The online version of this article (https://doi.org/10.1186/s12885-017-3896-y) contains supplementary material, which is available to authorized users.
The original version of this article was revised: we have been notified that the tagging of one of the author names was done incorrectly in the XML version of the paper. The online and pdf versions of this paper are not affected by the change. Incorrect name tagging: Last Name: Gerard Toussaint; First Name: L. Correct author name tagging: Last Name: Toussaint; Middle Name: Gerard; First Name: L.
A correction to this article is available online at https://doi.org/10.1186/s12885-018-5238-0.
The circadian clock is the basis for biological time keeping in eukaryotic organisms. The clock mechanism relies on biochemical signaling pathways to detect environmental stimuli and to regulate the expression of clock-controlled genes throughout the body. MAPK signaling pathways function in both circadian input and output pathways in mammals depending on the tissue; however, little is known about the role of p38 MAPK, an established tumor suppressor, in the mammalian circadian system. Increased expression and activity of p38 MAPK is correlated with poor prognosis in cancer, including glioblastoma multiforme; however, the toxicity of p38 MAPK inhibitors limits their clinical use. Here, we test if timed application of the specific p38 MAPK inhibitor VX-745 reduces glioma cell invasive properties in vitro.
The levels and rhythmic accumulation of active phosphorylated p38 MAPK in different cell lines were determined by western blots. Rhythmic luciferase activity from clock gene luciferase reporter cells lines was used to test the effect of p38 MAPK inhibition on clock properties as determined using the damped sine fit and Levenberg–Marquardt algorithm. Nonlinear regression and Akaike’s information criteria were used to establish rhythmicity. Boyden chamber assays were used to measure glioma cell invasiveness following time-of-day-specific treatment with VX-745. Significant differences were established using t-tests.
We demonstrate the activity of p38 MAPK cycles under control of the clock in mouse fibroblast and SCN cell lines. The levels of phosphorylated p38 MAPK were significantly reduced in clock-deficient cells, indicating that the circadian clock plays an important role in activation of this pathway. Inhibition of p38 MAPK activity with VX-745 led to cell-type-specific period changes in the molecular clock. In addition, phosphorylated p38 MAPK levels were rhythmic in HA glial cells, and high and arrhythmic in invasive IM3 glioma cells. We show that inhibition of p38 MAPK activity in IM3 cells at the time of day when the levels are normally low in HA cells under control of the circadian clock, significantly reduced IM3 invasiveness.
Glioma treatment with p38 MAPK inhibitors may be more effective and less toxic if administered at the appropriate time of the day.
Additional file 1: Full western blots of gels from Fig. 1. (DOXC 243 kb)
Additional file 2: Full western blots of gels from Fig. 5. (DOXC 200 kb)
Mohawk JA, Green CB, Takahashi JS. Central and peripheral circadian clocks in mammals. Annu Rev Neurosci. 2012;35:445–62. CrossRef
Dibner C, Schibler U, Albrecht U. The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu Rev Physiol. 2010;72:517–49. CrossRef
Koike N, Yoo SH, Huang HC, Kumar V, Lee C, Kim TK, Takahashi JS. Transcriptional architecture and chromatin landscape of the core circadian clock in mammals. Science. 2012;338:349–54. CrossRef
Ko CH, Takahashi JS. Molecular components of the mammalian circadian clock. Hum Mol Genet. 2006;15:271–7. CrossRef
Lande-Diner L, Boyault C, Kim JY, Weitz CJ. A positive feedback loop links circadian clock factor CLOCK-BMAL1 to the basic transcriptional machinery. Proc Natl Acad Sci U S A. 2013;110:16021–6. CrossRef
