Fra-1 induces apoptosis and neuroinflammation by targeting S100A8 to modulate TLR4 pathways in spinal cord ischemia/reperfusion injury

Abstract

Spinal cord ischemia/reperfusion injury (SCII) is a severe complication driven by apoptosis and neuroinflammation. An increase in the expression of c-Fos, a member of the AP-1 family, is known as a neuronal activation marker in SCII. The AP-1 family is composed of Jun, Fos, and is associated with the regulation of cytokines expression and apoptosis. Fra-1 is a member of the Fos family, however, the contribution of Fra-1 to SCII is still unclear. In our study, Fra-1 was highly upregulated especially in neurons and microglia and promoted apoptosis by changing the expression of Bax/Bcl-2 after SCII. Furthermore, we found that Fra-1 directly regulated the transcription expression of S100A8. We demonstrated that knockdown of Fra-1 alleviated S100A8 mediated neuronal apoptosis and inflammatory factor release, thus improved motor function after SCII. Interestingly, we showed that administration of TAK-242, the TLR4 inhibitor, to the ischemia/reperfusion (I/R) injury induced rats suppressed the activation of the ERK and NF-κB pathways, and further reduced Fra-1 expression. In conclusion, we found that Fra-1-targeted S100A8 was expressed the upstream of Fra-1, and the Fra-1/S100A8 interaction formed a feedback loop in the signaling pathways activated by SCII.

Keywords: apoptosis; feedback loop; ischemia/reperfusion; neuroinflammation; oxygen-glucose deprivation/reoxygenation; spinal cord ischemia/reperfusion injury.

FIGURE 1

The expression of Fra‐1 at different time points after spinal ischemia/reperfusion (I/R). (A) Schematic of the experimental design shows the timeline for grouping rats. At different time points (6, 12, 24, 36, 48 and 72 h) after I/R induction, the L4‐L6 segments of spinal cords were harvested from the rats. (B) qRT‐PCR‐assisted detection of the relative mRNA level of Fra‐1 after I/R (n = 3 rats/group). (C–E) Western blot‐assisted analysis of the protein expression levels of Fra‐1 and cleaved caspase‐3 after I/R (n = 3 rats/group). (F) Correlation analysis of Fra‐1 and cleaved caspase‐3 protein expression levels. All data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

Cellular distribution of Fra‐1 expression after spinal I/R (A) Schematic of the experimental design shows the timeline for grouping rats. Spinal cord ischemia model was established. After 48 h of I/R, tissue samples were collected. (B) In the ventral horn of the spinal cord, the distribution and expression of Fra‐1 (red) were detected by double immunofluorescence (IF) analysis with NeuN (green, neuron marker), Iba‐1 (green, microglial marker) and GFAP (green, astrocyte marker) 48 h after I/R (n = 3 rats/group). Scale bar = 100 μm. (C) Quantified Fra‐1 fluorescence intensity (INT/mm²) after I/R. (D) Quantified Fra‐1‐positive double‐labeled neurons, microglia and astrocytes in the spinal cord 48 h after I/R. All data represent the mean ± SEM. **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 3

Fra‐1 regulates apoptosis response in oxygen–glucose deprivation/reoxygenation (OGD/R)‐stressed VSC4.1 neurons. (A,B) CCK‐8‐assisted analysis of cell viability after OGD/R. (C,D) Western blot‐assisted analysis of the relative protein levels of Fra‐1 after transfection with si‐Fra‐1‐1, si‐Fra‐1‐2, si‐Fra‐1‐3 under OGD/R. (E,F) Western blot‐assisted analysis of the relative protein levels of cleaved caspase‐3, Bax and Bcl‐2 in VSC4.1 neurons after transfection with si‐Fra‐1 under OGD/R. (G,I) Western blot‐assisted analysis of the relative protein levels in VSC4.1 neurons after transfection with the plasmid used for Fra‐1 overexpression (OE‐Fra‐1) under OGD/R. (H) Immunofluorescence (IF) assays of cleaved caspase‐3 and Fra‐1 after OGD/R were performed. Scale bar = 100 μm. (J) Quantification of the cleaved caspase‐3 and Fra‐1 fluorescence intensity (INT/mm²). All data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 4

Knockdown of Fra‐1 alleviates spinal I/R induced injury in vivo. (A) Schematic of the experimental design shows the timeline for grouping rats. The rats were intrathecally injected sh‐Fra‐1 7 days before I/R induction. After 48 h of I/R, tissue samples were collected. (B,C) H&E staining‐assisted detection of the number of normal neurons in the ventral horn of the spinal cord (n = 3 rats/group). The black arrows indicate normal neurons. Scale bar = 100 μm. (D,E) Western blot‐assisted analysis of the relative levels of the Fra‐1,cleaved caspase‐3,Bax,Bcl‐2 after I/R (n = 5 rats/group). (F) Tarlov score analysis was used to evaluate neuromotor function in different groups of rats (n = 10 rats/group). Kruskal–Wallis test was used for comparison among multiple groups of data. (G) Limb motor function of rats was determined by examining paw patterns and stride length (n = 3 rats/group). (H) The stride length of the rats in different groups was analyzed. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 5

