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Background Intervertebral disc (IVD) degeneration is associated with chronic back pain. We previously demonstrated that the phosphatase pleckstrin homology domain and leucine‐rich repeat protein phosphatase (PHLPP) 1 was positively correlated with IVD degeneration and its deficiency decelerated IVD degeneration in both mouse IVDs and human nucleus pulposus (NP) cells. Small molecule PHLPP inhibitors may offer a translatable method to alleviate IVD degeneration. In this study, we tested the effectiveness of the two PHLPP inhibitors NSC117079 and NSC45586 in promoting a healthy NP phenotype. Methods Tail IVDs of 5‐month‐old wildtype mice were collected and treated with NSC117079 or NSC45586 under low serum conditions ex vivo. Hematoxylin & eosin staining was performed to examine IVD structure and NP cell morphology. The expression of KRT19 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human NP cells were obtained from patients with IVD degeneration. The gene expression of KRT19, ACAN, SOX9, and MMP13 was analyzed via real time qPCR, and AKT phosphorylation and the protein expression of FOXO1 was analyzed via immunoblot. Results In a mouse IVD organ culture model, NSC45586, but not NSC117079, preserved vacuolated notochordal cell morphology and KRT19 expression while suppressing cell apoptosis, counteracting the degenerative changes induced by serum deprivation, especially in males. Likewise, in degenerated human NP cells, NSC45586 increased cell viability and the expression of KRT19, ACAN, and SOX9 and reducing the expression of MMP13, while NSC117079 treatment only increased KRT19 expression. Mechanistically, NSC45586 treatment increased FOXO1 protein expression in NP cells, and inhibiting FOXO1 offset NSC45586‐induced regenerative potential, especially in males. Conclusions Our study indicates that NSC45586 was effective in promoting NP cell health, especially in males, suggesting that PHLPP plays a key role in NP cell homeostasis and that NSC45586 might be a potential drug candidate in treating IVD degeneration. Pharmacological inhibition of PHLPP via NSC45586 promotes nucleus pulposus cell health. NSC45586 is a potential drug candidate in treating IVD degeneration.

The phosphatidylinositol-3-OH kinase (PI3K)-Akt pathway is activated in cancer by genetic or epigenetic events and efforts are under way to develop targeted therapies. phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is the major brake of the pathway and a common target for inactivation in glioblastoma, one of the most aggressive and therapy-resistant cancers. To achieve potent inhibition of the PI3K-Akt pathway in glioblastoma, we need to understand its mechanism of activation by investigating the interplay between its regulators. We show here that PTEN modulates the PI3K-Akt pathway in glioblastoma within a tumor suppressor network that includes Na + /H + exchanger regulatory factor 1 (NHERF1) and pleckstrin-homology domain leucine-rich repeat protein phosphatases 1 (PHLPP1). The NHERF1 adaptor, previously characterized by our group as a PTEN ligand and regulator, shows also PTEN-independent Akt-modulating effects that led us to identify the PHLPP1/PHLPP2 Akt phosphatases as NHERF1 ligands. NHERF1 interacts via its PDZ domains with PHLPP1/PHLPP2 and scaffolds heterotrimeric complexes with PTEN. Functionally, PHLPP1 requires NHERF1 for membrane localization and growth-suppressive effects. PHLPP1 loss boosts Akt phosphorylation only in PTEN-negative cells and cooperates with PTEN loss for tumor growth. In a panel of low-grade and high-grade glioma patient samples, we show for the first time a significant disruption of all three members of the PTEN-NHERF1-PHLPP1 tumor suppressor network in high-grade tumors, correlating with Akt activation and patient's abysmal survival. We thus propose a PTEN-NHERF1-PHLPP PI3K-Akt pathway inhibitory network that relies on molecular interactions and can undergo parallel synergistic hits in glioblastoma.

