Int J Stem Cells.  2023 Aug;16(3):326-341. 10.15283/ijsc22046.

Wedelolactone Promotes the Chondrogenic Differentiation of Mesenchymal Stem Cells by Suppressing EZH2

• Qin W
• Yang L
• Chen X
• Ye S
• Liu A
• Chen D
• Hu K

• KMID: 2545232
• DOI: http://doi.org/10.15283/ijsc22046

Abstract

Background and Objectives
Osteoarthritis (OA) is a degenerative disease that leads to the progressive destruction of articular cartilage. Current clinical therapeutic strategies are moderately effective at relieving OA-associated pain but cannot induce chondrocyte differentiation or achieve cartilage regeneration. We investigated the ability of wedelolactone, a biologically active natural product that occurs in Eclipta alba (false daisy), to promote chondrogenic differentiation.
Methods and Results
Real-time reverse transcription–polymerase chain reaction, immunohistochemical staining, and immunofluorescence staining assays were used to evaluate the effects of wedelolactone on the chondrogenic differentiation of mesenchymal stem cells (MSCs). RNA sequencing, microRNA (miRNA) sequencing, and isobaric tags for relative and absolute quantitation analyses were performed to explore the mechanism by which wedelolactone promotes the chondrogenic differentiation of MSCs. We found that wedelolactone facilitates the chondrogenic differentiation of human induced pluripotent stem cell-derived MSCs and rat bone-marrow MSCs. Moreover, the forkhead box O (FOXO) signaling pathway was upregulated by wedelolactone during chondrogenic differentiation, and a FOXO1 inhibitor attenuated the effect of wedelolactone on chondrocyte differentiation. We determined that wedelolactone reduces enhancer of zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation of the promoter region of FOXO1 to upregulate its transcription. Additionally, we found that wedelolactone represses miR-1271-5p expression, and that miR-1271-5p post-transcriptionally suppresses the expression of FOXO1 that is dependent on the binding of miR-1271-5p to the FOXO1 3’-untranscribed region.
Conclusions
These results indicate that wedelolactone suppresses the activity of EZH2 to facilitate the chondrogenic differentiation of MSCs by activating the FOXO1 signaling pathway. Wedelolactone may therefore improve cartilage regeneration in diseases characterized by inflammatory tissue destruction, such as OA.

Keyword

Wedelolactone; Chondrogenic differentiation; EZH2; FOXO1; miR-1271-5p

Figure

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  Fig. 1 Wedelolactone promotes the differentiation of hiPSC-derived MSCs to chondrocytes in vitro. (A) Schematic procedure of the chondrogenic differentiation of MSCs induced by human iPSC-derived MSCs. (B) Image of the chondrogenic pellet differentiated from human iPSC-derived MSCs. (C) Gene expression analysis of the chondrogenic differentiation markers (collagen type II alpha-1 [COL2A1], SRY-box transcription factor 9 [SOX9], and aggrecan [ACAN]) in the chondrogenic pellet. (D) Immunohistochemistry image of the chondrogenic pellet stained with COL2A1 and ACAN. Scale bar=100 μm. Mean density was used to quantify the COL2A1 and ACAN contents in the chondrogenic pellet. (E) Immunofluorescence image of iPSC-derived MSCs under chondrogenic differentiation stained with SOX9, COL2A1, and ACAN. Scale bar=100 μm. Relative fluorescence intensity was used to quantify the expression levels of SOX9, COL2A1, and ACAN. Data are expressed as the mean±standard deviation (SD) (n=3). Statistical differences were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test: **p<0.01, ***p<0.001. EB: embryoid body, iMSCs: human iPSC-derived MSCs, Chon-dif: chondrogenic differentiation.

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  Fig. 2 Wedelolactone promotes the chondrogenic differentiation of rat BMSCs. (A) Schematic procedure of the chondrogenic differen-tiation of BMSCs. (B) Image of the chondrogenic pellet differentiated from BMSCs. (C) Gene expression analysis of the chondrogenic differentiation markers (COL2A1, SOX9, and ACAN) in the chondrogenic pellet. (D) Western blotting analysis of the chondrogenic differentiation markers (COL2A1 and SOX9) in the chondrogenic pellet. (E) Mean gray value was used to quantify the protein expression levels of COL2A1 and SOX9 in the chondrogenic pellet. (F) Schematic of the experimental outline. BMSCs treated with dimethyl sulfoxide (DMSO) or wedelolactone were mixed with the hydrogel and transplanted into the cartilage defect model, and the joint tissues were collected after 6 weeks. (G) Safranin-O fast green stains of joints after 6 weeks (n=5 rat per group). The Osteoarthritis Research Society International scoring system was used to grade the rat cartilage degeneration. Data are expressed as the mean±SD (n=3). Statistical differences were analyzed by one-way ANOVA followed by Dunnett’s test: **p<0.01, ***p<0.001.

