The LncRNA AK018453 regulates TRAP1/Smad signaling in IL-17-activated astrocytes: A potential role in EAE pathogenesis

Abstract

The pro-inflammatory cytokine interleukin 17 (IL-17), that is mainly produced by Th17 cells, has been recognized as a key regulator in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Reactive astrocytes stimulated by proinflammatory cytokines including IL-17 are involved in blood brain barrier destruction, inflammatory cells infiltration and spinal cord injury. However, the role of long non-coding RNAs (lncRNAs) induced by IL-17 in the pathogenesis of MS and EAE remains unknown. Herein, we found that an IL-17-induced lncRNA AK018453 promoted TGF-β receptor-associated protein 1 (TRAP1) expression and Smad-dependent signaling in mouse primary astrocytes. Knockdown of AK018453 significantly suppressed astrocytosis, attenuated the phosphorylation of Smad2/3, reduced NF-κB p65 and CBP/P300 binding to the TRAP1 promoter, and diminished pro-inflammatory cytokine production in the IL-17-treated astrocytes. AK018453 knockdown in astrocytes by a lentiviral vector in vivo dramatically inhibited inflammation and prevented the mice from demyelination in the spinal cord during the progression of EAE. Together, these results suggest that AK018453 regulates IL-17-dependent inflammatory response in reactive astrocytes and potentially promotes the pathogenesis of EAE via the TRAP1/Smad pathway. Targeting this pathway may have a therapeutic potential for intervening inflammatory demyelinating diseases.

Keywords: TGF-β receptor-associated protein 1; astrocytes; experimental autoimmune encephalomyelitis; lncRNA AK018453; multiple sclerosis.

FIGURE 1

TRAP1/Smad signal pathway is activated in EAE mice. (a) The transcriptional level of TGF‐β, TRAP1, and TGFβRII mRNAs in spinal cords of EAE mice was tested by real‐time PCR assay. (b) The protein level of TRAP1, TGFβRII, Smad4, p‐Smad2/3, and GFAP in spinal cords was measured by Western blot assay. The relative level TRAP1, TGFβRII, Smad4, and GFAP was determined by quantitative densitometry compared to GAPDH; the relative level of phosphorylation Smad2/3 (p‐Smad2/3) was determined by quantitative densitometry after normalized to corresponding non‐phosphorylation ones. The relative value of proteins in the NC group was considered to be 1 for comparison. (c) IFA was used to test the expression of GFAP (red), TRAP1 (green), and nuclear staining of DAPI (blue) in astrocytes from spinal cords of EAE mice. Scale bars, 20 μm. (d) The mRNA levels of TNF‐α, CXCL10, and MCP‐1 in spinal cords were determined by real‐time PCR assay. (e) The secretion levels of TNF‐α, CXCL10, and MCP‐1 in the sera of mice were detected by ELISA. Data are represented as the mean ± SEM. *p < .05, **p < .01, and ***p < .001 versus NC group (n = 10 mice/group)

FIGURE 2

TRAP1/Smad signaling is activated in primary mouse astrocytes by IL‐17. Primary mouse astrocytes were cultured in a serum‐free medium overnight, prior to stimulating with IL‐17 (50 ng/ml) at indicated time point. (a) The mRNA levels of TRAP1 and TGFβRII were determined by real‐time PCR assay. (b) The protein levels of TRAP1, TGFβRII, Smad4, p‐Smad2/3, Smad2/3, and GFAP in astrocytes stimulated with IL‐17 were measured by Western blot assay. (c) Co‐immunoprecipitation (co‐IP) assay was employed to measure the interaction of TRAP1 with Smad4 in astrocytes treated with IL‐17. (d) The mRNA levels of TGF‐β, TNF‐α, CXCL10, and MCP‐1 in astrocytes with IL‐17 were measured by real‐time PCR assay. (e) The secretion levels of TGF‐β, TNF‐α, CXCL10, and MCP‐1 were measured by ELISA in the supernatant from astrocytes treated with IL‐17. The data are from three independent experiments and represented as the mean ± SEM. *p < .05, **p < .01, and ***p < .001 versus 0 h group treated with IL‐17 (n = 3)