PH domain leucine-rich-repeats protein phosphatase (PHLPP) is a family of Ser/Thr protein phosphatases that serve as tumor suppressors by negatively regulating Akt. Our recent studies have demonstrated that the ubiquitin proteasome pathway has an important role in the downregulation of PHLPP in colorectal cancer. In this study, we show that the deubiquitinase USP46 stabilizes the expression of both PHLPP isoforms by reducing the rate of PHLPP degradation. USP46 binds to PHLPP and directly removes the polyubiquitin chains from PHLPP in vitro and in cells. Increased USP46 expression correlates with decreased ubiquitination and upregulation of PHLPP proteins in colon cancer cells, whereas knockdown of USP46 has the opposite effect. Functionally, USP46-mediated stabilization of PHLPP and the subsequent inhibition of Akt result in a decrease in cell proliferation and tumorigenesis of colon cancer cells in vivo . Moreover, reduced USP46 protein level is found associated with poor PHLPP expression in colorectal cancer patient specimens. Taken together, these results indentify a tumor suppressor role of USP46 in promoting PHLPP expression and inhibiting Akt signaling in colon cancer.

Background Pleckstrin homology (PH) domain leucine-rich repeat protein phosphatases (PHLPP) has been associated with several neurodegenerative diseases, however, few studies have investigated the role of PHLPP in Parkinson’s disease (PD). The present study aimed to answer this question through establishing a Parkinson’s disease (PD) model using the Phlpp1-/- and wild-type (WT) mice and testing their behavioral as well as molecular changes. Methods: MPTP was intraperitoneal injected into mice to generate a PD model. Neurobehavioral parameters, protein expression and inflammatory cytokines release were measured by the open filed test, the pole test, immunohistochemistry, immunoblotting, immunoprecipitation, and quantitative reverse transcription PCR. Results MPTP-induced neurobehavioral deficits were more significantly ameliorated in PHLPP-KO-MPTP mice compared to WT-MPTP mice. The survival rate of TH + neurons in the PHLPP-KO-MPTP group was higher than that in the WT-MPTP group (66% vs. 38%). Additionally, PHLPP1 knockout in KO-MPTP mice markedly reduced levels of IL-1β, IL-6, TNF-α, and iNOS, and increased levels of TGF-β compared to those of WT-MPTP mice. Furthermore, PHLPP1 was found to bind to NLRP3 and that PHLPP1 knockout inhibited MPTP-induced expression of IL-1β and caspase-1 in substantia nigra of PD model mice. Conclusion Our results demonstrates that PHLPP1 knockout in PD model is positively associated with the survival of TH + neurons by suppressing inflammatory response in substantia nigra, suggesting that PHLPP1 plays a critical role in the development of PD.

Abstract Background/Aims: Loss of endothelial barrier function plays an important role in the development of ventilator-induced lung injury (VILI). This study aimed to investigate the effects of miR135a on VILI in a model of mechanical stretch (MS)-induced human umbilical vein endothelial cell (HUVEC) injury. Methods: HUVECs were randomly assigned to 7 groups: blank, negative control (NC), NC+MS, miR135a over-expression (mi-miR135a), mi-miR135a + MS, miR135a silencing (si-miR135a) and si-miR135a + MS groups. MS was induced by subjecting cells to cyclic stretch at 20% stretch for 4 h. After 24 h, levels of reactive oxygen species (ROS) were measured by DCFH-DA fluorescence intensity. Apoptosis was measured using annexin V-FITC/propidium iodide assay with flow cytometry. Inflammatory cytokine levels were determined by ELISA. Barrier integrity was determined using FITC-conjugated dextran assay. Expression levels of PI3K, p-PI3K, Akt, p-Akt, Bcl-2 and Bax were examined using western blotting. The interaction between miR135a and PHLPP2 was evaluated by dual-luciferase reporter assay. Results: Our results showed that MS reduced cell numbers, increased the number of apoptotic cells, increased ROS, barrier dysfunction and inflammatory cytokines in HUVECs, and reduced p-PI3K and p-Akt expression; silencing of miR135a worsened MS-induced HUVEC injury. However, miR135a over-expression protected HUVECs against MS-induced increases in apoptotic cells, ROS, barrier dysfunction and inflammatory cytokines, which were accompanied by activation of the PI3K/Akt signaling pathway. Simultaneous silencing of miR135a and PHLPP2 partially salvaged the effects of miR135a silencing, and miR135a was found to interact with PHLPP2. Conclusion: miR135a may protect HUVECs from MS-induced injury by inhibiting PHLPP2 to activate PI3k/Akt signaling pathway.

