p300 arrests intervertebral disc degeneration by regulating the FOXO3/Sirt1/Wnt/β-catenin axis

Abstract

The transcription factor p300 is reportedly involved in age-associated human diseases, including intervertebral disc degeneration (IDD). In this study, we investigate the potential role and pathophysiological mechanism of p300 in IDD. Clinical tissue samples were collected from patients with lumbar disc herniation (LDH), in which the expression of p300, forkhead box O3 (FOXO3), and sirtuin 1 (Sirt1) was determined. Nucleus pulposus cells (NPCs) isolated from clinical degenerative intervertebral disc (IVD) tissues were introduced with oe-p300, oe-FOXO3, Wnt/β-catenin agonist 1, C646 (p300/CBP inhibitor), or si-p300 to explore the functional role of p300 in IDD and to characterize the relationship between p300 and the FOXO3/Sirt1/Wnt/β-catenin pathway. Also, we established a rat IDD model by inducing needle puncture injuries in the caudal IVDs for further verification of p300 functional role. We found that p300 was downregulated in the clinical tissues and NPCs of IDD. Overexpression of p300 promoted the proliferation and autophagy of NPCs while inhibiting cell apoptosis, which was associated with FOXO3 upregulation. p300 could increase the expression of FOXO3 by binding to the Sirt1 promoter, and thus, contributed to inactivation of the Wnt/β-catenin pathway. In vivo results further displayed that p300 slowed down the progression of IDD by disrupting the Wnt/β-catenin pathway through the FOXO3/Sirt1 axis. Taken together, we suggest that p300 can act to suppress IDD via a FOXO3-dependent mechanism, highlighting a potential novel target for treatment of IDD.

Keywords: Autophagy; FOXO3; Intervertebral disc degeneration; Nucleus pulposus cells; Sirt1; Wnt/β-catenin pathway; p300.

FIGURE 1

Overexpression of p300 alleviates IDD. (a) p300 expression in clinical NP tissues of healthy controls and patients with IDD as determined by RT‐qPCR (n = 58). *** p < 0.001 vs. healthy controls. (b) p300 protein expression in clinical NP tissues of healthy controls and patients with IDD as determined by Western blot assay. *** p < 0.001 vs. healthy controls. (c) p300 expression in human NPCs as determined by RT‐qPCR. *** p < 0.01 vs. controls. **** p < 0.001 vs. NPC + oe‐NC. (d) The protein expression of p300 in NPCs as determined by Western blot assay. *** p < 0.001 vs. controls. **** p < 0.0001 vs. NPC + oe‐NC. (e) The number of proliferating cells in cultured NPCs as observed by EdU assay. ** p < 0.01 vs. controls. **** p < 0.0001 vs. NPC + oe‐NC. (f) The proportion of apoptotic cells in cultured NPCs. ** p < 0.01 vs. controls

FIGURE 2

p300 promotes FOXO3 to inhibit apoptosis of NPCs. (a) FOXO3 expression in clinical NP tissues of healthy controls and patients with IDD as determined by RT‐qPCR. n = 58. *** p < 0.001 vs. healthy controls. (b) FOXO3 protein expression in clinical NP tissues of healthy controls and patients with IDD as determined by Western blot assay. ** p < 0.01 vs. healthy controls. (c) FOXO3 expression in human NPCs as determined by RT‐qPCR. (d) The protein expression of FOXO3 in NPCs as determined by Western blot assay. (e) The number of proliferating cells in cultured NPCs as observed by EdU assay, green fluorescence represents EdU positive staining and blue fluorescence represents DAPI (scale bar: 25 μm). (f) The proportion of apoptotic cells in cultured NPCs. (g) FOXO3 expression in NPCs after inhibition of p300 by C646. *** p < 0.001 vs. DMSO. (h) The protein expression of FOXO3 and p300 in NPCs treated with si‐p300 as determined by Western blot assay. (i) Pearson's correlation analysis on the correlation between the expression of p300 and FOXO3 in IVD tissues of IDD patients. The measurement data were expressed as mean ± standard deviation. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. Comparison between two groups was conducted using independent sample t‐test. Comparison among multiple groups was conducted using one‐way ANOVA, followed by Tukey post‐hoc test

FIGURE 3

p300 promotes FOXO3 expression and enhances autophagy of NPCs. (a) The ratio of LC3‐II/LC3‐I in clinical samples of healthy controls and patients with IDD as determined by Western blot assay. n = 58. (b) The protein expression of autophagy‐related factors in clinical samples of healthy controls and patients with IDD as determined by Western blot assay. (c) Observation of autophagy in human NPCs by TEM. (d) The ratio of LC3‐II/LC3‐I in NPCs as determined by Western blot assay. (e) The protein expression of autophagy‐related factors in NPCs as determined by Western blot assay. (f) Autophagic flux determined by mRFP‐GFP‐LC3 assay. The expression and location of LC3 in NPCs as examined by immunofluorescence staining. The measurement data were expressed as mean ± standard deviation. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. Comparison between two groups was conducted using independent sample t‐test. Comparison among multiple groups was conducted using one‐way ANOVA, followed by Tukey post‐hoc test