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  Fig. 3 Wedelolactone promotes the chondrogenic differentiation of MSCs by activating the FOXO pathway. (A) Scatter plot of the quantitation proteomics analysis showing differently expressed proteins in the chondrogenic pellet differentiated from human iPSC-derived MSCs treated with DMSO or wedelolactone. (B) The Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed proteins in the chondrogenic pellet differentiated from human iPSC-derived MSCs treated with DMSO or wedelolactone. (C) Heatmap of the quantitation proteomics analysis showing differently expressed proteins associated with FOXO signaling. (D) Gene expression analysis of FOXO1 after induction with different concentrations of wedelolactone. (E) Immunofluorescence staining of FOXO1 in the cartilage defect model after wedelolactone intervention. Scale bar=100 μm. Relative fluorescence intensity was used to quantify the expression of FOXO1. (F) Alcian blue and toluidine blue staining images of the chondrogenic differentiation of human iPSC-derived MSCs after different interventions. Quantification of the mean intensity of alcian blue and toluidine blue staining. (G) Gene expression analysis of the chondrogenic differentiation markers (COL2A1, SOX9, and ACAN) after FOXO1 knockdown. Data are expressed as the mean±SD (n=3). Statistical differences were analyzed by one-way ANOVA followed by Dunnett’s test: *p<0.05, **p<0.01, ***p<0.001.

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  Fig. 4 The promotion of chondrogenic differentiation by wedelolactone is weakened by FOXO1 inhibition. (A) Alcian blue and toluidine blue staining images of the chondrogenic differentiation of human iPSC-derived MSCs after different interventions. Quantification of the mean intensity of alcian blue and toluidine blue staining. (B) Gene expression analysis of the chondrogenic differentiation markers (COL2A1, SOX9, and ACAN) after wedelolactone and FOXO1 inhibitor (GSK126) intervention. Data are expressed as the mean±SD (n=3). Statistical differences were analyzed by one-way ANOVA followed by Dunnett’s test: *p<0.05, **p<0.01, ***p<0.001.

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  Fig. 5 Wedelolactone decreases EZH2-dependent trimethylation of H3K27 on the promoter region of FOXO1. (A) Chromatin immu-noprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) analysis of H3K27me3 enrichment in FOXO1 promoter after wedelolactone intervention in both human iPSC-derived MSCs and 293T cells. (B) Reverse transcription (RT)-qPCR analysis of FOXO1 in human iPSC-derived MSCs after FOXO1 inhibitor (GSK126) intervention. (C) Western blotting analysis of FOXO1 in human iPSC-derived MSCs after FOXO1 inhibitor (GSK126) intervention. (D) ChIP-qPCR analysis of EZH2 occupancy in FOXO1 promoter in both human iPSC-derived MSCs and 293T cells. (E) Western blotting analysis of FOXO1 in human iPSC-derived MSCs after EZH2 overexpression. (F) RT-qPCR analysis of FOXO1 in human iPSC-derived MSCs after EZH2 knockdown. (G) Western blotting analysis of FOXO1 in human iPSC-derived MSCs after EZH2 knockdown. (H) RT-qPCR analysis of FOXO1 in human iPSC-derived MSCs induced with wedelolactone after EZH2 knockdown.

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  Fig. 6 miR-1271-5P interferes with FOXO1 expression during chondrogenic differentiation. (A) Heatmap of miRNA-seq analysis, showing the differently expressed miRNAs in the chondrogenic pellet differentiated from human iPSC-derived MSCs, and human iPSC-derived MSCs treated with DMSO or wedelolactone. (B) Volcano plots showing the differently expressed miRNAs. Red color indicates the significantly upregulated miRNAs, blue color indicates the significantly downregulated miRNAs, and gray color indicates the genes with no differential expression. (C) Venn diagram overlap showing the number of upregulated miRNAs and miRNAs in the DMSO group compared to the human iPSC-derived MSCs and wedelolactone groups. (D) Venn diagram overlap showing the numbers of downregulated miRNAs and miRNAs in the DMSO group compared to the human iPSC-derived MSCs and wedelolactone groups. (E) miR-1271-5P target sequence in the 3’-untranslated region (UTR) of FOXO1 predicted by the TargetScan database. (F) MiR-1271-5P target 3’-untranslated region (UTR) of FOXO1 was confirmed by the luciferase reporter assay. (G) Western blotting detection of protein expression levels of FOXO1 in human iPSC-derived MSCs transfected with the miR-1271-5P mimic or inhibitor under chondrogenic differentiation. (H) RT-qPCR analysis of miR-1271-5P in human iPSC-derived MSCs after EZH2 knockdown. Statistical differences were analyzed by one-way ANOVA followed by Dunnett’s test: *p<0.05, **p<0.01, ***p<0.001.

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  Fig. 7 Schematic illustration of the working model.

Reference

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