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The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide

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Abstract

The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1β, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 μg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/β, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.

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Abbreviations

CK2:

casein kinase II

HSP:

heat shock protein

IFN:

interferon

IκB:

inhibitor of NF-κB

IKK:

IκB kinase

IL:

interleukin

IRF:

interferon regulatory factor

JNK:

c-Jun N-terminal kinase

LPS:

lipopolysaccharide

MAPK:

mitogen-activated protein kinase

NF-κB:

nuclear factor κB

TLR:

Toll-like receptor

TNF:

tumor necrosis factor

References

  1. Medzhitov, R. (2010) Inflammation 2010: new adventures of an old flame, Cell, 140, 771–776.

    Article  PubMed  CAS  Google Scholar 

  2. Morris, M. C., Gilliam, E. A., and Li, L. (2015) Innate immune programming by endotoxin and its pathological consequences, Front. Immunol., 5, 680.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Muralidharan, S., and Mandrekar, P. (2013) Cellular stress response and innate immune signaling: integrating path-ways in host defense and inflammation, J. Leukoc. Biol., 94, 1167–1184.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Takeda, K., and Akira, S. (2005) TLR signaling pathways, Semin. Immunol., 16, 3–9.

    Article  CAS  Google Scholar 

  5. Kumar, A., Takada, Y., Boriek, A. M., and Agarwal, B. B. (2004) Nuclear factor κB: its role in health and disease, J. Mol. Med. (Berl.), 82, 434–448.

    Article  CAS  Google Scholar 

  6. Glushkova, O. V., Parfenyuk, S. B., Khrenov, M. O., Novoselova, T. V., Lunin, S. M., Fesenko, E. E., and Novoselova, E. G. (2013) Inhibitors of TLR4, NF-κB, and SAPK/JNK signaling reduce the toxic effect of lipopolysaccharide on RAW 264.7 cells, J. Immunotoxicol., 10, 133–140.

    PubMed  CAS  Google Scholar 

  7. Litchfield, D. W. (2003) Protein kinase CK2: structure, regulation and role in cellular decisions of life and death, Biochem. J., 369, 1–15.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Montenarh, M. (2014) Protein kinase CK2 and angiogene-sis, Adv. Clin. Exp. Med., 23, 153–158.

    Article  PubMed  Google Scholar 

  9. Yu, M., Yeh, J., and Van Waes, C. (2006) Protein kinase casein kinase 2 mediates inhibitor-kappaB kinase and aber-rant nuclear factor-kappaB activation by serum factor(s) in head and neck squamous carcinoma cells, Cancer Res., 66, 6722–6731.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Venerando, A., Ruzzene, M., and Pinna, L. A. (2014) Casein kinase: the triple meaning of a misnomer, Biochem. J., 460, 141–156.

    Article  PubMed  CAS  Google Scholar 

  11. Romieu-Mourez, R., Landesman-Bollag, E., Seldin, D. C., and Sonenshein, G. E. (2002) Protein kinase CK2 pro-motes aberrant activation of nuclear factor-kappaB, trans-formed phenotype, and survival of breast cancer cells, Cancer Res., 62, 6770–6778.

    PubMed  CAS  Google Scholar 

  12. Dominguez, I., Sonenshein, G. E., and Seldin, D. C. (2009) Protein kinase CK2 in health and disease: CK2 and its role in Wnt and NF-kappaB signaling: linking develop-ment and cancer, Cell. Mol. Life Sci., 66, 1850–1857.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Volodina, Y. L., and Shtil, A. A. (2012) Casein kinase 2 is a universal regulator of cell survival, Mol. Biol., 46, 423–433.

    Article  CAS  Google Scholar 

  14. Christian, F., Smith, E. L., and Carmody, R. J. (2016) The regulation of NF-кB subunits by phosphorylation, Cells, 5; doi: 10.3390/cells5010012.

    Google Scholar 

  15. Bode, J. G., Ehlting, C., and Haussinger, D. (2012) The macrophage response towards LPS and its control through the p38(MAPK)-STAT3 axis, Cell. Signal., 24, 1185–1194.

    Article  PubMed  CAS  Google Scholar 

  16. Zarubin, T., and Han, J. (2005) Activation and signaling of the p38 MAP kinase pathway, Cell Res., 15, 11–18.

    Article  PubMed  CAS  Google Scholar 

  17. Cuadrado, A., and Nebreda, A. R. (2010) Mechanisms and functions of p38 MAPK signaling, Biochem. J., 429, 403–417.

    Article  PubMed  CAS  Google Scholar 

  18. Glushkova, O. V., Khrenov, M. O., Novoselova, T. V., Lunin, S. M., Fesenko, E. E., and Novoselova, E. G. (2015) The role of CK2 protein kinase in stress response of RAW 264.7 macrophages, Dokl. Biol. Sci., 464, 260–262.