FIGURE 3

Knockdown of TRAP1 decreases inflammatory reaction in activated astrocytes primary mouse astrocytes were infected with recombinant lentivirus containing TRAP‐shRNA and ctrl‐shRNA sequence, for 72 h, respectively. And then, astrocytes were incubated in a serum‐free medium overnight followed by IL‐17 (50 ng/ml) treatment for 6 h. (a) Western blot assay was utilized to measure the protein level of TRAP1, Smad4, p‐Smad2/3, and Smad2/3. (b) Co‐immunoprecipitation (co‐IP) assay was designed to determine the interaction of TRAP1 with Smad4. (c) The mRNA levels of TNF‐α, CXCL10, and MCP‐1 in astrocytes were detected by real‐time PCR assay. (d) The secretion levels of TNF‐α, MCP‐1, and CXCL10 in the supernatant of astrocytes were tested by ELISA. The data are from three independent experiments and represented as mean ± SEM. **p < .01 and ***p < .001 versus no IL‐17 group; ^# p < .05, ^## p < .01, and ^### p < .001 versus ctrl‐shRNA + IL‐17 group (n = 3)

FIGURE 4

Knockdown of TRAP1 alleviates EAE pathogenesis in mice. Mice were injected to recombinant lentiviruses carrying the promoter of GFAP and TRAP‐shRNA or ctrl‐shRNA sequence, for 7 days, prior to MOG_35–55 immunization for 21 days (n = 10 mice per group). (a) The clinical scores of EAE mice by TRAP‐shRNA and ctrl‐shRNA treatment. (b) Western blot assay was employed to detect the protein levels of TRAP1, Smad4, p‐Smad2/3, Smad2/3, and GFAP in the spinal cords. (c and d) The expression level of TNF‐α, CXCL10, and MCP‐1 in the spinal cords and peripheral blood of the TRAP‐shRNA and ctrl‐shRNA mice were measured by real‐time PCR assay and ELISA, respectively. The data are represented as mean ± SEM. ***p < .001 versus NC group; ^# p < .05 and ^## p < .01 versus ctrl‐shRNA group (n = 10 mice/group). (e) Hematoxylin and eosin (H&E) staining was employed to evaluate the infiltrations of inflammatory cells in spinal cords (scale bars, 50 μm). (f) Luxol fast blue (LFB) staining was used to investigate the medullary sheath damages from spinal cords (scale bars, 50 μm). Red box areas in the upper rows are presented enlarged underneath. (g) Electron microscope (EM) was employed to observe the severe disruption or mild loosening of the medullary sheath in spinal cords. Red arrow indicates the changes of the medullary sheath. Scale bars, 1 μm

FIGURE 5

LncRNA AK018453 is upregulated and regulates TRPA1 expression in activated astrocytes by IL‐17. (a) Comparative lncRNA array analysis indicated that differentially upregulated lncRNAs occurred in the primary astrocytes treated with IL‐17 (50 ng/ml) at 3 and 6 h (2.0‐fold higher than DMEM group). (b) Hot map showed some differentially‐expressed lncRNAs in astrocytes stimulated with IL‐17. Red box circled differentially co‐upregulated lncRNA AK018453. (c) Real‐time PCR assay was employed to verify lncRNA AK018453 expression in primary mouse astrocytes treated by IL‐17 and spinal cords from EAE mice. Results were represented as mean ± SEM. **p < .01 and ***p < .001 versus 0 h group. ^# p < .05 and ^## p < .01 versus NC group (n = 3). (d and e) RIP assay was performed to investigate the interaction of AK018453 with NF‐κB p65 and CBP/P300 in astrocytes stimulated with IL‐17 for 6 h, or prior to infection by lentiviruses AK018453‐shRNA (AK‐shRNA) and ctrl‐shRNA for 72 h. The data were from three independent experiments. **p < .01 and ***p < .001 versus DMEM. ^# p < .05 versus ctrl‐shRNA + IL‐17 group (n = 3). (f and g) ChIP assay analysis of NF‐κB p65, CBP/P300, H3K27ac, and RNA pol II enrichment on the promoter of TRAP1 gene in astrocytes stimulation with IL‐17 for 6 h, or prior to infection by lentiviruses AK‐shRNA and ctrl‐shRNA for 72 h. The data were from three independent experiments and represented as mean ± SEM. *p < .05, **p < .01, ***p < .001 versus DMEM; ^# p < .05 and ^## p < .01 versus ctrl‐shRNA + IL‐17 group (n = 3)