Guillaumond F, Dardente H, Giguere V, Cermakian N. Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors. J Biol Rhythm. 2005;20:391–403. CrossRef
Goldsmith CS, Bell-Pedersen D. Diverse roles for MAPK signaling in circadian clocks. Adv Genet. 2013;84:1–39. CrossRef
Obrietan K, Impey S, Storm DR. Light and circadian rhythmicity regulate MAP kinase activation in the suprachiasmatic nuclei. Nat Neurosci. 1998;1:693–700. CrossRef
Pizzio GA, Hainich EC, Ferreyra GA, Coso OA, Golombek DA. Circadian and photic regulation of ERK, JNK and p38 in the hamster SCN. Neuroreport. 2003;14:1417–9. CrossRef
Cermakian N, Pando MP, Thompson CL, Pinchak AB, Selby CP, Gutierrez L, Wells DE, Cahill GM, Sancar A, Sassone-Corsi P. Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases. Curr Biol. 2002;12:844–8. CrossRef
Yoshitane H, Honma S, Imamura K, Nakajima H, Nishide SY, Ono D, Kiyota H, Shinozaki N, Matsuki H, Wada N, et al. JNK regulates the photic response of the mammalian circadian clock. EMBO Rep. 2012;13:455–61. CrossRef
Bennett LD, Beremand P, Thomas TL, Bell-Pedersen D. Circadian activation of the mitogen-activated protein kinase MAK-1 facilitates rhythms in clock-controlled genes in Neurospora crassa. Eukaryot Cell. 2013;12:59–69. CrossRef
Williams JA, Su HS, Bernards A, Field J, Sehgal A. A circadian output in drosophila mediated by neurofibromatosis-1 and Ras/MAPK. Science. 2001;293:2251–6. CrossRef
Lamb TM, Goldsmith CS, Bennett L, Finch KE, Bell-Pedersen D. Direct transcriptional control of a p38 MAPK pathway by the circadian clock in Neurospora crassa. PLoS One. 2011;6:e27149. CrossRef
Vitalini MW, de Paula RM, Goldsmith CS, Jones CA, Borkovich KA, Bell-Pedersen D. Circadian rhythmicity mediated by temporal regulation of the activity of p38 MAPK. Proc Natl Acad Sci U S A. 2007;104:18223–8. CrossRef
Hayashi Y, Sanada K, Hirota T, Shimizu F, Fukada Y. p38 mitogen-activated protein kinase regulates oscillation of chick pineal circadian clock. J Biol Chem. 2003;278:25166–71. CrossRef
Chik CL, Mackova M, Price D, Ho AK. Adrenergic regulation and diurnal rhythm of p38 mitogen-activated protein kinase phosphorylation in the rat pineal gland. Endocrinology. 2004;145:5194–201. CrossRef
Ko ML, Shi L, Tsai JY, Young ME, Neuendorff N, Earnest DJ, Ko GY. Cardiac-specific mutation of clock alters the quantitative measurements of physical activities without changing behavioral circadian rhythms. J Biol Rhythm. 2011;26:412–22. CrossRef
Zarubin T, Han J. Activation and signaling of the p38 MAP kinase pathway. Cell Res. 2005;15:11–8. CrossRef
Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in cancer development. Nature rev. Cancer. 2009;9:537–49. PubMed
Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, Appella E, Fornace AJ Jr. Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J. 1999;18:6845–54. CrossRef
Han J, Sun P. The pathways to tumor suppression via route p38. Trends Biochem Sci. 2007;32:364–71. CrossRef
Loesch M, Chen G. The p38 MAPK stress pathway as a tumor suppressor or more? Front. Bioscience. 2008;13:3581–93.
del Barco BI, Nebreda AR. Roles of p38 MAPKs in invasion and metastasis. Biochem Soc Trans. 2012;40:79–84. CrossRef
Handra-Luca A, Lesty C, Hammel P, Sauvanet A, Rebours V, Martin A, Fagard R, Flejou JF, Faivre S, Bedossa P, et al. Biological and prognostic relevance of mitogen-activated protein kinases in pancreatic adenocarcinoma. Pancreas. 2012;41:416–21. CrossRef
Lee JC, Kumar S, Griswold DE, Underwood DC, Votta BJ, Adams JL. Inhibition of p38 MAP kinase as a therapeutic strategy. Immunopharmacology. 2000;47:185–201. CrossRef
Yang K, Liu Y, Liu Z, Liu J, Liu X, Chen X, Li C, Zeng Y. p38γ overexpression in gliomas and its role in proliferation and apoptosis. Sci Rep 2013;3:2089.