    Article  PubMed  CAS  Google Scholar 

  19. Allende, J. E., and Allende, C. C. (1995) Protein kinase CK2: an enzyme with multiple substrates and a puzzling regulation, FASEB J., 9, 313–323.

    Article  PubMed  CAS  Google Scholar 

  20. Kato, T., Jr., Delhase, M., Hoffmann, A., and Karin, M. (2003) CK2 is a C-terminal IkappaB kinase responsible for NF-kappaB activation during the UV response, Mol. Cell, 12, 829–839.

    Article  PubMed  CAS  Google Scholar 

  21. Scaglioni, P. P., Yung, T. M., Cai, L. F., Erdjument-Bromage, H., Kaufman, A. J., Singh, B., Teruya-Feldstein, J., Tempst, P., and Pandolfi, P. P. (2006) A CK2-dependent mechanism for degradation of the PML tumor suppressor, Cell, 126, 269–283.

    Article  PubMed  CAS  Google Scholar 

  22. Tsuchiya, Y., Asano, T., Nakayama, K., Kato, T., Jr., Karin, M., and Kamata, H. (2010) Nuclear IKKbeta is an adaptorprotein for IkappaBalpha ubiquitination and degradation in UV-induced NF-kappaB activation, Mol. Cell, 39, 570–582.

    Article  PubMed  CAS  Google Scholar 

  23. Terazawa, S., Mori, S., Nakajima, H., Yasuda, M., and Imokawa, G. (2015) The UVB-stimulated expression of transglutaminase 1 is mediated predominantly via the NFκB signaling pathway: new evidence of its significant attenuation through the specific interruption of the p38/MSK1/NFкBp65 Ser276 axis, PLoS One, 10, e0136311.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Kumar, S., Boehm, J., and Lee, J. C. (2003) p38 MAP kinases: key signaling molecules as therapeutic targets for inflammatory diseases, Nat. Rev. Drug Discov., 2, 717–726.

    Article  PubMed  CAS  Google Scholar 

  25. Singh, N. N., and Ramji, D. P. (2008) Protein kinase CK2, an important regulator of the inflammatory response, J. Mol. Med. (Berl.), 86, 887–897.

    Article  CAS  Google Scholar 

  26. Huang, J., Chen, Z., Li, J., Chen, Q., Li, J., Gong, W., Huang, J., Liu, P., and Huang, H. (2017) Protein kinase CK2α catalytic subunit ameliorates diabetic renal inflam-matory fibrosis via NF-κB signaling pathway, Biochem. Pharmacol., 132, 102–117.

    Article  PubMed  CAS  Google Scholar 

  27. Yang, X.-D., Huang, B., Li, M., Lamb, A., Kelleher, N. L., and Chen, L.-F. (2009) Negative regulation of NF-κB action by Set9-mediated lysine methylation of the RelA subunit, EMBO J., 28, 1055–1066.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Zhong, H., Voll, R. E., and Ghosh, S. (1998) Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent inter-action with the coactivator CBP/p300, Mol. Cell, 1, 661–671.

    Article  PubMed  CAS  Google Scholar 

  29. Vermeulen, L., De Wilde, G., Van Damme, P., Vanden Berghe, W., and Haegeman, G. (2003) Transcriptional acti-vation of the NF-κB p65 subunit by mitogen-and stress-activated protein kinase-1 (MSK1), EMBO J., 22, 1313–1324.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Brasier, A. R., Tian, B., Jamaluddin, M., Kalita, M. K., Garofalo, R. P., and Lu, M. (2011) RelA Ser276 phospho-rylation-coupled Lys310 acetylation controls transcription-al elongation of inflammatory cytokines in respiratory syn-cytial virus infection, J. Virol., 85, 11752–11769.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Nowak, D. E., Tian, B., Jamaluddin, M., Boldogh, I., Vergara, L. A., Choudhary, S., and Brasier, A. R. (2008) RelA Ser276 phosphorylation is required for activation of a subset of NF-κB-dependent genes by recruiting cyclin-dependent kinase 9/cyclin T1 complexes, Mol. Cell. Biol., 28, 3623–3638.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Nihira, K., Ando, Y., Yamaguchi, T., Kagami, Y., Miki, Y., and Yoshida, K. (2010) Pim-1 controls NF-kappaB sig-nalling by stabilizing RelA/p65, Cell Death Differ., 17, 689–698.

