PP2ACα deficiency impairs early cortical development through inducing DNA damage in neuroprojenitor cells.

The role of protein phosphatase 2ACα (PP2ACα) in brain development is poorly understood. To understand the function of PP2ACα in neurogenesis, we inactivated Pp2acα gene in the central nervous system (CNS) of mice by Cre/LoxP system and generated the PP2ACα deficient mice (designated as the Pp2acα-/- mice). PP2ACα deletion results in DNA damage in neuroprogenitor cells (NPCs), which impairs memory formation and cortical neurogenesis. We first identify that PP2ACα can directly associate with Ataxia telangiectasia mutant kinase (ATM) and Ataxia telangiectasia/Rad3-related kinase (ATR) in neocortex and NPCs. Importantly, the P53 and hypermethylated in cancer 1 (HIC1) function complex, the newly found down-stream executor of the ATR/ATM cascade, will be translocated into nuclei and interact with homeodomain interacting protein kinase 2 (HIPK2) to respond to DNA damage. Notably, HICI plays a direct transcriptional regulatory role in HIPK2 gene expression. The interplay among P53, HIC1 and HIPK2 maintains DNA stability in neuroprogenitor cells. Taken together, our findings highlight a new role of PP2ACα in regulating early neurogenesis through maintaining DNA stability in neuroprogenitor cells. The P53/HIC/HIPK2 regulation loop, directly targeted by the ATR/ATM cascade, is involved in DNA repair in neuroprogenitor cells.

Wip1 knockout inhibits neurogenesis by affecting the Wnt/ß-catenin signaling pathway in focal cerebral ischemia in mice.

Neurogenesis correlates closely with the recovery of neural function after brain ischemia but the critical proteins and signaling pathways involved remain unclear. The phosphatase WIP1 has been shown to regulate neurogenesis in models of aging. However, it is not known if WIP1 affects neurogenesis and functional recovery after brain ischemia. To explore these questions, we performed permanent middle cerebral artery occlusion (MCAO) in mice and performed BrdU labeling, neurobehavioral testing, western blotting, and immunofluorescence staining. We found that ischemia induced WIP1 expression in the area bordering the injury. Compared to wild-type mice, the knockout of the Wip1 gene inhibited neurological functional recovery, reduced the expression of doublecortin, and inactivated the Wnt/ß-Catenin signaling pathway in cerebral ischemia in mice. Pharmacological activation of the Wnt/ß-Catenin signaling pathway compensated for the Wip1 knockout-induced deficit in neuroblast formation in animals with MCAO. These findings indicate that WIP1 is essential for neurogenesis after brain injury by activating the Wnt/ß-Catenin signaling pathway.

PPM1B depletion in U2OS cells supresses cell growth through RB1-E2F1 pathway and stimulates bleomycin-induced cell death.

PPM1B is a metal-dependent serine/threonine protein phosphatase, with a similar structure and function to the well-known oncogene in breast cancer, PPM1D (WIP1). However, clinical significance of PPM1B as a pharmacological target in cancer therapy has not been explored. To test if PPM1B can be a drug target in the cellular proliferation and death pathway, the lentiviral PPM1B shRNA was stably expressed in cancer cell lines and its regulatory function in the RB1-E2F1 pathway was examined. We found that PPM1B depletion suppressed cellular proliferation of U2OS cells, accompanied by hyper-phosphorylation of RB1 and up-regulation of E2F1 target genes, p27 and caspase 7. Notably, PPM1B depletion significantly sensitised U2OS cells to bleomycin-induced cell death at a minimal effective concentration. Our results suggest that PPM1B plays a negative role in the activation of the p38-RB1-E2F1 pathway and that targeting PPM1B could be useful in certain types of cancer by stimulating chemotherapy-induced cell death.

Wild-type p53-induced Phosphatase 1 Deficiency Exacerbates Myocardial Infarction-induced Ischemic Injury.

BACKGROUND: Myocardial infarction (MI) is a major disease burden. Wild-type p53-induced phosphatase 1 (Wip1) has been studied extensively in the context of cancer and the regulation of different types of stem cells, but the role of Wip1 in cardiac adaptation to MI is unknown. We investigated the significance of Wip1 in a mouse model of MI. METHODS: The study began in June 2014 and was completed in July 2016. We compared Wip1-knockout (Wip1-KO) mice and wild-type (WT) mice to determine changes in cardiac function and survival in response to MI. The heart weight/body weight (HW/BW) ratio and cardiac function were measured before MI. Mouse MI was established by ligating the left anterior descending (LAD) coronary artery under 1.5% isoflurane anesthesia. After MI, survival of the mice was observed for 4 weeks. Cardiac function was examined by echocardiography. The HW/BW ratio was analyzed, and cardiac hypertrophy was measured by wheat germ agglutinin staining. Hematoxylin and eosin (H&E) staining was used to determine the infarct size. Gene expression of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) was assessed by quantitative real-time polymerase chain reaction (qPCR), and the levels of signal transducers and activators of transcription 3 (stat3) and phosphor-stat3 (p-stat3) were also analyzed by Western blotting. Kaplan-Meier survival analysis, log-rank test, unpaired t-test, and one-way analysis of variance (ANOVA) were used for statistical analyses. RESULTS: Wip1-KO mice had a marginally increased HW/BW ratio and slightly impaired cardiac function before LAD ligation. After MI, Wip1-deficient mice exhibited increased mortality (57.14% vs. 29.17%; n = 24 [WT], n = 35 [Wip1-KO], P< 0.05), increased cardiac hypertrophy (HW/BW ratio: 7 days: 7.25 ± 0.36 vs. 5.84 ± 0.18, n = 10, P< 0.01, and 4 weeks: 6.05 ± 0.17 vs. 5.87 ± 0.24, n = 10, P > 0.05; cross-sectional area: 7 days: 311.80 ± 8.29 vs. 268.90 ± 11.15, n = 6, P< 0.05, and 4 weeks: 308.80 ± 11.26 vs. 317.00 ± 13.55, n = 6, P > 0.05), and reduced cardiac function (ejection fraction: 7 days: 29.37 ± 1.38 vs. 34.72 ± 1.81, P< 0.05, and 4 weeks: 19.06 ± 2.07 vs. 26.37 ± 2.95, P< 0.05; fractional shortening: 7 days: 13.72 ± 0.71 vs. 16.50 ± 0.94, P< 0.05, and 4 weeks: 8.79 ± 1.00 vs. 12.48 ± 1.48, P< 0.05; n = 10 [WT], n = 15 [Wip1-KO]). H&E staining revealed a larger infarct size in Wip1-KO mice than in WT mice (34.79% ± 2.44% vs. 19.55% ± 1.48%, n = 6, P< 0.01). The expression of IL-6 and p-stat3 was downregulated in Wip1-KO mice (IL-6: 1.71 ± 0.27 vs. 4.46 ± 0.79, n = 6, P< 0.01; and p-stat3/stat3: 1.15 ± 0.15 vs. 1.97 ± 0.23, n = 6, P< 0.05). CONCLUSION: The results suggest that Wip1 could protect the heart from MI-induced ischemic injury.

