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PP2A inhibitors arrest G2/M transition through JNK/Sp1-dependent down-regulation of CDK1 and autophagy-dependent up-regulation of p21

ABSTRACT: Protein phosphatase 2A (PP2A) plays an important role in the control of the cell cycle. We previously reported that the PP2A inhibitors, cantharidin and okadaic acid (OA), efficiently repressed the growth of cancer cells. In the present study, we found that PP2A inhibitors arrested the cell cycle at the G2 phase through a mechanism that was dependent on the JNK pathway. Microarrays further showed that PP2A inhibitors induced expression changes in multiple genes that participate in cell cycle transition. To verify whether these expression changes were executed in a PP2A-dependent manner, we targeted the PP2A catalytic subunit (PP2Ac) using siRNA and evaluated gene expression with a microarray. After the cross comparison of these microarray data, we identified that CDK1 was potentially the same target when treated with either PP2A inhibitors or PP2Ac siRNA. In addition, we found that the down-regulation of CDK1 occurred in a JNK-dependent manner. Luciferase reporter gene assays demonstrated that repression of the transcription of CDK1 was executed through the JNK-dependent activation of the Sp1 transcription factor. By constructing deletion mutants of the CDK1 promoter and by using ChIP assays, we identified an element in the CDK1 promoter that responded to the JNK/Sp1 pathway after stimulation with PP2A inhibitors. Cantharidin and OA also up-regulated the expression of p21, an inhibitor of CDK1, via autophagy rather than PP2A/JNK pathway. Thus, this present study found that the PP2A/JNK/Sp1/CDK1 pathway and the autophagy/p21 pathway participated in G2/M cell cycle arrest triggered by PP2A inhibitors. Quality-checked RNAs were then transcribed with the First-Strand cDNA Synthesis Kit(Agilent)and their expression data obtained using Agilent 4×44 K Human Whole-Genome 60-mer oligonucleotide microarrays according to the protocols by the manufacturer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE114288 | GEO | 2018/12/06

REPOSITORIES: GEO

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Project description:Protein phosphatase 2A (PP2A) is one of the most common serine/threonine phosphatases in mammalian cells and primarily functions to regulate cell signaling, glycolipid metabolism, and apoptosis. Its PP2A catalytic subunit (PP2Ac) plays an important role in its function. Nonetheless, at present, there are only a few reports on the regulatory role of PP2Ac in pancreatic β-cells under lipotoxicity. Mouse pancreatic insulinoma (MIN6) cells were transfected by lentiviruses to generate PP2Ac knockdown cells and incubated with palmitate (PA) to establish the lipotoxicity model. Serine/Threonine Phosphatase Assay System kit, Cell Counting Kit-8 (CCK-8), flow cytometry, enzyme-linked immunosorbent assay (ELISA), Western Blotting (WB) and other techniques were used to measure PP2A activity, cell viability, apoptosis, oxidative stress, and insulin secretion. An animal model of lipotoxicity with knockdown of PP2Ac was established by a high-fat diet (HFD) after using adeno-associated viruses (AAV) to interfere with PP2Ac expression in mouse pancreatic tissues. Serine/Threonine Phosphatase Assay System kit, ELISA, pancreatic tissue immunofluorescence and other techniques were used to measure PP2A activity in pancreatic tissue, serum insulin level and the proliferation of mouse pancreatic β-cells. We found that PP2Ac knockdown inhibited lipotoxicity-induced PP2A hyperactivation, increased the resistance of pancreatic β-cells to lipotoxicity, decreased PA-induced apoptosis in MIN6 cells, protected the function of both the endoplasmic reticulum (ER) and mitochondria, and improved insulin secretion. By mRNA sequencing and Western blotting analysis, we hypothesized that the protective effects of PP2Ac knockdown in MIN6 cells may be mediated by the MAPK pathway. Moreover, the results obtained from animal experiments suggest that the specific knockdown of pancreatic PP2Ac could effectively attenuate HFD-induced insulin resistance and reduce the compensatory proliferation of pancreatic β-cells in mice. The present study revealed the effects and mechanisms of interfering with PP2Ac gene expression on pancreatic β-cells in vivo and in vitro, which might provide insights for the treatment of type 2 diabetes in the clinic.

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PP2A inhibitors arrest G2/M transition through JNK/Sp1-dependent down-regulation of CDK1 and autophagy-dependent up-regulation of p21

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