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

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.

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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.

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Correspondence to Martha K. Cathcart.

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Responsible Editor: John Di Battista.

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

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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

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

Keywords

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