The PI3K/AKT pathway is frequently activated in endometrial carcinoma. BMI‐1 (B‐lymphoma Mo‐MLV insertion region 1) protein affects expression of PTEN (phosphatase and tensin homolog) in some cancers, but its significance for endometrial tumorigenesis is not known. The objective of this study was to determine the relationship between BMI‐1 and expression of factors affecting AKT (protein kinase B) phosphorylation level in endometrial cancer. The expression of proteins and mRNAs was investigated in endometrial cancer specimens and samples of non‐neoplastic endometrial tissue by Western blot and RT‐PCR, respectively. The impact of BMI‐1 down‐regulation on AKT phosphorylation and expression of genes coding for several phosphatases were studied in HEC1A cells. The results showed that BMI‐1 depletion caused increase in PHLPP1 and PHLPP2 (PH domain and leucine‐rich repeat protein phosphatases 1/2) expression and decrease in phospho‐AKT (pAKT) level. In more advanced tumours with higher metastatic potential, the expression of BMI‐1 was lower compared to tumours less advanced and without lymph node metastasis. There were significant inverse correlations between BMI‐1 and PHLPPs, especially PHLPP1 in normal endometrial samples. The inverse correlation between BMI‐1 and PHLPP1/PHLPP2 expression was observed in PTEN positive but not PTEN negative cancers. Low PHLPP2 expression in tumours predicted poorer overall survival. BMI‐1 impacts on AKT phosphorylation level in endometrial cells by regulation of PHLPP expression.

Neutrophils are known to adopt dynamic and distinct functional phenotypes involved in the modulation of inflammation and immune homeostasis. However, inter-cellular signaling mechanisms that govern neutrophil polarization dynamics are not well understood. Employing a novel model of PHLPP deficient mice, we examined how neutrophils deficient in PHLPP may uniquely modulate immune defense and the host response during acute colitis. We found that PHLPP mice were protected from dextran sodium sulfate (DSS)-induced septic colitis characterized by minimal body weight-loss, alleviated colon tissue destruction and reduced clinical symptoms. PHLPP neutrophils have enhanced immune homeostasis as compared to WT neutrophils, reflected in enhanced migratory capacity toward chemoattractants, and reduced expression of inflammatory mediators due to elevated phosphorylation of AKT, STAT1, and ERK. Further, adoptive transfer of PHLPP deficient neutrophils to WT mice is sufficient to potently alleviate the severity of DSS-induced colitis. Our data reveal that PHLPP deficient neutrophils can be uniquely reprogrammed to a state conducive to host inflammation resolution. As a consequence, PHLPP neutrophils can effectively transfer immune homeostasis in mice subjected to acute colitis. Our findings hold significant and novel insights into the mechanisms by which neutrophils can be effectively reprogrammed into a homeostatic state conducive for treating acute injuries such as septic colitis.

De-ubiquitinating enzymes (DUBs) can reverse the modifications catalyzed by ubiquitin ligases and as such are believed to be important regulators of a variety of cellular processes. Several members of this protein family have been associated with human cancers; however, there is little evidence for a direct link between deregulated de-ubiquitination and neoplastic transformation. Ubiquitin C-terminal hydrolase (UCH)-L1 is a DUB of unknown function that is overexpressed in several human cancers, but whether it has oncogenic properties has not been established. To address this issue, we generated mice that overexpress UCH-L1 under the control of a ubiquitous promoter. Here, we show that UCH-L1 transgenic mice are prone to malignancy, primarily lymphomas and lung tumors. Furthermore, UCH-L1 overexpression strongly accelerated lymphomagenesis in Eμ- myc transgenic mice. Aberrantly expressed UCH-L1 boosts signaling through the Akt pathway by downregulating the antagonistic phosphatase PHLPP1, an event that requires its de-ubiquitinase activity. These data provide the first in vivo evidence for DUB-driven oncogenesis and suggest that UCH-L1 hyperactivity deregulates normal Akt signaling.

