Role of ERK map kinase and CRM1 in IL-1beta-stimulated release of HMGB1 from cortical astrocytes

Abstract

Reactive astrocytes are traditionally thought to impede brain plasticity after stroke. However, we previously showed that reactive astrocytes may also contribute to stroke recovery, partly via the release of a nuclear protein called high-mobility group box 1 (HMGB1). Here, we investigate the mechanisms that allow stimulated astrocytes to release HMGB1. Exposure of rat primary astrocytes to IL-1beta for 24 h elicited a dose-dependent HMGB1 response. Immunostaining and western blots of cell lysates showed increased intracellular levels of HMGB1. Western blots confirmed that IL-1beta induced a release of HMGB1 into astrocyte conditioned media. MAP kinase signaling was involved. Levels of phospho-ERK were increased by IL-1beta, and the MEK/ERK inhibitor U0126 decreased HMGB1 upregulation in the stimulated astrocytes. Since HMGB1 is a nuclear protein, the role of the nuclear protein exporter, chromosome region maintenance 1 (CRM1), was assessed as a candidate mechanism for linking MAP kinase signaling to HMGB1 release. IL-1beta increased CRM1 expression in concert with a translocation of HMGB1 from nucleus into cytoplasm. Blockade of IL-1beta-stimulated HMGB1 release with the ERK inhibitor U0126 was accompanied by a downregulation of CRM1. Our findings reveal that IL-1beta stimulates the release of HMGB1 from activated astrocytes via ERK MAP kinase and CRM1 signaling. These data suggest a novel pathway by which inflammatory cytokines may enhance the ability of reactive astrocytes to release prorecovery mediators after stroke.

Figure 1

(A) Immunohistochemical staining demonstrated that HMGB1 expression is enhanced after IL-1b treatment in our purified astroglia culture system. Cells were stained for the astrocytic marker glial fibrillary acidic protein (GFAP; green), anti-HMGB1 antibody (red). Scale bar indicates 100 μm. (B) IL-1b (10-1000 pg/ml) significantly enhanced the expression of HMGB1 in astrocytes after 24 hrs. Values are means ± SEM of four independent experiments. *P<0.05, **P<0.01 compared with 0 pg/ml treatment controls.

Figure 2

(A) Treatment with IL-1b (100 pg/ml) for 24 hrs significantly induced the release of HMGB1 into extracellular media. Values are means ± SEM of six independent experiments. *P<0.05 compared with 0 pg/ml treatment controls. IL-1b (100 pg/ml) did not affect (B) WST cell proliferation, (C) LDH release or (D) MTT cell viability after 24 hrs. Values are means ± SEM of four independent experiments.

Figure 3

(A) IL-1b (10-1000 pg/ml) induced phosphorylation in ERK1/2 without affecting total ERK1/2 levels after 1 hr in astrocytes. (B) The MEK inhibitor U0126 (0.1-10 μM) effectively reduced IL-1b -induced ERK phosphorylation after 1 hr in a dose-dependent manner. (C) The IL-1b (100 pg/ml) stimulated elevation of HMGB1 after 24 hrs in astrocytes was attenuated after inhibition of ERK signaling with U0126 (1, 10 μM). Values are means ± SEM of four independent experiments. **P<0.01 compared with no-treated control, ^##P<0.01 compared with IL-1b (100 pg/ml)-treated group.

Figure 4

IL-1b (100 pg/ml) significantly enhanced the expression of CRM1 in astrocytes after 24 hrs, and the elevation of CRM1 in astrocytes was attenuated by U0126 (10 μM). Values are means ± SEM of six independent experiments. *P<0.05 compared with no-treated control, ^#P<0.05 compared with IL-1b (100 pg/ml)-treated group.

Figure 5

IL-1b (100 pg/ml) increased the immunostaining of CRM1 in astrocytes after 24 hrs. HMGB1 staining also showed evidence of translocation from nucleus into cytoplasm. Both CRM1 upregulation and HMGB1 translocation was inhibited by U0126 (10 μM). Cells were stained for the anti-CRM1 antibody (green), anti-HMGB1 antibody (red), and a nuclear stain DAPI (blue). Scale bar indicates 20 μm.

Figure 6

(A) Western blots showed that overall expression levels of HMGB1 were increased at 24 hrs after IL-1b stimulation. Cytosolic fractions showed evidence of HMGB1 translocation. (B) Densitometry quantitation of western blots demonstrated that cytosolic levels of HMGB1 were significantly increases by IL-1b. This response was significantly blocked by either inhibition of ERK signaling with U0126 (10 μM) or leptomycin B (LMB, 20 nM) which blocks the binding of CRM1 to proteins containing the nuclear export signal. Values are means ± SEM of four independent experiments. *P<0.05 compared with non-treated controls, ^#P<0.05, ^##P<0.01 compared with IL-1b (100 pg/ml)-treated group.

Figure 7

IL-1b -stimulated HMGB1 release from astrocytes was attenuated by U0126 (10 μM) and LMB (20 nM). Values are means ± SEM of four independent experiments. *P<0.05, **P<0.01 compared with non-treated control, ^##P<0.01 compared with IL-1b (100 pg/ml)-treated group.