Aims 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). Methods 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. Results 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. Conclusions 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.
KRAB domain-associated protein 1 (KAP1) is highly expressed in atherosclerotic plaques. Here, we studied the role of KAP1 in atherosclerosis development using a cell model of endothelial dysfunction induced by oxidative low-density lipoprotein (OxLDL). The phosphorylation and protein levels of KAP1 were similar between OxLDL-treated and non-treated endothelial cells (ECs). KAP1 depletion significantly inhibited the production of OxLDL-enhanced reactive oxygen species and the expression of adhesion molecules in ECs. Treatment with OxLDL promoted the proliferation and migration of ECs, which was also confirmed by the elevated levels of the proliferative markers c-Myc and PCNA, as well as the migratory marker MMP-9. However, these effects were also abrogated by KAP1 depletion. Moreover, the depletion of KAP1 in OxLDL-treated ECs resulted in decreases in the LOX-1 level and increases in eNOS expression. Generally, the data suggest that strategies targeting KAP1 depletion might be particularly useful for the prevention or treatment of atherosclerosis.
The roles of Caveolin-1 (Cav-1) and Wnt/β-catenin signaling pathways in cerebral ischemia-reperfusion (I/R) injury are well established. Translocation of β-catenin into the nucleus is critical in regulating the apoptosis, repair, and nerve regeneration of neurons in the ischemic brain. Caveolin (Cav)-1 scaffold domain (residues 95-98) has been reported to interact with β-catenin (residues 330-337). However, the contribution of the Cav-1/β-catenin complex to I/R injury is currently unknown. To investigate the mechanism underlying the involvement of the Cav-1/β-catenin complex in the subcellular translocation of β-catenin and its subsequent effects on cerebral I/R injury, we treated ischemic brains with ASON (Cav-1 antisense oligodeoxynucleotides) or FTVT (a competitive peptide antagonist of the interaction Cav-1 and β-catenin). Our study showed that the binding of Cav-1 to β-catenin after I/R injury prevented the nuclear accumulation of β-catenin. The disruption of the Cav-1/β-catenin complex with ASON or FTVT after I/R injury significantly increased nuclear β-catenin. ASON and FTVT reduced the Ser33, Ser37 Thr41 phosphorylation of β-catenin, contributing to its proteasomal degradation; however, it increased the Tyr333 phosphorylation relating to its nuclear translocation. The above results indicated that the formation of the Cav-1/ β-catenin complex anchored the cytoplasmic β-catenin after I/R injury. Besides, both ASON and FTVT could attenuate neuronal death in ischemic brains. Our study suggests that targeting the interaction of Cav-1 with β-catenin could be a novel therapeutic strategy to protect against neuronal damage during cerebral injury.
Aims: To explore the function of phosphorylation of KAP1 (p-KAP1) at serine-824 site (S824) in the proliferation and apoptosis of endogenous neural stem cells (NSCs) after cerebral ischemic/reperfusion (I/R). Methods: 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 were analyzed by immunoprecipitation. In the rats MCAO model, we performed the lentiviral-mediated p-KAP1 mutants to verify the proliferation of endogenous NSCs and the colocalization of PCNA and CUL4A by immunofluorescence.Results: 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. With the lentiviral AAV2/9-mediated p-KAP1(S824), there was increased endogenous NSCs proliferation, PCNA expression, p-KAP1 binding to PCNA, and improved neurological function in the rat MCAO model.Conclusions: Our findings confirmed that simulated p-KAP1(S824) improved the survival and proliferation of endogenous NSCs. The underlying mechanism involved in that highly expressed p-KAP1(S824) promoted to bind to PCNA, inhibiting the binding of CUL4A to PCNA. This reduced CUL4A-mediated ubiquitination degradation to increase the stability of PCNA and promote survival and proliferation of NSCs.
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