The PP2A phosphatase is often inactivated in cancer and is considered as a tumour suppressor. A new pathway controlling PP2A activity in mitosis has been recently described. This pathway includes the Greatwall (GWL) kinase and its substrates endosulfines. At mitotic entry, GWL is activated and phosphorylates endosulfines that then bind and inhibit PP2A. We analysed whether GWL overexpression could participate in cancer development. We show that GWL overexpression promotes cell transformation and increases invasive capacities of cells through hyperphosphorylation of the oncogenic kinase AKT. Interestingly, AKT hyperphosphorylation induced by GWL is independent of endosulfines. Rather, GWL induces GSK3 kinase dephosphorylation in its inhibitory sites and subsequent SCF-dependent degradation of the PHLPP phosphatase responsible for AKT dephosphorylation. In line with its oncogenic activity, we find that GWL is often overexpressed in human colorectal tumoral tissues. Thus, GWL is a human oncoprotein that promotes the hyperactivation of AKT via the degradation of its phosphatase, PHLPP, in human malignancies. In order to form a tumour, cancer cells have to overcome the controls that normally prevent cells from dividing too often. These controls include an enzyme called PP2A, which inhibits cell division by regulating the activity of other proteins. In a normal cell, PP2A may be temporarily deactivated from time to time to enable the cell to divide. However, PP2A is permanently deactivated in many cancer cells, which allows these cells to divide many times in quick succession. A protein called Greatwall is involved in deactivating PP2A in healthy cells, but it was not clear whether increases in Greatwall activity can promote the formation of tumours. Here, Vera et al. use a variety of cell biology techniques to address this question. The experiments show that increasing the amount of Greatwall in human and mouse cells can promote some of the transformations needed for these cells to become cancerous. Also, Greatwall increases the growth of tumours in mice. These effects are caused by the over-activation of a protein called AKT, which is already known to promote the formation of many cancers. Vera et al. show that Greatwall regulates AKT activity using a different pathway to how it influences PP2A activity. Further experiments revealed that many human tumours, especially those from patients with colon cancer, produce excessive amounts of Greatwall protein. Together, these findings show that Greatwall can promote the development of cancer. A future challenge is to understand how this works and to find out whether high levels of Greatwall are a common feature of other human cancers.

PHLPP (PH domain leucine-rich repeats protein phosphatase) represents a family of novel Ser/Thr protein phosphatases. Two highly related isoforms in this family, PHLPP1 and PHLPP2, have been identified to serve as negative regulators of Akt and protein kinase C by dephosphorylating the kinases directly. In this study, we examined the expression pattern of both PHLPP isoforms in colorectal cancer specimens and the adjacent normal mucosa using immunohistochemical staining. We found that the expression of PHLPP1 or PHLPP2 isoform was lost or decreased in 78 and 86% of tumor tissues, respectively. Stable overexpression of either PHLPP isoform in colon cancer cells decreased the rate of cell proliferation and sensitized the cells to growth inhibition induced by the phosphoinositide-3 kinase inhibitor, LY294002, whereas knockdown of either PHLPP isoform by shRNA promoted the proliferation of DLD1 cells. In addition, we demonstrated that the PHLPP-mediated growth inhibition in colon cancer cells was largely rescued by overexpression of a constitutively active Akt. Moreover, reexpression of either PHLPP isoform in HCT116 cells inhibited tumor growth in vivo . Taken together, our results strongly support a tumor suppressor role of PHLPP in colon cancer.