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

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.

Fig. 1

a IκBα degradation in rHSP27-treated macrophages. PMA-differentiated THP-1 cells were treated with either LPS (10 ng/mL), rHSP27 (9.6 μM), or rC1 (9.6 μM) for the times indicated. Equal amounts of whole cell lysates were subject to Western blotting with an antibody directed against IκBα. β-Actin was used as a loading control. b Nuclear translocation of the NF-κB p65 subunit in response to rHSP27. PMA-differentiated THP-1 cells were treated with media alone (control), rC1 (9.6 μM), rHSP27 (9.6 μM), or LPS (10 ng/mL) for 30 min. The cells were then stained with an antibody to p65 (green) and the nuclear stain Hoechst (blue). Scale bar = 10 μm. c NF-κB activity in rHSP27-treated macrophages. NF-κB reporter assays were performed using THP-1 Blue macrophages. PMA-differentiated cells were treated with LPS (10 ng/mL), Polymyxin B (10 μg/mL), rHSP27 (9.6 μM), or C1 (9.6 μM) for 24 h. Cell supernatants were then analyzed for presence of secreted embryonic alkaline phosphatase (SEAP) as a reporter of NF-κB activation. One-way ANOVA *p < 0.05; n = 3–4. d Reduction of rHSP27-induced NF-κB activity in the presence of BAY 11-7082, an inhibitor of IκBα phosphorylation. Inhibition of NF-κB signaling was achieved by pre-treating the cells for 1 h with BAY 11-7082 (10 μM). One-way ANOVA ***p < 0.001; n = 3

Fig. 2

Focused gene expression profiling of PMA differentiated THP-1 cells treated with rHSP27. NF-κB pathway-focused qPCR arrays were used to screen the expression of genes involved in this pathway. THP-1 macrophages were treated with rHSP27 (9.6 μM) supplemented with polymyxin B (PMB, 10 μg/mL). The delta delta Ct method was then used to calculate the fold change of genes that are up/down-regulated in rHSP27-treated macrophages compared to control. Genes with fold change >5 and a p value < 0.05 were considered to be significant and are shown on a logarithmic scale

Fig. 3

a GM-CSF mRNA levels rise in rHSP27-treated macrophages. PMA-differentiated THP-1 cells were treated with media (control), rC1 (9.6 μM), or rHSP27 (9.6 μM) ± BAY 11-7082 (10 μM) for 24 h. Inhibition of NF-κB signaling was achieved by pre-treating the cells for 1 h with BAY 11-7082 (10 μM). All treatments were supplemented with Polymyxin B (PMB, 10 μg/mL). One-way ANOVA *p < 0.001); n = 3. b GM-CSF is secreted by THP-1 macrophages in response to rHSP27. PMA-differentiated THP-1 cells were treated with media, LPS (10 ng/mL), rHSP27 (9.6 μM), or rC1 (9.6 μM) ± BAY 11-7082 (10 μM) for 24 h. Inhibition of NF-κB signaling was achieved by pre-treating the cells for 1 h with BAY 11-7082 (10 μM). Polymyxin B (PMB, 10 μg/mL) was used as indicated. One-way ANOVA *p < 0.001; n = 3. c IL-10 is secreted by THP-1 macrophages in response to rHSP27. THP-1 macrophages were treated with media, LPS (10 ng/mL), rHSP27 (9.6 μM), or rC1 (9.6 μM) ± BAY 11-7082 (10 μM) for 24 h. Inhibition of NF-κB signaling was achieved by pre-treating the cells for 1 h with BAY 11-7082 (10 μM). Polymyxin B (PMB, 10 μg/mL) was used as indicated. One-way ANOVA *p < 0.001; n = 3

Fig. 4

Schematic diagram representing the transcriptional outcome of HSP27-mediated activation of NF-κB in THP-1 macrophages. Given the previously described anti-atherogenic effects of HSP27, it is speculated that the net effect of these transcriptional regulations is likely tipping the balance towards anti-inflammatory effects and suppression of atherosclerosis