Monitor activation status of interferon-stimulated signaling pathways
Li Xu Tim Sanchez Mary Buchanan Chao-Feng Zheng
Stratagenes family of PathDetect cis-reporting systems now includes the GAS and ISRE reporting systems, to better study the activation of signal transduction pathways stimulated by interferon - a, - b, or - g. The pISRE-Luc cis-reporter plasmid contains direct repeats of the interferon-stimulated response element (ISRE) found in the promoter of the 54-kDa interferon-stimulated gene (ISG54); the pGAS-Luc cis-reporter plasmid contains direct repeats of the gamma-activating sequence from the promoter of the guanylate-binding protein. In these plasmids, a basic promoter element (TATA box), and the direct repeats of ISRE or GAS, control expression of the luciferase gene. The pISRE-Luc and pGAS-Luc reporter plasmids monitor the activation status of signaling molecules and transcription factors that mediate the function of interferons.
Interferons (IFNs) are soluble proteins that act as local hormones, inhibit viral replication, and activate certain host defense responses. These interesting proteins modulate the expression of many genes and elicit a wide range of biological functions, including tissue repair, activation of various blood cells involved in antibody production, inflammation, and host defense against viruses.2,3 IFNs have also been used as therapeutic agents against many viral diseases, including acquired immune-deficiency syndrome (AIDS).1,2,3
IFN-a and IFN-b are interferons synthesized by many cell types following viral infection and have two major functions: They inhibit viral replication by activating cellular genes whose products are involved in the destruction of viral RNA and in the inhibition of protein translation; and they induce expression of MHC class I molecules by most cells in the body, except virus-infected cells. In contrast, type II interferon (IFN-g) is produced mainly by effector T cells after the induction of the adaptive immune response.2,3
The actions of interferons are mediated by members of the class II cytokine receptor family. Stimulation of cells with IFNs results in the dimerization of their receptors and the activation of the associated protein tyrosine kinases, including JAKs and Tyks. These events lead to the activation and nuclear localization of a group of transcription factors known as signal transducers and activators of transcription (STATs).3 STATs can then bind to cis-acting DNA elements present in the promoters of various IFN-inducible genes and modulate their expression.3
At this time, six different STATs have been cloned and characterized from mammalian cells. These STATs can form homo- and heterodimers and bind to very similar yet different symmetrical, dyad sequences.3 When cells are stimulated with IFN-a or IFN-b, a DNA-binding complex is formed that consists of STAT1, STAT2, and another protein known as p48. This complex binds to the enhancer element, IFN-stimulated response element (ISRE). In contrast, the STAT1 homodimer, which binds to the unique gamma-activated sequence (GAS), is formed in response to IFN-g.3 The mechanism of the activation and nuclear localization of STATs is still not fully understood. To allow interferon-related regulation of transcription to be better characterized, Stratagene has introduced two new PathDetect cis-reporting systems that use the cis-acting enhancer elements ISRE and GAS.
All PathDetect cis-reporting systems feature a cis-reporter plasmid that contains the luciferase reporter gene driven by the basic promoter element (TATA box) joined to direct repeats of cis-acting DNA elements.4 When a plasmid expressing a gene of interest is cotransfected into mammalian cells with a cis-reporter plasmid, increased luciferase expression indicates either direct or indirect activation of the transcription factors that bind to the cis-acting elements in the plasmids. The cis-reporting plasmids can be used to evaluate the effect of uncharacterized genes, growth factors, drug candidates, or the effects of extracellular stimuli on these transcriptional elements.
The expression of a group of human genes, the interferon-stimulated genes (ISGs), is induced by IFN-a and IFN-b, the type I interferons.5,6 One of these genes, termed ISG54, because it encodes a 54-kDa protein, has a cis-acting element (TAGTTTCACTTTCCC, nucleotides -101 to -87) in its promoter.7 This element is responsible for the inducible expression of the ISG54 gene and is referred to as IFN-stimulated response element (ISRE).7 To create the pISRE-Luc plasmid, we inserted five direct repeats of this ISRE upstream of the basic promoter element (TATA box) and luciferase gene of the PathDetect cis-reporter plasmid backbone.