Demuth T, Reavie LB, Rennert JL, Nakada M, Nakada S, Hoelzinger DB, Beaudry CE, Henrichs AN, Anderson EM, Berens ME. MAP-ing glioma invasion: mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival. Mol Cancer Ther. 2007;6:1212–22. CrossRef
Sooman L, Lennartsson J, Gullbo J, Bergqvist M, Tsakonas G, Johansson F, Edqvist PH, Ponten F, Jaiswal A, Navani S, et al. Vandetanib combined with a p38 MAPK inhibitor synergistically reduces glioblastoma cell survival. Med Oncol. 2013;30:638. CrossRef
Hammaker D, Firestein GS. "Go upstream, young man": lessons learned from the p38 saga. Ann Rheum Dis. 2010;69(Suppl 1):i77–82. CrossRef
Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM, Hong HK, Oh WJ, Yoo OJ, et al. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U S A. 2004;101:5339–46. CrossRef
Bae K, Jin X, Maywood ES, Hastings MH, Reppert SM, Weaver DR. Differential functions of mPer1, mPer2, and mPer3 in the SCN circadian clock. Neuron. 2001;30:525–36. CrossRef
Farnell YF, Shende VR, Neuendorff N, Allen GC, Earnest DJ. Immortalized cell lines for real-time analysis of circadian pacemaker and peripheral oscillator properties. Eur J Neurosci. 2011;33:1533–40. CrossRef
Ramanathan C, Khan SK, Kathale ND, Xu H, Liu AC. Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters. J Vis Exp. 2012;67:4234.
Koo S, Martin GS, Schulz KJ, Ronck M, Toussaint LG. Serial selection for invasiveness increases expression of miR-143/miR-145 in glioblastoma cell lines. BMC Cancer. 2012;12:143. CrossRef
Allen G, Rappe J, Earnest DJ, Cassone VM. Oscillating on borrowed time: diffusible signals from immortalized suprachiasmatic nucleus cells regulate circadian rhythmicity in cultured fibroblasts. J Neurosci. 2001;21:7937–43. CrossRef
Balsalobre A, Damiola F, Schibler UA. Serum shock induces circadian gene expression in mammalian tissue culture cells. Cell. 1998;93:929–37. CrossRef
Farnell YZ, Allen GC, Nahm SS, Neuendorff N, West JR, Chen WJ, Earnest DJ. Neonatal alcohol exposure differentially alters clock gene oscillations within the suprachiasmatic nucleus, cerebellum, and liver of adult rats. Alcohol Clin Exp Res. 2008;32:544–52. CrossRef
Nahm SS, Farnell YZ, Griffith W, Earnest DJ. Circadian regulation and function of voltage-dependent calcium channels in the suprachiasmatic nucleus. J Neurosci. 2005;25:9304–8. CrossRef
Yagita K, Yamanaka I, Koinuma S, Shigeyoshi Y, Uchiyama Y. Mini screening of kinase inhibitors affecting period-length of mammalian cellular circadian clock. Acta Histochem Cytochem. 2009;42:89–93. CrossRef
Lowrey PL, Shimomura K, Antoch MP, Yamazaki S, Zemenides PD, Ralph MR, Menaker M, Takahashi JS. Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science. 2000;288:483–92. CrossRef
Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, Young MW. The drosophila clock gene double-time encodes a protein closely related to human casein kinase I epsilon. Cell. 1998;94:97–107. CrossRef
Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young MW. double-time is a novel drosophila clock gene that regulates PERIOD protein accumulation. Cell. 1998;94:83–95. CrossRef
Natarajan SR, Doherty JB. p38 MAP kinase inhibitors: evolution of imidazole-based and pyrido-pyrimidin-2-one lead classes. Curr Top Med Chem. 2005;5:987–1003. CrossRef
Natarajan SR, Wisnoski DD, Singh SB, Stelmach JE, O'Neill EA, Schwartz CD, Thompson CM, Fitzgerald CE, O'Keefe SJ, Kumar S, et al. p38 MAP kinase inhibitors. Part 1: design and development of a new class of potent and highly selective inhibitors based on 3,4-dihydropyrido[3,2-d]pyrimidone scaffold. Bioorg Med Chem Lett. 2003;13:273–6. CrossRef
