Skip to main content

Vimentin is a target of PKCβ phosphorylation in MCP-1-activated primary human monocytes

Inflammation Research volume 62, pages 991–1001 (2013)Cite this article

Abstract

Objective and design

We designed a study to detect downstream phosphorylation targets of PKCβ in MCP-1-induced human monocytes.

Methods

Two-dimensional gel electrophoresis was performed for monocytes treated with MCP-1 in the presence or absence of PKCβ antisense oligodeoxyribonucleotides (AS-ODN) or a PKCβ inhibitor peptide, followed by phospho- and total protein staining. Proteins that stained less intensely with the phospho-stain, when normalized to the total protein stain, in the presence of PKCβ AS-ODN or the PKCβ inhibitor peptide, were sequenced.

Results

Of the proteins identified, vimentin was consistently identified using both experimental approaches. Upon 32P-labeling and vimentin immunoprecipitation, increased phosphorylation of vimentin was observed in MCP-1 treated monocytes as compared to the untreated monocytes. Both PKCβ AS-ODN and the PKCβ inhibitor reduced MCP-1-induced vimentin phosphorylation. The IP of monocytes with anti-vimentin antibody and immunoblotting with a PKCβ antibody revealed that increased PKCβ becomes associated with vimentin upon MCP-1 activation. Upon MCP-1 treatment, monocytes were shown to secrete vimentin and secretion depended on PKCβ expression and activity.

Conclusions

We conclude that vimentin, a major intermediate filament protein, is a phosphorylation target of PKCβ in MCP-1-treated monocytes and that PKCβ phosphorylation is essential for vimentin secretion. Our recently published studies have implicated vimentin as a potent stimulator of the innate immune receptor Dectin-1 as reported by Thiagarajan et al. (Cardiovasc Res 99:494–504, 2013). Taken together our findings suggest that inhibition of PKCβ regulates vimentin secretion and, thereby, its interaction with Dectin-1 and downstream stimulation of superoxide anion production. Thus, PKCβ phosphorylation of vimentin likely plays an important role in propagating inflammatory responses.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Thiagarajan PS, Yakubenko VP, Elsori DH, Yadav SP, Willard B, Tan CD, et al. Vimentin is an endogenous ligand for the pattern recognition receptor Dectin-1. Cardiovasc Res. 2013;99:494–504.

    PubMed  Article  CAS  Google Scholar 

  2. Luster AD. Chemokines–chemotactic cytokines that mediate inflammation. N Engl J Med. 1998;338:436–45.

    PubMed  Article  CAS  Google Scholar 

  3. Osterud B, Bjorklid E. Role of monocytes in atherogenesis. Physiol Rev. 2003;83:1069–112.

    PubMed  CAS  Google Scholar 

  4. Rollins BJ. Chemokines. Blood. 1997;90:909–28.

    PubMed  CAS  Google Scholar 

  5. Carnevale KA, Cathcart MK. Protein kinase C beta is required for human monocyte chemotaxis to MCP-1. J Biol Chem. 2003;278:25317–22.

    PubMed  Article  CAS  Google Scholar 

  6. Liebler JM, Kunkel SL, Allen RM, Burdick MD, Strieter RM. Interferon-gamma stimulates monocyte chemotactic protein-1 expression by monocytes. Mediators Inflamm. 1994;3:27–31.

    PubMed  Article  CAS  Google Scholar 

  7. Rollins BJ, Stier P, Ernst T, Wong GG. The human homolog of the JE gene encodes a monocyte secretory protein. Mol Cell Biol. 1989;9:4687–95.

    PubMed  CAS  Google Scholar 

  8. Yu X, Dluz S, Graves DT, Zhang L, Antoniades HN, Hollander W, et al. Elevated expression of monocyte chemoattractant protein 1 by vascular smooth muscle cells in hypercholesterolemic primates. Proc Natl Acad Sci USA. 1992;89:6953–7.

    PubMed  Article  CAS  Google Scholar 

  9. Marmur JD, Poon M, Rossikhina M, Taubman MB. Induction of PDGF-responsive genes in vascular smooth muscle. Implications for the early response to vessel injury. Circulation. 1992;86:III53–60.

    PubMed  CAS  Google Scholar 

  10. Rollins BJ, Yoshimura T, Leonard EJ, Pober JS. Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE. Am J Pathol. 1990;136:1229–33.

    PubMed  CAS  Google Scholar 

  11. Strieter RM, Wiggins R, Phan SH, Wharram BL, Showell HJ, Remick DG, et al. Monocyte chemotactic protein gene expression by cytokine-treated human fibroblasts and endothelial cells. Biochem Biophys Res Commun. 1989;162:694–700.