    Article  PubMed  CAS  Google Scholar 

  33. Lawrence, T., Bebien, M., Liu, G. Y., Nizet, V., and Karin, M. (2005) IKKalpha limits macrophage NF-kappaB acti-vation and contributes to the resolution of inflammation, Nature, 434, 1138–1143.

    Article  PubMed  CAS  Google Scholar 

  34. Arun, P., Brown, M., Ehsanian, R., Chen, Z., and Van Waes, C. (2009) Nuclear NF-κB p65 phosphorylation at serine 276 by protein kinase A contributes to the malignant phenotype of head and neck cancer, Clin. Cancer Res., 15, 5974–5984.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Bu, Y., Li, X., He, Y., Huang, C., Shen, Y., Cao, Y., Huang, D., Cai, C., Wang, Y., Wang, Z., Liao, D. F., and Cao, D. (2016) A phosphomimetic mutant of RelA/p65 at Ser536 induces apoptosis and senescence: an implication for tumor-suppressive role of Ser536 phosphorylation, Int. J. Cancer, 138, 1186–1198.

    Article  PubMed  CAS  Google Scholar 

  36. Bu, Y., Cai, G., Shen, Y., Huang, C., Zeng, X., Cao, Y., Cai, C., Wang, Y., Huang, D., Liao, D. F., and Cao, D. (2016) Targeting NF-κB RelA/p65 phosphorylation over-comes RITA resistance, Cancer Lett., 383, 261–271.

    Article  PubMed  CAS  Google Scholar 

  37. Cui, Y., and Guo, G. (2016) Immunomodulatory function of the tumor suppressor p53 in host immune response and the tumor microenvironment, Int. J. Mol. Sci., 17, E1942.

    Article  PubMed  CAS  Google Scholar 

  38. Pal, S., Bhattacharjee, A., Ali, A., Mandal, N. C., Mandal, S. C., and Pal, M. (2014) Chronic inflammation and can-cer: potential chemoprevention through nuclear factor kappa B and p53 mutual antagonism, J. Inflam. (Lond.), 11, 23.

    Article  CAS  Google Scholar 

  39. Martinez, F. O., and Gordon, S. (2014) The M1 and M2 paradigm of macrophage activation: time for reassessment, F1000Prime Rep., 6, 13.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Bzowska, M., Nogiec, A., Bania, K., Zygmunt, M., Zarebski, M., Dobrucki, J., and Guzik, K. (2017) Involvement of cell surface 90 kDa heat shock protein (HSP90) in pattern recognition by human monocyte-derived macrophages, J. Leukoc. Biol., 102, 763–774.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  41. Glushkova, O. V., Novoselova, T. V., Khrenov, M. O., Parfenyuk, S. B., Lunin, S. M., Fesenko, E. E., and Novoselova, E. G. (2010) Role of heat shock protein hsp90 in formation of protective reactions in acute toxic stress, Biochemistry (Moscow), 75, 702–707.

    Article  CAS  Google Scholar 

  42. Moss, J. E., Aliprantis, A. O., and Zychlinsky, A. (1999) The regulation of apoptosis by microbial pathogens, Int. Rev. Cytol., 187, 203–259.

    Article  PubMed  CAS  Google Scholar 

  43. Rosadini, C. V., and Kagan, J. C. (2016) Early innate immune responses to bacterial LPS, Curr. Opin. Immunol., 44, 14–19.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Authors and Affiliations

  1. Institute of Cell Biophysics, Russian Academy of Sciences, 142290, Pushchino, Moscow Region, Russia

    O. V. Glushkova, S. B. Parfenyuk, T. V. Novoselova, M. O. Khrenov, S. M. Lunin & E. G. Novoselova

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  1. O. V. Glushkova
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  2. S. B. Parfenyuk
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  6. E. G. Novoselova
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Correspondence to O. V. Glushkova.

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Original Russian Text © O. V. Glushkova, S. B. Parfenyuk, T. V. Novoselova, M. O. Khrenov, S. M. Lunin, E. G. Novoselova, 2018, published in Biokhimiya, 2018, Vol. 83, No. 6, pp. 949-959.

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Glushkova, O.V., Parfenyuk, S.B., Novoselova, T.V. et al. The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide. Biochemistry Moscow 83, 746–754 (2018). https://doi.org/10.1134/S0006297918060123

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  • DOI: https://doi.org/10.1134/S0006297918060123

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