Kim SM, Neuendorff N, Chapkin RS, Earnest DJ. Role of inflammatory signaling in the differential effects of saturated and poly-unsaturated fatty acids on peripheral circadian clocks. EBioMedicine. 2016;7:100–11. CrossRef
Yeung YT, McDonald KL, Grewal T, Munoz L. Interleukins in glioblastoma pathophysiology: implications for therapy. Br J Pharmacol. 2013;168:591–606. CrossRef
Yagita K, Tamanini F, van Der Horst GT, Okamura H. Molecular mechanisms of the biological clock in cultured fibroblasts. Science. 2001;292:278–81. CrossRef
Menger GJ, Allen GC, Neuendorff N, Nahm SS, Thomas TL, Cassone VM, Earnest DJ. Circadian profiling of the transcriptome in NIH/3T3 fibroblasts: comparison with rhythmic gene expression in SCN2.2 cells and the rat SCN. Physiol Genomics. 2007;29:280–9. CrossRef
Shimizu K, Okada M, Nagai K, Fukada Y. Suprachiasmatic nucleus circadian oscillatory protein, a novel binding partner of K-Ras in the membrane rafts, negatively regulates MAPK pathway. J Biol Chem. 2003;278:14920–5. CrossRef
Menet JS, Rodriguez J, Abruzzi KC, Rosbash M. Nascent-Seq reveals novel features of mouse circadian transcriptional regulation. elife. 2012;1:e00011. CrossRef
Hirota T, Lewis WG, Liu AC, Lee JW, Schultz PG, Kay SA. A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta. Proc Natl Acad Sci U S A. 2008;105:20746–51. CrossRef
Fabian MA, Biggs WH 3rd, Treiber DK, Atteridge CE, Azimioara MD, Benedetti MG, Carter TA, Ciceri P, Edeen PT, Floyd M, et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol. 2005;23:329–36. CrossRef
Hasegawa M, Cahill GM. Regulation of the circadian oscillator in Xenopus retinal photoreceptors by protein kinases sensitive to the stress-activated protein kinase inhibitor, SB 203580. J Biol Chem. 2004;279:22738–46. CrossRef
Chansard M, Molyneux P, Nomura K, Harrington ME. Fukuhara C: c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms. Neurosci. 2007;145:812–23. CrossRef
Kon N, Sugiyama Y, Yoshitane H, Kameshita I, Fukada Y. Cell-based inhibitor screening identifies multiple protein kinases important for circadian clock oscillations. Commun Integr Biol. 2015;8:e982405. CrossRef
Hai T, Curran T. Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci U S A. 1991;88:3720–4. CrossRef
Schindler JF, Monahan JB, Smith WG. p38 pathway kinases as anti-inflammatory drug targets. J Dent Res. 2007;86:800–11. CrossRef
Kiessling S, Beaulieu-Laroche L, Blum ID, Landgraf D, Welsh DK, Storch KF, Labrecque N, Cermakian N. Enhancing circadian clock function in cancer cells inhibits tumor growth. BMC Biol. 2017;15:13. CrossRef
Miyazaki K, Wakabayashi M, Hara Y, Ishida N. Tumor growth suppression in vivo by overexpression of the circadian component, PER2. Genes Cells. 2010;15:351–8. CrossRef
Relogio A, Thomas P, Medina-Perez P, Reischl S, Bervoets S, Gloc E, Riemer P, Mang-Fatehi S, Maier B, Schafer R, et al. Ras-mediated deregulation of the circadian clock in cancer. PLoS Genet. 2014;10:e1004338. CrossRef
Kostaras X, Cusano F, Kline GA, Roa W, Easaw J, Team APCT. Use of dexamethasone in patients with high-grade glioma: a clinical practice guideline. Curr Oncol. 2014;21:E493–503. CrossRef
- Inhibition of p38 MAPK activity leads to cell type-specific effects on the molecular circadian clock and time-dependent reduction of glioma cell invasiveness
Charles S. Goldsmith
Sam Moon Kim
L. Gerard Toussaint
David J. Earnest
- BioMed Central
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