    PubMed  Article  CAS  Google Scholar 

  12. Schwartz CJ, Valente AJ, Sprague EA, Kelley JL, Nerem RM. The pathogenesis of atherosclerosis: an overview. Clin Cardiol. 1991;14:I1–16.

    PubMed  Article  CAS  Google Scholar 

  13. Sozzani S, Locati M, Zhou D, Rieppi M, Luini W, Lamorte G, et al. Receptors, signal transduction, and spectrum of action of monocyte chemotactic protein-1 and related chemokines. J Leukoc Biol. 1995;57:788–94.

    PubMed  CAS  Google Scholar 

  14. Wilson HM, Barker RN, Erwig LP. Macrophages: promising targets for the treatment of atherosclerosis. Curr Vasc Pharmacol. 2009;7:234–43.

    PubMed  Article  CAS  Google Scholar 

  15. Sozzani S, Luini W, Molino M, Jilek P, Bottazzi B, Cerletti C, et al. The signal transduction pathway involved in the migration induced by a monocyte chemotactic cytokine. J Immunol. 1991;147:2215–21.

    PubMed  CAS  Google Scholar 

  16. Kuziel WA, Morgan SJ, Dawson TC, Griffin S, Smithies O, Ley K, et al. Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2. Proc Natl Acad Sci USA. 1997;94:12053–8.

    PubMed  Article  CAS  Google Scholar 

  17. Fujita T, Asai T, Andrassy M, Stern DM, Pinsky DJ, Zou YS, et al. PKCbeta regulates ischemia/reperfusion injury in the lung. J Clin Invest. 2004;113:1615–23.

    PubMed  CAS  Google Scholar 

  18. Aragay AM, Mellado M, Frade JM, Martin AM, Jimenez-Sainz MC, Martinez AC, et al. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2. Proc Natl Acad Sci USA. 1998;95:2985–90.

    PubMed  Article  CAS  Google Scholar 

  19. Penton-Rol G, Polentarutti N, Luini W, Borsatti A, Mancinelli R, Sica A, et al. Selective inhibition of expression of the chemokine receptor CCR2 in human monocytes by IFN-gamma. J Immunol. 1998;160:3869–73.

    PubMed  CAS  Google Scholar 

  20. Murphy PM, Baggiolini M, Charo IF, Hebert CA, Horuk R, Matsushima K, et al. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev. 2000;52:145–76.

    PubMed  CAS  Google Scholar 

  21. Shyy YJ, Hsieh HJ, Usami S, Chien S. Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium. Proc Natl Acad Sci USA. 1994;91:4678–82.

    PubMed  Article  CAS  Google Scholar 

  22. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, et al. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest. 1990;85:1260–6.

    PubMed  Article  CAS  Google Scholar 

  23. Yan SF, Harja E, Andrassy M, Fujita T, Schmidt AM. Protein kinase C beta/early growth response-1 pathway: a key player in ischemia, atherosclerosis, and restenosis. J Am Coll Cardiol. 2006;48:A47–55.

    PubMed  Article  CAS  Google Scholar 

  24. Harja E, Bucciarelli LG, Lu Y, Stern DM, Zou YS, Schmidt AM, et al. Early growth response-1 promotes atherogenesis: mice deficient in early growth response-1 and apolipoprotein E display decreased atherosclerosis and vascular inflammation. Circ Res. 2004;94:333–9.

    PubMed  Article  CAS  Google Scholar 

  25. Harja E, Chang JS, Lu Y, Leitges M, Zou YS, Schmidt AM, et al. Mice deficient in PKCbeta and apolipoprotein E display decreased atherosclerosis. FASEB J. 2009;23:1081–91.

    PubMed  Article  CAS  Google Scholar 

  26. Kumagai K, Itoh K, Hinuma S, Tada M. Pretreatment of plastic petri dishes with fetal calf serum. A simple method for macrophage isolation. J Immunol Methods. 1979;29:17–25.

    PubMed  Article  CAS  Google Scholar 

  27. Cathcart MK, Morel DW, Chisolm GM 3rd. Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic. J Leukoc Biol. 1985;38:341–50.

    PubMed  CAS  Google Scholar 

  28. Keightley JA, Shang L, Kinter M. Proteomic analysis of oxidative stress-resistant cells: a specific role for aldose reductase overexpression in cytoprotection. Mol Cell Proteomics. 2004;3:167–75.

    PubMed  CAS  Google Scholar 

  29. Kinter M, Sherman NE. Protein sequencing and identification using tandem mass spectrometry. New York: John Wiley; 2000:xvi, p. 301.

  30. Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969;244:4406–12.

    PubMed  CAS  Google Scholar 

  31. Mor-Vaknin N, Punturieri A, Sitwala K, Markovitz DM. Vimentin is secreted by activated macrophages. Nat Cell Biol. 2003;5:59–63.

