Guanabenz Downregulates Inflammatory Responses via eIF2α Dependent and Independent Signaling
Abstract
:1. Introduction
2. Results
2.1. Guanabenz-Driven Suppression of Inflammatory Gene Expression
2.2. Prediction of the Pathways and Genes Involved in the Responses to Guanabenz
2.3. Guanabenz-Driven Downregulation of Egr2 and Csf2 in Lipopolysaccharide (LPS)-Stimulated Cells
2.4. Potential Linkage of Egr2 and Csf2 to IL1β, TNFα, and Cox2
2.5. Direct Linkage of eif2α Signaling to IL6, Cox2, and Csf2
3. Discussion
4. Experimental Section
4.1. Cell Culture
4.2. Microarray
4.3. qPCR and Western Blot Analysis
4.4. RNA Silencing Using siRNAs
4.5. Statistical Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Pathway | Ratio | p |
---|---|---|
Lysosome | 3.27 | 0.0007 |
T cell receptor signaling pathway | 3.29 | 0.0011 |
Hematopoietic cell lineage | 3.70 | 0.0011 |
Cytokine-cytokine receptor interaction | 2.38 | 0.0011 |
Fc gamma R-mediated phagocytosis | 3.39 | 0.0017 |
Rheumatoid arthritis | 3.39 | 0.0026 |
Chemokine signaling pathway | 2.42 | 0.0044 |
MAPK signaling pathway | 2.06 | 0.0077 |
ErbB signaling pathway | 2.84 | 0.0154 |
NOD-like receptor signaling pathway | 3.45 | 0.0154 |
Gene | Description | p1 | C1 | C2 | C3 | L1 | L2 | L3 | G1 | G2 | G3 |
---|---|---|---|---|---|---|---|---|---|---|---|
F3 | coagulation factor III | −0.997 | |||||||||
Has1 | hyaluronan synthase1 | −0.992 | |||||||||
Il12a | interleukin 12a | −0.979 | |||||||||
Egr2 | early growth response 2 | −0.967 | |||||||||
Csf2 | colony stimulating factor 2 | −0.964 | |||||||||
Serpinb2 | serine/cysteine peptidase inhibitor, B2 | −0.946 | |||||||||
Slc20a1 | solute carrier family 20, member 1 | −0.317 |
Gene | Accession Number | Forward Primer | Backward Primer |
---|---|---|---|
Mouse Primers | |||
IL1β | NM_008361 | 5′-GCCCATCCTCTGTGACTCAT-3′ | 5′-AGGCCACAGGTATTTTGTCG-3′ |
IL6 | NM_031168 | 5′-TTCCATCCAGTTGCCTTCTT-3′ | 5′-TCCACGATTTCCCAGAGAAC-3′ |
TNFα | NM_013693 | 5′-GAACTGGCAGAAGAGGCACT-3′ | 5′-AGGGTCTGGGCCATAGAACT-3′ |
Cox2 | AF378830 | 5′-CCCCCACAGTCAAAGACACT-3′ | 5′-CTCATCACCCCACTCAGGAT-3′ |
Csf2 | NM_009969 | 5′-GAGGCCATCAAAGAAGCCCT-3′ | 5′-AAATTGCCCCGTAGACCCTG-3′ |
eIF2α | NM_026114 | 5′-GAATGTACTCCAGATTGGCTGACTAC-3′ | 5′-CCTCAATGTGAAGACCTGTATCGA-3′ |
Egr2 | NM_010118 | 5′-GATCTGCATGCGAAACTTCA-3′ | 5′-CACTGCTCTTCCGTTCCTTC-3′ |
GAPDH | NM_008084 | 5′-TGCACCACCAACTGCTTAG-3′ | 5′-GGATGCAGGGATGATGTTC-3′ |
Human Primers | |||
IFNγ | NM_000619 | 5′-TTCAGCTCTGCATCGTTTTG-3′ | 5′-TCTTTTGGATGCTCTGGTCA-3′ |
IL2 | NM_000586 | 5′-GCAACTCCTGTCTTGCATTG-3′ | 5′-GCCTTCTTGGGCATGTAAAA-3′ |
IL13 | NM_002188 | 5′-GTACTGTGCAGCCCTGGAAT-3′ | 5′-TTTACAAACTGGGCCACCTC-3′ |
TNFα | NM_000594 | 5′-CAGAGGGCCTGTACCTCATC-3′ | 5′-GGAAGACCCCTCCCAGATAG-3′ |
GAPDH | NM_001289745 | 5′-GCACCGTCAAGGCTGAGAAC-3′ | 5′-ATGGTGGTGAAGACGCCAGT-3′ |
Mouse siRNAs | |||
Csf2 (GM-CSF) | 5′-GCGGAAGACAAACGAGAGA-3′ | 5′-GCCUGAAGAUAUUCGAGCA-3′ | |
5′-AUGAAGAGGUAGAAGUCGU-3′ | 5′-CCAGCUACUACCAGACAUA-3′ | ||
eIF2α (eIF2s1) | 5′-UCGAGCAGAUAUUGAAGUA-3′ | 5′-CAUGAUUCUUCUUAGUGAA-3′ | |
5′-UGUCACAAGUUAAAGCCAA-3′ | 5′-GAACUCAAUGGGCAAGUAA-3′ | ||
Egr2 | 5′-CGACCUCGAAAGUACCCUA-3′ | ||
Control #1 (NC1) for Egr2 | 5′-UGUACUGCUUACGAUUCGG-3′ | ||
Control #2 (NC2) for Csf2 & eIF2α | 5′-UGGUUUACAUGUCGACUAA-3′ | 5′-UGGUUUACAUGUUGUGUGA-3′ | |
5′-UGGUUUACAUGUUUUCUGA-3′ | 5′-UGGUUUACAUGUUUUCCUA-3′ |
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Takigawa, S.; Chen, A.; Nishimura, A.; Liu, S.; Li, B.-Y.; Sudo, A.; Yokota, H.; Hamamura, K. Guanabenz Downregulates Inflammatory Responses via eIF2α Dependent and Independent Signaling. Int. J. Mol. Sci. 2016, 17, 674. https://doi.org/10.3390/ijms17050674
Takigawa S, Chen A, Nishimura A, Liu S, Li B-Y, Sudo A, Yokota H, Hamamura K. Guanabenz Downregulates Inflammatory Responses via eIF2α Dependent and Independent Signaling. International Journal of Molecular Sciences. 2016; 17(5):674. https://doi.org/10.3390/ijms17050674
Chicago/Turabian StyleTakigawa, Shinya, Andy Chen, Akinobu Nishimura, Shengzhi Liu, Bai-Yan Li, Akihiro Sudo, Hiroki Yokota, and Kazunori Hamamura. 2016. "Guanabenz Downregulates Inflammatory Responses via eIF2α Dependent and Independent Signaling" International Journal of Molecular Sciences 17, no. 5: 674. https://doi.org/10.3390/ijms17050674