S100A8 is a direct target of Fra‐1. (A) Schematic of the experimental design shows the timeline for grouping rats. At different time points (6, 12, 24, 36, 48, and 72 h) after I/R induction, tissue samples were collected. (B,C) Western blot‐assisted analysis and quantification of S100A8 at different time points after I/R (n = 3 rats/group). (D) Correlation analysis of Fra‐1 and S100A8 protein expression. (E) We selected three putative Fra‐1 binding sites of the S100A8 promoter (P1–P3). (F) ChIP‐qPCR‐assisted analysis of S100A8 promoter fragments containing the binding site for Fra‐1. (G) The rats were intrathecally injected sh‐Fra‐1 7 days before I/R induction. After 48 h of I/R, tissue samples were collected. (H,I) Western blot‐assisted analysis and quantification of S100A8 protein expression after intrathecal injection of sh‐Fra‐1 (n = 5 rats/group). (J) qRT‐PCR‐assisted detection of the relative mRNA level of S100A8 in rat spinal cord samples (n = 5 rats/group). (K) Spinal cord ischemia model was established. After 48 h of I/R, tissue samples were collected. (L) In the ventral horn of the spinal cord, the distribution and expression of S100A8 (red) were detected by double immunofluorescence (IF) analysis with NeuN (green, neuron marker), Iba‐1 (green, microglial marker) and GFAP (green, astrocyte marker) after I/R (n = 3 rats/group). Scale bar = 100 μm. (M) Quantified S100A8‐positive double‐labeled neurons, microglia and astrocytes in the spinal cord after I/R. All data represent the mean ± SEM. **p < 0.01, ****p < 0.0001, ns, not significant.

FIGURE 6

Fra‐1/S100A8‐mediated TLR4 signaling promotes ERK1/2 and NF‐κB pathways activation and IL‐1β production after spinal I/R. (A) Schematic of the experimental design shows the timeline for grouping rats. The rats were intrathecally injected sh‐S100A8 7 days before I/R induction. After 48 h of I/R, tissue samples were collected. (B) The rats were intrathecally injected rS100A8, and tissue samples were collected 48 h later. (C) The rats were intrathecally injected rS100A8 before I/R induction. After 48 h of I/R, tissue samples were collected. (D) The rats were intrathecally injected sh‐Fra‐1 7 days before I/R induction. After 48 h of I/R, tissue samples were collected. (E) The rats were intrathecally injected sh‐Fra‐1 7 days before administration of rS100A8 and intrathecally injected rS100A8 before I/R induction. After 48 h of I/R, tissue samples were collected. (F‐M) Western blot‐assisted analysis and quantification of the protein levels of S100A8, cleaved caspase‐3, TLR4, p‐ERK/ERK, p‐NF‐κB/NF‐κB and IL‐1β (n = 5 rats/group). (N) Immunofluorescence (IF) assays of IL‐1β expression in different groups were performed (n = 3 rats/group). Scale bar =100 μm. (O) Quantification of the IL‐1β fluorescence intensity (INT/mm²). All data represent the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 7

TAK‐242 attenuates S100A8‐induced apoptosis and activation of ERK1/2 and NF‐κB pathways and IL‐1β production, and activated TLR4/NF‐κB and ERK1/2 pathways promote Fra‐1 expression after I/R. (A) Schematic of the experimental design shows the timeline for grouping rats. The rats were intrathecally injected rS100A8, and tissue samples were collected 48 h later. (B) The rats were intraperitoneally injected TAK‐242 30 min before administration of rS100A8 and intrathecally injected rS100A8 before I/R induction. After 48 h of I/R, tissue samples were collected. (C) TUNEL staining of the ventral horn of the spinal cord showing apoptotic cells (n = 3 rats/group). Scale bar = 100 μm. (D) Quantification of TUNEL‐positive cells after I/R. (E,F) Western blot‐assisted analysis and quantification of the protein levels of p‐ERK/ERK, p‐NF‐κB/NF‐κB, IL‐1β, and Fra‐1 (n = 5 rats/group). All data represent the mean ± SEM. **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 8

Schematic illustration of a proposed working model wherein Fra‐1‐mediated S100A8/TLR4/NF‐κB and ERK signaling and the generated feedback loop in the regulation of apoptosis and neuroinflammation during spinal I/R. I/R stress promotes Fra‐1 expression mainly in neurons of spinal cord. Fra‐1, as the transcription factor, translocates into the nucleus and binds S100A8 gene to activate S100A8 transcription. Upon recognition of S100A8 that possibly secreted by neurons following spinal I/R insult, microglia initiate the TLR4 signaling that activates NF‐κB and ERK pathways. NF‐κB pathway induces the production of the inflammatory cytokine IL‐1β. The NF‐κB and ERK pathways, in turn, promotes Fra‐1 expression. TAK‐242, as a TLR4 receptor inhibitor, inhibits the responses activated by S100A8 binding with the TLR4 receptor, which rescues NF‐κB and ERK activity as well as the interactions of microglia with neuron. Therefore, Fra‐1, through S100A8/TLR4 pathway, forms a feedback loop to promote sustained activation of microglia and induces microglia‐mediated neuroinflammation and apoptosis after spinal I/R.