Notch1 signaling promoted BMP2-induced endochondral ossification by simultaneously down-regulating Sox9 transcription and increasing VEGFA expression. (IMAGE)
Caption
(A) The binding of NICD1 to RBPjk. The interaction between NICD1 and RBPjk was testified by co-immunoprecipitation. NICD1 protein was pulled down by RBPjk antibody, and the expression of NICD1 was detected by Western blot analysis after protein precipitation, taking IgG as the control group. (B) RBPjk mediated regulation of target gene expression. The RBPjk protein-DNA complex was pulled down by RBPjk antibody, the expression of RBPjk was identified in the Input group, taking IgG as the control group, RBPjk were detected by Western blot after protein precipitation (IP group), and RBPjk protein-DNA complex was subjected to CHIP-seq. (C) CHIP-seq indicated the binding of the RBPjk complex between Sox9 and VEGFA promoters. CHIP-Seq analysis showed the binding sites of Sox9 were in the promoter region from 112781993 to 112782331 (a) and the binding sites of VEGFA were in the promoter region from 46031275 to 46031455 and from 46031827 to 46032374 (b). (D) Quantitative analysis of CHIP products. CHIP products were subjected to quantitative PCR analysis, the corresponding primers were designed according to the CHIP-Seq sites, and relative expression of the Sox9 promoter sequence and VEGFA promoter sequence were shown. (E) Sox9 promoted chondrogenic differentiation marker expression, inhibited endochondral ossification marker expression, and did not influence VEGFA expression. AdSox9 was used to overexpress Sox9 in BMP2-mediated chondrogenic differentiation. At indicated time points, Western blot assays were used for detecting the chondrogenic markers Sox9 and Col2a1, the endochondral ossification markers Runx2, Col1a1, and ONP, and the angiogenic differentiation marker VEGFA. Cropped blots were presented. (F) Quantitative analysis of Western blot results. Overexpression of Sox9 in BMP2-induced chondrogenic differentiation of MSCs promoted chondrogenic differentiation marker expression, inhibited endochondral ossification marker expression, and did not influence VEGFA expression. One-way analysis of variance; ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 versus the AdGFP group; ####p < 0.0001, ###p < 0.001, and ##p < 0.01 versus the indicated group; ns, p > 0.05. BMP2, bone morphogenetic protein 2; CHIP-seq, chromatin immunoprecipitation followed by sequencing; Col1a1, collagen type I alpha 1 chain; Col2a1, collagen type II alpha 1 chain; MSC, mesenchymal stem cell; NICD1, Notch1 intracellular domain; Notch1, Notch receptor 1; RBPjk, recombination signal-binding protein for immunoglobulin kappa J region; Runx2, RUNX family transcription factor 2; Sox9; SRY-box transcription factor 9; VEGFA, vascular endothelial growth factor A.
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Genes & Diseases
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