To test the specificity of the pISRE-Luc plasmid, we analyzed the response of the synthetic promoter by transfecting the plasmid into HeLa cells, then treating cells with increasing amounts of IFN-a (Figure 1, solid squares) and IFN-b (Figure 1, solid circles). For H eLa cells transfected with the pISRE-Luc plasmid and treated with 200 to 10,000 U/ml of IFN-a 9- to 16-fold increase in luciferase activity was seen. As expected, IFN-b treatment of the cells also stimulated luciferase expression from the pISRE-Luc plasmid up to 11-fold (Figure 1, solid circles). Although the pISRE-Luc plasmid was somewhat responsive to IFN-g (Figure 1, solid triangles), the luciferase activity was considerably higher for HeLa cells treated with IFN-a and IFN-b (Figure 1, solid squares and solid circles). This weak response of the pISRE-Luc plasmid to IFN-g could be attributed to the similarity of the ISRE and GAS sequences; it is also likely that types I and II interferons use a few common components to transmit their signals to the cell nucleus. However, neither IFN-a nor IFN-b induced luciferase expression from HeLa cells transfected with the pCIS-CK plasmid, a control plasmid that differs from the cis-reporter plasmids only in that it lacks an enhancer sequence (Figure 1, open squares and open circles). We conclude that the pISRE-Luc reporter plasmid is effective for monitoring the signaling pathways activated by type I interferons.
The human gene encoding guanylate-binding protein (GBP) is induced in fibroblasts by INF-g within 15 minutes of treatment. An IFN gamma-activating sequence (GAS) has been identified in the GBP promoter (nucleotides -123 to -103).1 To construct the pGAS-Luc plasmid, we inserted four direct repeats of this GAS enhancer sequence upstream of the TATA box and luciferase gene of the PathDetect cis-reporter plasmid backbone.4
To evaluate the pGAS-Luc plasmid, we performed a series of transfection experiments. For HeLa cells transfected with the pGAS-Luc plasmid, then treated with 20 to 1,000 U/ml of IFN-g (Figure 2, solid triangles), luciferase activity, increased 4- to 10-fold, as compared to cells transfected with the pGAS-Luc plasmid but receiving no interferon treatment (Figure 2, open circles). Neither IFN-a nor IFN-b treatment of HeLa cells transfected with pGAS-Luc increased luciferase expression significantly (Figure 2, solid squares and solid circles). IFN-g did not induce luciferase expression from HeLa cells transfected with the pCIS-CK control plasmid (Figure 2, open triangles). Therefore, the direct repeats of GAS in the pGAS-Luc plasmid are responsible for the inducible expression of luciferase, and the reporter plasmid pGAS-Luc can be used to monitor the signaling pathways activated by IFN-g.
Both the pISRE-Luc and pGAS-Luc plasmids functioned in other cell lines as well. Luciferase expression in Chinese hamster ovary (CHO) cells transfected with the pISRE-Luc plasmid, for example, increased by over 8-fold after treatment with 2,000 U/ml IFN-b (data not shown). However, due to the varying biochemical composition of cell lines, we expect that the luciferase levels resulting from transfection of these plasmids into different cell lines will also vary.
Stratagenes new pISRE-Luc and pGAS-Luc cis-reporter plasmid scan be used to monitor the activation of signal pathways by IFN-a, -b, or -g. Upon transfection of the pISRE-Luc plasmid into HeLa cells and subsequent treatment with IFN-a or IFN-b, a significant increase in luciferase activity was seen. Similarly, increased luciferase expression levels resulted from IFN-g treatment of HeLa cells transfected with the pGAS-Luc plasmid. Therefore, the activation status of signaling molecules and transcription factors can be monitored with these two new PathDetect cis-reporter plasmids.
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