School of Medical Technology, Xuzhou Key Laboratory of Laboratory Diagnostics, Xuzhou Medical University, Xuzhou, 221004, China.
Department of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221002, China.
Stem Cell Res Ther. 2022 Jul 7;13(1):290. doi: 10.1186/s13287-022-02962-5.
To explore the function of phosphorylation of KAP1 (p-KAP1) at the serine-824 site (S824) in the proliferation and apoptosis of endogenous neural stem cells (NSCs) after cerebral ischemic/reperfusion (I/R).
The apoptosis and proliferation of C17.2 cells transfected with the p-KAP1-expression plasmids and the expression of proliferation cell nuclear antigen (PCNA) and p-KAP1 were detected by immunofluorescence and Western blotting after the Oxygen Glucose deprivation/reperfusion model (OGD/R). The interaction of p-KAP1 and CUL4A with PCNA was analyzed by immunoprecipitation. In the rats MCAO model, we performed the adeno-associated virus (AAV) 2/9 gene delivery of p-KAP1 mutants to verify the proliferation of endogenous NSCs and the colocalization of PCNA and CUL4A by immunofluorescence.
The level of p-KAP1 was significantly down-regulated in the stroke model in vivo and in vitro. Simulated p-KAP1(S824) significantly increased the proliferation of C17.2 cells and the expression of PCNA after OGD/R. Simulated p-KAP1(S824) enhanced the binding of p-KAP1 and PCNA and decreased the interaction between PCNA and CUL4A in C17.2 cells subjected to OGD/R. The AAV2/9-mediated p-KAP1(S824) increased endogenous NSCs proliferation, PCNA expression, p-KAP1 binding to PCNA, and improved neurological function in the rat MCAO model.
Our findings confirmed that simulated p-KAP1(S824) improved the survival and proliferation of endogenous NSCs. The underlying mechanism is that highly expressed p-KAP1(S824) promotes binding to PCNA, and inhibits the binding of CUL4A to PCNA. This reduced CUL4A-mediated ubiquitination degradation to increase the stability of PCNA and promote the survival and proliferation of NSCs.
探讨 KAP1（p-KAP1）丝氨酸 824 位磷酸化（S824）在脑缺血/再灌注（I/R）后内源性神经干细胞（NSCs）增殖和凋亡中的作用。
采用氧葡萄糖剥夺/复灌（OGD/R）模型，观察转染 p-KAP1 表达质粒的 C17.2 细胞凋亡和增殖情况，免疫荧光法和 Western blot 检测增殖细胞核抗原（PCNA）和 p-KAP1 的表达，免疫沉淀法分析 p-KAP1 与 CUL4A 与 PCNA 的相互作用。采用腺相关病毒（AAV）2/9 基因转染 p-KAP1 突变体，观察其在大鼠大脑中动脉闭塞（MCAO）模型中对内源性 NSCs 增殖的影响，通过免疫荧光法观察 PCNA 和 CUL4A 的共定位。
体内和体外脑缺血模型中 p-KAP1 水平明显下调。模拟 p-KAP1（S824）可显著增加 C17.2 细胞在 OGD/R 后的增殖和 PCNA 的表达。模拟 p-KAP1（S824）增强了 C17.2 细胞 OGD/R 后 p-KAP1 与 PCNA 的结合，减少了 PCNA 与 CUL4A 的相互作用。AAV2/9 介导的 p-KAP1（S824）增加了内源性 NSCs 的增殖、PCNA 的表达、p-KAP1 与 PCNA 的结合，并改善了 MCAO 大鼠的神经功能。
本研究证实模拟 p-KAP1（S824）可改善内源性 NSCs 的存活和增殖。其机制可能是高表达的 p-KAP1（S824）促进与 PCNA 的结合，抑制 CUL4A 与 PCNA 的结合，减少 CUL4A 介导的泛素化降解，增加 PCNA 的稳定性，促进 NSCs 的存活和增殖。