WIP1 phosphatase is a critical regulator of adipogenesis through dephosphorylating PPARγ serine 112.

WIP1, as a critical phosphatase, plays many important roles in various physiological and pathological processes through dephosphorylating different substrate proteins. However, the functions of WIP1 in adipogenesis and fat accumulation are not clear. Here, we report that WIP1-deficient mice show impaired body weight growth, dramatically decreased fat mass, and significantly reduced triglyceride and leptin levels in circulation. This dysregulation of adipose development caused by the deletion of WIP1 occurs as early as adipogenesis. In contrast, lentivirus-mediated WIP1 phosphatase overexpression significantly increases the adipogenesis of pre-adipocytes via an enzymatic activity-dependent mechanism. PPARγ is a master gene of adipogenesis, and the phosphorylation of PPARγ at serine 112 strongly inhibits adipogenesis; however, very little is known about the negative regulation of this phosphorylation. Here, we show that WIP1 phosphatase plays a pro-adipogenic role by interacting directly with PPARγ and dephosphorylating p-PPARγ S112 in vitro and in vivo.

WIP1 Phosphatase Plays a Critical Neuroprotective Role in Brain Injury Induced by High-Altitude Hypoxic Inflammation.

The hypobaric hypoxic environment in high-altitude areas often aggravates the severity of inflammation and induces brain injury as a consequence. However, the critical genes regulating this process remain largely unknown. The phosphatase wild-type p53-induced phosphatase 1 (WIP1) plays important roles in various physiological and pathological processes, including the regulation of inflammation in normoxia, but its functions in hypoxic inflammation-induced brain injury remain unclear. Here, we established a mouse model of this type of injury and found that WIP1 deficiency augmented the release of inflammatory cytokines in the peripheral circulation and brain tissue, increased the numbers of activated microglia/macrophages in the brain, aggravated cerebral histological lesions, and exacerbated the impairment of motor and cognitive abilities. Collectively, these results provide the first in vivo evidence that WIP1 is a critical neuroprotector against hypoxic inflammation-induced brain injury.

Myeloid-Specific Gene Deletion of Protein Phosphatase 2A Magnifies MyD88- and TRIF-Dependent Inflammation following Endotoxin Challenge.

Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the α isoform of the catalytic subunit of PP2A (PP2ACα). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2Afl/fl) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-α, KC, IL-6, and IL-10 were significantly increased in lyM-PP2Afl/fl mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-κB pathway (IKKα/ß, NF-κB p65) in bone marrow-derived macrophages (BMDMs) from knockout mice. Heightened NF-κB activation was not associated with degradation of IκBα; instead, enhanced phosphorylation of the NF-κB p65 subunit and p38 phosphorylation-mediated TNF-α mRNA stabilization appear to contribute to the increased TNF-α expression. In addition, increased IL-10 expression appears to be due to PP2ACα-knockout-induced IKKα/ß hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-ß/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/ß production compared with control BMDMs. Serum IFN-ß levels from PP2ACα-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain-containing adaptor inducing IFN-ß-dependent pathways.

Wip1 phosphatase modulates both long-term potentiation and long-term depression through the dephosphorylation of CaMKII.

Synaptic plasticity is an important mechanism that underlies learning and cognition. Protein phosphorylation by kinases and dephosphorylation by phosphatases play critical roles in the activity-dependent alteration of synaptic plasticity. In this study, we report that Wip1, a protein phosphatase, is essential for long-term potentiation (LTP) and long-term depression (LTD) processes. Wip1-deletion suppresses LTP and enhances LTD in the hippocampus CA1 area. Wip1 deficiency-induced aberrant elevation of CaMKII T286/287 and T305 phosphorylation underlies these dysfunctions. Moreover, we showed that Wip1 modulates CaMKII dephosphorylation. Wip1(-/-) mice exhibit abnormal GluR1 membrane expression, which could be reversed by the application of a CaMKII inhibitor, indicating that Wip1/CaMKII signaling is crucial for synaptic plasticity. Together, our results demonstrate that Wip1 phosphatase plays a vital role in regulating hippocampal synaptic plasticity by modulating the phosphorylation of CaMKII.