Part:BBa_K3338005

Interleukin-6 Promoter mutagenized (IL-6 P_mut)

Usage and Biology

The human interleukin-6 promoter originally controls the expression of the cytokine Il-6 that is involved in the regulation of the acute-phase response to injury and infection but also other processes like hematopoiesis and embryonal development (Heinrich el al. 2003). The regulation of transcription exerted by the IL-6 promoter is mainly controlled by several cis-acting response elements present within the promoter region including binding motifs for NF-κB, NF-IL6, CREB, C/EBP, AP-1 and AP-2 (Xiao et al. 2004, Beetz et al. 2000). The regulation of the IL-6 promoter depends on the cell type and the stimulus. This means that different transcription factors are needed under different conditions. In our study we used LPS to induce IL-6 promoter activation. In this case the transcription factors NF-κB and AP1 play important roles (Xiao et al. 2004, Liu et al. 2018). The activation of the promoter is achieved by the cooperative binding of NF-κB and c-Jun (AP-1) (Xiao et al. 2004). AP-1- and NF-κB-translocation to the nucleus is triggered downstream of Toll like receptor (TLR) signaling cascades involving TRIF, MyD88, RIPK1 and TAK1. Toll like receptors are activated by a variety of pathogen-associated molecular patterns (Kawai and Akira 2007).

In our study it was tested for a LPS-sensitivity for use in our inflammatory toxin sensor. Unfortunately, the mutagenized promoter is not sensitive to LPS supplementation to the medium of Hela cells.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1347
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 220
  Illegal BamHI site found at 395
  Illegal XhoI site found at 1581
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 340
  Illegal BsaI.rc site found at 387
  Illegal BsaI.rc site found at 696
  

Characterization

To characterize our mutated IL-6 promoter we combined it with the reporter gene hGLuc and cloned it into the mammalian expression vector pEGFP-C2 (BBa_K3338020). The vector was linearized with AseI and HindIII leading to the excision of the CMV-EGFP-cassette. The interleukin-6 promoter and the downstream hGLuc reporter were synthesizes by IDT with 20 bp overhangs matching the ends of the linearized plasmid. The assembly was achieved using the NEBuilder® HiFi DNA Assembly Cloning kit from NEB. The sequence was verified using sanger sequencing. The final plasmid is depicted in figure 1.

Figure 1: Vector map of IL-6_mut-hGLuc in pEGFP-C2.

The final vector was transfected into HeLa cells. The cells were subsequently treated with different amounts of LPS for 3 h. 24 h and 48 h after LPS-treatment, the supernatants were removed to perform a luciferase assay. The results are shown in figure 2.

Figure 2: Relative activity of IL6mut promoter in HeLa cells 24 hours (A) and 48 hours (B) after treatment with different concentrations of LPS for 3 hours, normalized to untreated control (0 µg/mL LPS). Data shown represents mean ± SEM of n=4 biological replicates. Statistical analysis was performed by unpaired t-test in comparison to untreated control, significance level: 10 %, significance is indicated by asterisk.

It can be clearly seen that the mutagenized IL-6 promoter does not show induction after LPS stimulation. This could be because the mutation was introduced in a necessary region of the IL-6 promoter. However, this was not predicted by transcription factor binding site prediction programms.

Summary

In conclusion the mutagenized IL-6 promoter is not applicable for our inflammatory toxin sensor because for the sensor it is crucial that the promoter senses LPS outside the cell.

References

Beetz, A., Peter, R. U., Oppel, T., Kaffenberger, W., Rupec, R. A., Meyer, M., van Beuningen, D., Kind, P., & Messer, G. (2000). NF-kappaB and AP-1 are responsible for inducibility of the IL-6 promoter by ionizing radiation in HeLa cells. International journal of radiation biology, 76(11), 1443–1453.

Heinrich, P. C., Behrmann, I., Haan, S., Hermanns, H. M., Müller-Newen, G., & Schaper, F. (2003). Principles of interleukin (IL)-6-type cytokine signalling and its regulation. The Biochemical journal, 374(Pt 1), 1–20.

Kawai, T., & Akira, S. (2007). Signaling to NF-kappaB by Toll-like receptors. Trends in molecular medicine, 13(11), 460–469.

Liu, X., Yin, S., Chen, Y., Wu, Y., Zheng, W., Dong, H., Bai, Y., Qin, Y., Li, J., Feng, S., & Zhao, P. (2018). LPS‑induced proinflammatory cytokine expression in human airway epithelial cells and macrophages via NF‑κB, STAT3 or AP‑1 activation. Molecular medicine reports, 17(4), 5484–5491.

Xiao, W., Hodge, D. R., Wang, L., Yang, X., Zhang, X., & Farrar, W. L. (2004). NF-kappaB activates IL-6 expression through cooperation with c-Jun and IL6-AP1 site, but is independent of its IL6-NFkappaB regulatory site in autocrine human multiple myeloma cells. Cancer biology & therapy, 3(10), 1007–1017.