    PubMed  Article  CAS  Google Scholar 

  32. Steinberg TH, Agnew BJ, Gee KR, Leung WY, Goodman T, Schulenberg B, et al. Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology. Proteomics. 2003;3:1128–44.

    PubMed  Article  CAS  Google Scholar 

  33. Herrmann H, Fouquet B, Franke WW. Expression of intermediate filament proteins during development of Xenopus laevis. I. cDNA clones encoding different forms of vimentin. Development. 1989;105:279–98.

    PubMed  CAS  Google Scholar 

  34. Biddle D, Spandau DF. Expression of vimentin in cultured human keratinocytes is associated with cell—extracellular matrix junctions. Arch Dermatol Res. 1996;288:621–4.

    PubMed  Article  CAS  Google Scholar 

  35. Eckes B, Dogic D, Colucci-Guyon E, Wang N, Maniotis A, Ingber D, et al. Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts. J Cell Sci. 1998;111(Pt 13):1897–907.

    PubMed  CAS  Google Scholar 

  36. Nieminen M, Henttinen T, Merinen M, Marttila-Ichihara F, Eriksson JE, Jalkanen S. Vimentin function in lymphocyte adhesion and transcellular migration. Nat Cell Biol. 2006;8:156–62.

    PubMed  Article  CAS  Google Scholar 

  37. Rius C, Aller P. Vimentin expression as a late event in the in vitro differentiation of human promonocytic cells. J Cell Sci. 1992;101(Pt 2):395–401.

    PubMed  CAS  Google Scholar 

  38. Correia I, Chu D, Chou YH, Goldman RD, Matsudaira P. Integrating the actin and vimentin cytoskeletons. Adhesion-dependent formation of fimbrin-vimentin complexes in macrophages. J Cell Biol. 1999;146:831–42.

    PubMed  Article  CAS  Google Scholar 

  39. Barberis L, Pasquali C, Bertschy-Meier D, Cuccurullo A, Costa C, Ambrogio C, et al. Leukocyte transmigration is modulated by chemokine-mediated PI3Kgamma-dependent phosphorylation of vimentin. Eur J Immunol. 2009;39:1136–46.

    PubMed  Article  CAS  Google Scholar 

  40. Brown MJ, Hallam JA, Colucci-Guyon E, Shaw S. Rigidity of circulating lymphocytes is primarily conferred by vimentin intermediate filaments. J Immunol. 2001;166:6640–6.

    PubMed  CAS  Google Scholar 

  41. Gonzales M, Weksler B, Tsuruta D, Goldman RD, Yoon KJ, Hopkinson SB, et al. Structure and function of a vimentin-associated matrix adhesion in endothelial cells. Mol Biol Cell. 2001;12:85–100.

    PubMed  Article  CAS  Google Scholar 

  42. Kreuzer J, Denger S, Schmidts A, Jahn L, Merten M, von Hodenberg E. Fibrinogen promotes monocyte adhesion via a protein kinase C dependent mechanism. J Mol Med. 1996;74:161–5.

    PubMed  Article  CAS  Google Scholar 

  43. Chu YW, Runyan RB, Oshima RG, Hendrix MJ. Expression of complete keratin filaments in mouse L cells augments cell migration and invasion. Proc Natl Acad Sci USA. 1993;90:4261–5.

    PubMed  Article  CAS  Google Scholar 

  44. Gilles C, Polette M, Piette J, Delvigne AC, Thompson EW, Foidart JM, et al. Vimentin expression in cervical carcinomas: association with invasive and migratory potential. J Pathol. 1996;180:175–80.

    PubMed  Article  CAS  Google Scholar 

  45. Sommers CL, Heckford SE, Skerker JM, Worland P, Torri JA, Thompson EW, et al. Loss of epithelial markers and acquisition of vimentin expression in adriamycin- and vinblastine-resistant human breast cancer cell lines. Cancer Res. 1992;52:5190–7.

    PubMed  CAS  Google Scholar 

  46. Hu L, Lau SH, Tzang CH, Wen JM, Wang W, Xie D, et al. Association of Vimentin overexpression and hepatocellular carcinoma metastasis. Oncogene. 2004;23:298–302.

    PubMed  Article  CAS  Google Scholar 

  47. Geisler N, Hatzfeld M, Weber K. Phosphorylation in vitro of vimentin by protein kinases A and C is restricted to the head domain. Identification of the phosphoserine sites and their influence on filament formation. Eur J Biochem. 1989;183:441–7.

    PubMed  Article  CAS  Google Scholar 

  48. O’Connor CM, Gard DL, Lazarides E. Phosphorylation of intermediate filament proteins by cAMP-dependent protein kinases. Cell. 1981;23:135–43.

