Glucocorticoid receptor-mediated suppression of activator protein-1 activation and matrix metalloproteinase expression after spinal cord injury

Abstract

Post-traumatic inflammatory reaction may contribute to progressive tissue damage after spinal cord injury (SCI). Two key transcription factors, nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1), are activated in inflammation. An increase in NF-kappaB binding activity has been shown in the injured spinal cord. We report activation of AP-1 after SCI. Electrophoretic mobility shift assay showed that AP-1 binding activity increased after SCI, starting at 1 hr, peaking at 8 hr, and declining to basal levels by 7 d. Methylprednisolone (MP) is the only therapeutic agent approved by the Food and Drug Administration for treating patients with acute traumatic SCI. MP reduced post-traumatic AP-1 activation. RU486, a glucocorticoid receptor (GR) antagonist, reversed MP inhibition of AP-1 activation. Immunostaining showed an increase in the expression of the Fos-B and c-Jun components of AP-1 in the injured cord. A c-fos antisense oligodeoxynucleotide (ODN) inhibited AP-1, but not NF-kappaB, activation after SCI. AP-1 and NF-kappaB can transactivate genes encoding matrix metalloproteinase-1 (MMP-1) and MMP-9. Western blotting and immunostaining show increased expression of MMP-1 and MMP-9 in the injured cord. MP inhibited MMP-1 and MMP-9 expression after SCI. RU486 reversed this MP effect. The c-fos antisense ODN, however, failed to suppress MMP-1 or MMP-9 expression. These findings demonstrate that MP may suppress post-traumatic inflammatory reaction by inhibiting both the AP-1 and NF-kappaB transcription cascades via a GR mechanism. Expression of inflammatory genes such as MMP-1 and MMP-9 that are transactivated jointly by AP-1 and NF-kappaB may not be suppressed by inhibiting only AP-1 activity.

Fig. 1.

A, Time-dependent changes in AP-1 binding activity after SCI based on EMSA. Lanes 1,Normal control; 2, sham-operated control;3, 1 hr; 4, 4 hr; 5, 8 hr;6, 1 d; 7, 3 d; and8, 7 d after SCI. Note progressive increase in AP-1 binding activity starting at 1 hr and peaking at 8 hr. AP-1 activation was still evident 3 d after injury. B, Effect of MP and RU486 on AP-1 binding activity after SCI. Rats were treated with or without MP (30 mg/kg, i.v.; 15 min after injury). In some MP-treated animals, pretreatment with RU486 (15 mg/kg, i.p.; 30 min before injury) was given. Lanes 1, Free probe; 2,sham-operated control; 3, normal control;4, SCI; 5, SCI + MP; and6, SCI + RU486 + MP. Note MP inhibition of post-traumatic AP-1 activation. This MP effect was reversed by RU486 pretreatment. Data shown in A and B are representative of three separate experiments with similar results.

Fig. 2.

Increase in Fos-B and c-Jun immunoreactivity after SCI. A, In a sham-operated cord segment, little Fos-B immunoreactivity was detected. B,Fos-B immunoreactivity was intense in a section of the injured cord 7 mm rostral to the epicenter; 8 hr after SCI. C,Preabsorption with Fos-B in a section adjacent to the section shown inB resulted in the disappearance of Fos-B immunoreactivity. D, E, Fos-B immunoreactivity was detected in both the gray (D) and white (E) matter in the injured cord, 5 mm distal to the epicenter. F,G, c-Jun expression was also detected in the gray (F) and white (G) matter, in areas corresponding to D and E. Note that Fos-B and c-Jun immunoreactivity could be localized in the cytoplasm and more intensely in the nucleus of cells with morphology suggestive of neurons in the gray matter (D,F) and glial cells in the white matter (F, G). Results shown are representative of two separate experiments with similar results. Scale bars: A–C, 150 μm; D, F, 75 μm; E,G, 100 μm.

Fig. 3.

Effect of c-fos antisense and sense ODN on AP-1 and NF-κB binding activity. Rats were treated with c-fos antisense or sense ODN or vehicle (lipofectin only) 16 hr before SCI. The injured cord segment was sampled for AP-1 or NF-κB binding activity by EMSA 8 hr after SCI. Lanes 1, Lipofectin; 2, c-fos sense ODN; and 3, c-fos antisense ODN. Notec-fos antisense ODN treatment blocked post-traumatic increase in AP-1 (top panel) but not NF-κB (bottom panel) binding activity. Data shown are representative of three separate experiments with similar results.

Fig. 4.

MMP-1 expression after SCI. A,Sham-operated control showing little MMP-1 immunoreactivity.B, Intense MMP-1 expression 1 d after SCI in a section 5 mm rostral to the epicenter. C, MP suppression of MMP-1 expression in a cord section corresponding to Bfrom an animal treated with MP. D, RU486 reversal of MP inhibition of MMP-1 expression after SCI in a section corresponding toB in an animal pretreated with RU486 and post-treated with MP. Results shown are representative of three separate experiments with similar results. Scale bar, 100 μm for all panels.

Fig. 5.

MMP-9 expression after SCI. A,Sham-operated control. B, Increased expression of MMP-9 1 d after SCI in a cord section 5 mm rostral to the epicenter.C, MP suppression of MMP-9 expression in a cord section corresponding to B from an animal treated with MP.D, RU486 reversal of MP inhibition of MMP-9 expression after SCI in a section corresponding to B in an animal pretreated with RU486 and post-treated with MP. Results shown are representative of three separate experiments with similar results. Scale bar, 100 μm for all panels.

Fig. 6.

Western blot analysis showing effects of MP and RU486 on MMP-1 and MMP-9 expression after SCI. A, MMP-1.B, MMP-9. The injured cord segment was sampled for Western blot analysis 1 d after SCI. For both A andB, Lanes 1, Normal control;2, sham-operated control; 3, SCI;4, SCI + MP; and 5, RU486 + SCI + MP. Note MP suppression of MMP-1 and MMP-9 expression after SCI and RU486 reversal of the MP effect. Each lane represents a sample obtained from one individual rat. Data shown are representative of three separate experiments with similar results.

Fig. 7.

The effect of c-fos antisense and sense ODNs on MMP-1 and MMP-9 expression after SCI. Rats were treated with vehicle (lipofectin), c-fos sense, or antisense ODN 16 hr before SCI. The injured cord segment was sampled for Western blotting 1 d after SCI. A, MMP-1. B,MMP-9. For both A and B, Lanes 1, Lipofectin vehicle; 2, sense ODN; and3, antisense ODN. Note no difference in intensity of MMP-1 or MMP-9 expression among the three groups. Each lane represents a sample obtained from one individual rat. Data shown are representative of three separate experiments with similar results.