Alpha smooth muscle actin (α-SMA) is a protein marker of myofibroblast differentiation, which occurs de novo in wound healing, tissue fibrosis and desmoplasia in cancers. While this is a transient event in successful healing, its persistence characterizes chronic fibrosis, which can progress to organ failure. Thus myofibroblast differentiation is an indicator of active fibrosis, which can be used to monitor tissue/organ fibrosis. Many of these diseases lack effective therapy, thus there is a need for development of novel therapies. A simple, reliable and high throughput system (HTS) to screen for compounds to control myofibroblast differentiation would be helpful in this effort.
Reporter Assay to Quantify Transcriptional Activation of α-SMA
Recently, a collection of constructs has been developed for the readout of transcriptional activation of the aSMA gene. These plasmids consist of either a human or rat α-SMA (Acta2 gene) promoter fused to a luciferase reporter gene. Activation of the α-SMA promoter induces expression of luciferase, which can be measured by monitoring luminescence as an indicator of myofibroblast differentiation. In addition, truncated and select mutated promoter regions have also been fused to luciferase for assessment of transcriptional regulation of this gene. These mutations include alterations in consensus binding elements for Smad3, C/EBPβ, and NKX2.5 all of which are involved in the regulation of α-SMA gene expression. These constructs have been validated and used in published work to help elucidate the genes and proteins involved in regulation of α-SMA expression. Together, this collection of constructs can be used to find new transcription factors involved in the regulation of α-SMA gene expression, and can be used as a readout for active fibrosis in an HTS setting to screen for anti-fibrotic agents. Their use in the appropriate stable reporter cell lines can provide the HTS system needed for screening drug targets related to fibrotic diseases and desmoplastic reactions in cancer.
- Plasmid 1: pGal3-αSMAp-luc-C/EBPmut: Rat α-SMA promoter with a mutation in the C/EBPβ consensus domain (1)
- Plasmid 2: pGal3-αSMAp-luc: Wild type rat α-SMA promoter fused to luciferase (1)
- Plasmid 3: pGL3-α-SMAp-TCEm: Rat α-SMA promoter with a mutation in the TCE site where GKLF binds, and is fused to luciferase (2)
- Plasmid 4: pGL420-hαSMAp-luc: Human α-SMA promoter from -1698 to +87 fused to luciferase (3)
- Plasmid 5: pGL420-hαSMAp-GBEm: Human α-SMA promoter with a mutated Gli binding site fused to luciferase (3)
- Plasmid 6: αSMAp-luc-SBEm1: Rat α-SMA promoter with SBE1 site mutated to C524T, A525C and C528A fused to luciferase (4)
- Plasmid 7: αSMAp-lucSBEm2: Rat α-SMA promoter with SBE2 site mutated to C15T, A16G and G17C fused to luciferase (4)
- Plasmid 8: αSMAp-lucSBEcontrol: Rat α-SMA promoter with mutations C294A, T295G, and G296A fused to luciferase (4)
- Hu B, Zhe W, Jin H, Hashimoto N, Liu T and Phan S. CCAAT/Enhancer-binding protein β isoforms and the regulation of α-smooth muscle actin gene expression by IL-1β. Journal of Immunology. 173:4661-4668.
- Hu B, Zhe W, Liu T, Ullenbruch MR, Jin H, and Phan SH. Gut-enriched Kruppel-like factor interaction with Smad3 inhibits myofibroblast differentiation. 2007. American Journal of Respiratory Cell and Molecular Biology. 36:78-84.
- Hu B, Liu J, Wu Z, Liu T, Ullenbruch MR, Ding L, Henke CA, Bitterman PB, and Phan SH. Reemergence of hedgehog mediates epithelial-mesenchymal crosstalk in pulmonary fibrosis. April 2015. American Journal of Respiratory Cell and Molecular Biology. 52(4):418-428.
- Hu B, Wu Z, and Phan SH. Smad3 mediates transforming growth factor-β-induced α-smooth muscle actin expression.