    PubMed  Article  Google Scholar 

  49. Huang CK, Devanney JF, Kennedy SP. Vimentin, a cytoskeletal substrate of protein kinase C. Biochem Biophys Res Commun. 1988;150:1006–11.

    PubMed  Article  CAS  Google Scholar 

  50. Ivaska J, Vuoriluoto K, Huovinen T, Izawa I, Inagaki M, Parker PJ. PKCepsilon-mediated phosphorylation of vimentin controls integrin recycling and motility. EMBO J. 2005;24:3834–45.

    PubMed  Article  CAS  Google Scholar 

  51. Murti KG, Kaur K, Goorha RM. Protein kinase C associates with intermediate filaments and stress fibers. Exp Cell Res. 1992;202:36–44.

    PubMed  Article  CAS  Google Scholar 

  52. Owen PJ, Johnson GD, Lord JM. Protein kinase C-delta associates with vimentin intermediate filaments in differentiated HL60 cells. Exp Cell Res. 1996;225:366–73.

    PubMed  Article  CAS  Google Scholar 

  53. Spudich A, Meyer T, Stryer L. Association of the beta isoform of protein kinase C with vimentin filaments. Cell Motil Cytoskeleton. 1992;22:250–6.

    PubMed  Article  CAS  Google Scholar 

  54. Xu B, deWaal RM, Mor-Vaknin N, Hibbard C, Markovitz DM, Kahn ML. The endothelial cell-specific antibody PAL-E identifies a secreted form of vimentin in the blood vasculature. Mol Cell Biol. 2004;24:9198–206.

    PubMed  Article  CAS  Google Scholar 

  55. Huet D, Bagot M, Loyaux D, Capdevielle J, Conraux L, Ferrara P, et al. SC5 mAb represents a unique tool for the detection of extracellular vimentin as a specific marker of Sezary cells. J Immunol. 2006;176:652–9.

    PubMed  CAS  Google Scholar 

  56. Mahesh B, Leong HS, McCormack A, Sarathchandra P, Holder A, Rose ML. Autoantibodies to vimentin cause accelerated rejection of cardiac allografts. Am J Pathol. 2007;170:1415–27.

    PubMed  Article  CAS  Google Scholar 

  57. Bynagari-Settipalli YS, Chari R, Kilpatrick L, Kunapuli SP. Protein kinase C—possible therapeutic target to treat cardiovascular diseases. Cardiovasc Hematol Disord Drug Targets. 2010;10:292–308.

    PubMed  Article  CAS  Google Scholar 

  58. Rask-Madsen C, King GL. Proatherosclerotic mechanisms involving protein kinase C in diabetes and insulin resistance. Arterioscler Thromb Vasc Biol. 2005;25:487–96.

    PubMed  Article  CAS  Google Scholar 

  59. Liu Y, Lei S, Gao X, Mao X, Wang T, Wong GT, et al. PKCbeta inhibition with ruboxistaurin reduces oxidative stress and attenuates left ventricular hypertrophy and dysfunction in rats with streptozotocin-induced diabetes. Clin Sci (Lond). 2012;122:161–73.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Meenakshi Shukla for isolating primary human monocytes for our study. Our study was sponsored by NIH grants HL051068, HL61971 and HL087018 to M.K.C and National Center for Research resources, CTSA 1UL1RR024989.

Conflict of interest

The author(s) declare that they have no competing interests.

Author information

Author notes

  1. Ayse C. Akbasli

    Present address: Scientific Research Projects Coordination Unit, Istanbul University, Istanbul, Turkey

  2. Michael T. Kinter

    Present address: Free Radical Biology and Aging Research Program, University of Oklahoma Health Sciences Center, Oklahoma, OK, USA

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH, 44195, USA

    Praveena S. Thiagarajan, Ayse C. Akbasli, Michael T. Kinter, Belinda Willard & Martha K. Cathcart

  2. School of Biomedical Sciences, Kent State University, Kent, OH, USA

    Praveena S. Thiagarajan & Martha K. Cathcart

  3. Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA

    Martha K. Cathcart

Authors
  1. Praveena S. Thiagarajan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ayse C. Akbasli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael T. Kinter
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Belinda Willard
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martha K. Cathcart
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martha K. Cathcart.

Additional information

Responsible Editor: John Di Battista.

Praveena S. Thiagarajan and Ayse C. Akbasli contributed equally.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Thiagarajan, P.S., Akbasli, A.C., Kinter, M.T. et al. Vimentin is a target of PKCβ phosphorylation in MCP-1-activated primary human monocytes. Inflamm. Res. 62, 991–1001 (2013). https://doi.org/10.1007/s00011-013-0657-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00011-013-0657-5

Keywords

  • Chemotaxis
  • Inflammation
  • Monocytes
  • MCP-1
  • PKCβ
  • Vimentin