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Extracellular HSP27 acts as a signaling molecule to activate NF-κB in macrophages

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Cell Stress and Chaperones Aims and scope

Abstract

Heat shock protein 27 (HSP27) shows attenuated expression in human coronary arteries as the extent of atherosclerosis progresses. In mice, overexpression of HSP27 reduces atherogenesis, yet the precise mechanism(s) are incompletely understood. Inflammation plays a central role in atherogenesis, and of particular interest is the balance of pro- and anti-inflammatory factors produced by macrophages. As nuclear factor-kappa B (NF-κB) is a key immune signaling modulator in atherogenesis, and macrophages are known to secrete HSP27, we sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling in macrophages. Treatment of THP-1 macrophages with rHSP27 resulted in the degradation of an inhibitor of NF-κB, IκBα, nuclear translocation of the NF-κB p65 subunit, and increased NF-κB transcriptional activity. Treatment of THP-1 macrophages with rHSP27 yielded increased expression of a variety of genes, including the pro-inflammatory factors, IL-1β, and TNF-α. However, rHSP27 also increased the expression of the anti-inflammatory factors IL-10 and GM-CSF both at the mRNA and protein levels. Our study suggests that in macrophages, activation of NF-κB signaling by rHSP27 is associated with upregulated expression and secretion of key pro- and anti-inflammatory cytokines. Moreover, we surmise that it is the balance in expression of these mediators and antagonists of inflammation, and hence atherogenesis, that yields a favorable net effect of HSP27 on the vessel wall.

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Acknowledgments

This work was supported by operating grants MOP 80204 from the Canadian Institute for Health Research (CIHR) and T6335 from the Heart and Stroke Foundation of Ontario. CIHR and Medtronic collectively provide EOB with a peer-reviewed Research Chair (URC #57093; IGO 94418). SS was supported by an Ontario Graduate Scholarship and a CIHR IGH Women’s Health Council Masters Award. TS was supported by a studentship from the Heart and Stroke Foundation of Ontario and by a CIHR Frederick Banting and Charles Best Canada Graduate Doctoral Award. JR was supported by a CIHR Frederick Banting and Charles Best Canada Graduate Doctoral Award. CMC was supported by a postdoctoral fellowship from le Fonds de Recherche en Santé du Québec (FRSQ) and the Ernest and Margaret Ford cardiology endowed research fellowship from the University of Ottawa Heart Institute.

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Authors and Affiliations

  1. University of Ottawa Heart Institute, Ottawa, ON, Canada

    Samira Salari, Tara Seibert, Yong-Xiang Chen, Tieqiang Hu, Chunhua Shi, Xiaoling Zhao, Charles M. Cuerrier, Joshua E. Raizman & Edward R. O’Brien

  2. Division of Cardiology, Libin Cardiovascular Institute of Alberta, Room C823, Foothills Medical Centre, 1403—29th Street NW, Calgary, AB, T2N 2T9, Canada

    Edward R. O’Brien

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  1. Samira Salari
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  2. Tara Seibert
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  3. Yong-Xiang Chen
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Correspondence to Edward R. O’Brien.

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Samira Salari and Tara Seibert contributed equally to this work

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Supplemental Fig. 1

rHSP27 is not phosphorylated. a Western blot demonstrating rHSP27 is not phosphorylated b LC-MS/MS confirmed rHSP27 is not phosphorylated (PPT 184 kb)

Supplemental Fig. 2

Gel filtration of rHSP27 and rC1 to demonstrate molecular size in 1× PBS buffer (pH = 7.4). The size of rHSP27 and rC1 was measured by gel filtration. The size of most of rHSP27 (>90 %) is between 1,000—5,000 kD with <10 % in the range of 150 kD. The size of recombinant C1 appears to be around 100 kD (PPT 40 kb)

Supplemental Fig. 3

a Assays of cell viability and cytotoxicity. THP1 cells that were treated with rHSP27 (9.6 μM) or rC1 (9.6 μM) show no changes in viability or membrane integrity as demonstrated by MTT (a) and LDH (b) assays, respectively (PSD 2915 kb)

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Salari, S., Seibert, T., Chen, YX. et al. Extracellular HSP27 acts as a signaling molecule to activate NF-κB in macrophages. Cell Stress and Chaperones 18, 53–63 (2013). https://doi.org/10.1007/s12192-012-0356-0

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