FIGURE 4

FOXO3 binds to Sirt1 promoter to promote Sirt1 expression in IDD. (a) The interaction network for FOXO3 target genes. (b) The statistical plot for the core value of the core genes. A gene with more interaction genes has a higher core degree value. The X‐axis represents the degree value, and the Y‐axis represents the gene name. (c) The FOXO3 binding site in the Sirt1 promoter and its mutant sequences. (d) The binding relationship between FOXO3 and Sirt1‐promoter as examined by dual‐luciferase reporter gene assay. *** p < 0.001 vs. pGL3 SIRT1‐promoter‐WT. **** p < 0.0001 vs. pGL3 SIRT1‐promoter‐WT. (e) ChIP‐PCR for FOXO3 enrichment in the SIRT1 promoter. (f) Sirt1 expression in clinical NP tissues of healthy controls and patients with IDD as determined by RT‐qPCR. n = 58. *** p < 0.001 vs. healthy controls. (g) Sirt1 protein expression in clinical NP tissues of healthy controls and patients with IDD as determined by Western blot assay. (h) The expression of Sirt1 in NPCs as determined by RT‐qPCR. (i) Sirt1 protein expression in NPCs as determined by Western blot assay. The measurement data were expressed as mean ± standard deviation. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. Comparison was conducted using one‐way ANOVA, followed by Tukey post‐hoc test

FIGURE 5

FOXO3 promotes Sirt1 expression to inhibit apoptosis and promote autophagy of NPCs. (a) The proportion of apoptotic cells in cultured NPCs. (b) Observation of autophagosomes in human NPCs by TEM. (c) The changes and ratio of LC3‐II/LC3‐I in NPCs. (d) The expression of autophagy‐related factors in NPCs as determined by Western blot assay. The measurement data were expressed as mean ± standard deviation. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. Statistical comparison was conducted using one‐way ANOVA, followed by Tukey post‐hoc test

FIGURE 6

p300‐mediated FOXO3 promotes Sirt1 to inhibit apoptosis of NPCs through inactivation of the Wnt/β‐catenin pathway. (a) Protein expression changes of p300, FOXO3 and Sirt1 in NPCs with different treatment as determined by Western blot assay. (b) The expression of Wnt/β‐catenin pathway‐related factors in NPCs after treatment with Wnt/β‐catenin agonist 1 as determined by Western blot assay. (c) The number of proliferating cells in cultured NPCs as observed by EdU staining (scale bar: 25 μm), green fluorescence represents EdU positive staining and blue fluorescence represents DAPI. (d) The proportion of apoptotic cells in NPCs. * p < 0.05 vs. NPCs treated with oe‐NC + DMSO. # p < 0.05 vs. NPCs treated with oe‐p300 + DMSO. $ p < 0.05 vs. NPCs treated with oe‐Sirt1 + DMSO. (e) Immunofluorescence staining for determination of the nucleation of β‐catenin in NPCs (scale bar: 25 μm), red fluorescence represents β‐catenin positive staining and blue fluorescence represents DAPI. (f) The autophagy in human NPCs as observed by TEM. (g) The protein expression of autophagy‐related factors in NPCs after treatment with Wnt/β‐catenin agonist 1 as determined by Western blot assay. (h) The protein expression of LC3‐II and LC3‐1 and the changes in the ratio of LC3‐II/LC3‐1 in NPCs after treatment with Wnt/β‐catenin agonist 1 as determined by Western blot assay. The measurement data were expressed as mean ± standard deviation. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. Statistical comparison was conducted using one‐way ANOVA, followed by Tukey post‐hoc test

FIGURE 7

Overexpression of p300 regulates the Wnt/β‐catenin pathway to inhibit IDD in rats. (a) LC3‐II/LC3‐I ratio in the IVD tissues of IDD rats as determined by Western blot assay. (b) Changes of protein expression of p300, FOXO3 and Sirt1 in the IVD tissues of IDD rats as determined by Western blot assay. (c) Changes of protein expression of Wnt/β‐catenin pathway‐related factors in the IVD tissues of IDD rats as determined by Western blot assay. (d) IDD in rats determined by the MRI method with Pfirrmann grading. (e) Autophagosomes in NPCs of rat IVD tissues as observed by TEM. (f) Safranin O‐fast green staining for the pathological changes of IVD tissues. (g) TUNEL staining for the apoptosis of rat IVD tissues (scale bar: 50 μm), green fluorescence represents TUNEL staining and blue fluorescence represents DAPI. The measurement data were expressed as mean ± standard deviation. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. Statistical comparison was conducted using one‐way ANOVA, followed by Tukey post‐hoc test. There were 6 rats in each group