FIGURE 6

Knockdown of AK018453 decreases the activation of TRAP1/Smad pathway and the production of inflammation cytokines in IL‐17‐activated astrocytes. Primary mouse astrocytes were infected with recombinant lentivirus carrying GFAP promoter and AK‐shRNA or ctrl‐shRNA, for 72 h, respectively, followed by IL‐17 (50 ng/ml) treatment for 6 h. (a) Western blot assay was arranged to detect the protein level of TRAP1, Smad4, p‐Smad2/3, and Smad2/3. (b) Co‐immunoprecipitation (co‐IP) assay was employed to measure the interaction of TRAP1 with Smad4. (c) The mRNA levels of TNF‐α, CXCL10, and MCP‐1 in astrocytes were tested by real‐time PCR assay. (d) The secretion levels of TNF‐α, CXCL10, and MCP‐1 in the supernatant of astrocytes were measured by ELISA. The data are from three independent experiments and represented as mean ± SEM. *p < .05 and ***p < .001 versus no IL‐17 group; ^# p < .05 and ^## p < .01 versus ctrl‐shRNA + IL‐17 group (n = 3)

FIGURE 7

AK018453 knockdown lessens the pathology of EAE mice. On day 7 after administration with recombinant lentivirus carrying GFAP promoter and AK‐shRNA or ctrl‐shRNA sequence, respectively, mice were immunized with or without MOG_35–55 for 21 days. (a) The level of lncRNA AK018453 was determined by real‐time PCR assay. ***p < .001 versus NC group; ^# p < .05 versus ctrl‐shRNA group (n = 10 mice/group). (b) The clinical scores for EAE mice infected with AK‐shRNA or ctrl‐shRNA (n = 10 mice per group). ***p < .001 versus NC group; ^## p < .01 versus ctrl‐shRNA group. (c) Western blot assay was used to determine the protein levels of TRAP1, Smad4, p‐Smad2/3, Smad2/3, and GFAP in the spinal cords. (d and e) The production of TNF‐α, CXCL10, and MCP‐1 in the spinal cords and peripheral blood from the AK‐shRNA and ctrl‐shRNA mice was measured by real‐time PCR assay and ELISA assay, respectively. The data are represented as mean ± SEM. ***p < .001 versus NC group; ^# p < .05, ^## p < .01, and ^### p < .001 versus ctrl‐shRNA group (n = 10 mice/group). (f) Hematoxylin and eosin (H&E) staining was designed to observe the infiltrations of inflammatory cells in spinal cords (scale bars, 50 μm). (g) Luxol fast blue (LFB) staining was utilized to test the medullary sheath damages from spinal cords (scale bars, 50 μm). Red box areas in the upper rows are presented enlarged underneath. (h) Electron microscope (EM) was employed to investigate the severe disruption or mild loosening of the medullary sheath in spinal cords. Red arrow shows the changes of the medullary sheath. Scale bars, 1 μm

FIGURE 8

Schematic representation of lncRNA AK018453 contributing to the pathology of EAE mice via regulating TRAP1/Smad pathway. Response to IL‐17 stimulation, AK018453 is upregulated in astrocytes, which regulates epigenetically the expression of TRAP1 through interacting with CBP/P300, in turn promotes the production of pro‐inflammatory cytokines such as TNF‐α, CXCL10, and MCP‐1, thus aggravating the EAE progression