( New luciferase vectors for studying s...)Signal Transduction Reporting Systems Using Cis-Acting Enhancer,,,Elements

New luciferase vectors for studying signal transduction pathways

Li Xu Tim Sanchez Chao-Feng Zheng
Stratagene Cloning Systems, Inc.

Enhancer elements are the convergent points for many intracellular signal transduction pathways. For simple, rapid and convenient assessment of the in vivo activation of these pathways, Stratagene has designed the PathDetect in vivo signal transduction pathway cis-reporting systems. Each of the cis-reporter plasmids contains the luciferase reporter gene driven by a basic promoter element (TATA box) joined to tandem repeats of AP-1, CRE, SRE, NF-kB or SRF binding elements. When a plasmid expressing the gene of interest is cotransfected into mammalian cells with a cis-reporter plasmid, increased luciferase activity indicates transcription activation. The PathDetect cis-reporting systems can be used for studying the in vivo effects of a new gene, growth factor or drug candidate on signaling pathways. The effects of extracellular stimuli can also be studied with these systems.

Extracellular signals trigger the sequential activation of a series of intracellular signaling molecules, such as protein kinases and phosphatases.^1,2 The final step in the activation of many pathways is the binding of an activated transcription factor to specific enhancer elements found in the promoters of various cellular genes. This binding modulates transcription of these genes. The transcription level of cellular genes reflects the activation status of the involved signaling events. Transcription and expression levels are typically monitored by Northern and Western blotting. To determine the activation of the promoter, and hence signaling events, we have designed vectors that use a reporter gene in place of the cellular gene. The reporter gene encodes the firefly luciferase enzyme that can be monitored easily, inexpensively and quantitatively. Reporter enzyme assays replace tedious blotting procedures and are widely used due to their convenience, sensitivity and ability to generate reliable in vivo data.³

PathDetect Cis-Reporting Systems

figure 1

Each of the PathDetect in vivo signal transduction pathway cis-reporting systems includes one of five cis- reporter plasmids and a positive control plasmid. Positive control plasmids express gene products known to activate the signaling pathway converging at the corresponding enhancer elements. The five reporter plasmids (figure 1) of the PathDetect cis-reporting systems contain tandem repeats of one of the following enhancer elements: cyclic AMP response element (CRE), serum response element (SRE), nuclear factor kB (NF-kB), activator protein 1 (AP-1) or serum response factor (SRF). Together with a TATA box, these enhancer elements control the expression of the luciferase reporter gene located downstream.

Since most enhancer elements can be regulated by more than one transcription activator, each enhancer element can be used to monitor more than one signal transduction pathway. When a plasmid expressing a gene of interest is cotransfected into mammalian cells along with one of these cis-reporter plasmids, increased luciferase activity indicates transcription activation. The PathDetect cis-reporting systems are useful tools for assessing the potential involvement of a gene product in a pathway. For more specific assessment, the PathDetect trans-reporting systems can be used.⁴

Response to Overexpression of Known Activators

figure 2

To test the performance of these plasmids (figure 2), we cotransfected each of the individual reporter plasmids into HeLa cells together with an expression vector of a known activator, either MEK kinase (MEKK) or the catalytic subunit of cAMP-dependent protein kinase (PKA). The empty vector without an insert was used as a negative control.^5,6 The overexpression of both MEKK and PKA stimulated luciferase expression from pAP1-Luc and pSRE-Luc plasmids. However, pCRE-Luc responded only to PKA and pNFkB-Luc only to MEKK. These results are consistent with previous reports that both MEKK and PKA are capable of activating the expression of genes containing SRE and AP-1 or NF-kB binding elements in their promoter, while the CRE element found in somatostatin promoter is very specific to PKA.^7,8-12 MEKK, a protein kinase known to activate the stress signaling pathways and the MAP kinase pathway, also activates signaling molecules converging at NF-kB, as measured with pNFkB-Luc.

As true for all transfection experiments, conditions must be optimized to achieve high activation levels. The variables to consider include the choice of cell line, transfection method and protocol. In addition, the amounts of reporter vector and expression vector encoding the gene of interest must be optimized.

Conclusions

Stratagenes PathDetect cis-reporter plasmids were constructed for studying the in vivo activation of signal transduction pathways. These vectors contain the luciferase reporter gene driven by a basic promoter element (TATA box) joined to tandem repeats of CRE, SRE, AP-1, NF-kB or SRF binding elements. When a cis-reporter plasmid and an experimental mammalian expression construct are cotransfected into mammalian cells, increased luciferase activity indicates transcription activation and the involvement of the gene product in signaling pathways converging at these cis-acting elements. The PathDetect in vivo signal transduction pathway cis- reporting systems can be used for studying the in vivo effects of a new gene, growth factor or drug candidate. When more specific assessment of a gene product is desired, the PathDetect trans-reporting systems can be used.

REFERENCES

1. Karin, M., and Hunter, T. (1995) Curr. Biol. 5: 747-757.

2. Treisman, R. (1996) Curr. Opin. Cell Biol. 8: 205-215.

3. Wood, K.V. (1995) Curr. Opin. Biotechnol. 5: 50-58.

4. Xu, L., and Zheng, C.-F. (1997) Strategies 10:1-3.

5. Lange-Carter, C., et al. (1993) Science 260: 315-319.

6. Uhler, M.D., et al. (1986) Proc. Natl. Acad. Sci. USA 83: 1300-1304.

7. Galang, C.K., et al. (1994) Oncogene 9: 2913-2921.

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