attractive screening tools. Our results demonstrate that our assay works well in a highthroughput 96-well format suitable for fast turnaround (24 h), high efficiency, highly-automated systems, and that the COL1A2luc cell line can be successfully used to quantify gene responsiveness quickly, easily, and accurately. This tool will allow sensitive and precise analysis of fibrosis-inducing and blocking agents and provide proof-of-principle for rationalizing testing of potential lead compounds in pre-clinical and clinical trials.
Acknowledgements The authors would like to thank Dr. Nina Aroutiounova and Ms. Sari Yakubovich for technical assistance. This work was supported by the St. Boniface Hospital Foundation (M.P.C.). R.A.B. is a recipient of a doctoral research award from Canadian Institutes of Health Research and Research Manitoba.
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