PH Domain Leucine-rich Repeat Protein Research Articles

MEOX2 Participates in Hepatic Stellate Cells-Induced Liver Fibrosis by Regulating the PI3K/AKT Signaling Pathway.

Hepatic stellate cells (HSCs) serve as the crucial accelerating factor in the progression of liver fibrosis (LF). In contrast to HSCs, adult-derived human liver stem/progenitor cells (ADHLSCs) exhibit greater potency in terms of differentiation and proliferation, rendering them highly applicable in LF treatment. The objective of this study is to identify new therapeutic targets for LF by comparing differentially expressed genes (DEGs) between ADHLSCs and HSCs. We investigated DEGs between ADHLSCs and HSCs using the GSE49995 dataset obtained from the Gene Expression Omnibus (GEO) database, aiming to identify new therapeutic targets for LF. Subsequently, we activated HSCs to delve deeper into the mesenchyme homeobox 2 (MEOX2), PH domain Leucine-rich repeat protein phosphatase (PHLPP), and Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in LF progression, employing platelet-derived growth factor (PDGF), and conducted infection with Overexpression (OE)-MEOX2 and shRNA-MEOX2 (sh-MEOX2) lentiviruses. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay, while cell proliferation was evaluated through 5-ethynyl-2'-deoxyuridine (EdU) staining and flow cytometry. Relative mRNA expression levels were determined via qPCR. Western blot analysis was performed to measure protein expression levels, and the regulatory role of MEOX2 was investigated using dual luciferase reporter assays. We identified 332 DEGs that were down-regulated and 201 DEGs that were up-regulated between ADHLSCs and HSCs. Notably, MEOX2 expression in ADHLSCs was significantly reduced. These DEGs primarily participated in the collagen-containing extracellular matrix and the PI3K/AKT signaling pathway. MEOX2 could inhibit cancer cell proliferation via the PI3K/AKT signaling pathway. Additionally, the JASRPAR2022 database predicted the target gene PHLPP of MEOX2. Our results indicated that OE-MEOX2 significantly inhibited HSCs' cell vitality and proliferation. Further analysis revealed that MEOX2 binds to PHLPP promoters, thereby up-regulating its transcription. This action led to the inhibition of p-AKT expression, consequently reducing HSC proliferation and slowing the progression of LF. MEOX2 up-regulates PHLPP expression and inhibits AKT phosphorylation, thereby reducing the cell activity and proliferation ability of HSCs and inhibiting the progression of LF.

Hyperoxia-Induced miR-195 Causes Bronchopulmonary Dysplasia in Neonatal Mice.

Background: Exposure to hyperoxia is an important factor in the development of bronchopulmonary dysplasia (BPD) in preterm newborns. MicroRNAs (miRs) have been implicated in the pathogenesis of BPD and provide a potential therapeutic target. Methods: This study was conducted utilizing a postnatal animal model of experimental hyperoxia-induced murine BPD to investigate the expression and function of miR-195 as well as its molecular signaling targets within developing mouse lung tissue. Results: miR-195 expression levels increased in response to hyperoxia in male and female lungs, with the most significant elevation occurring in 40% O2 (mild) and 60% O2 (moderate) BPD. The inhibition of miR-195 improved pulmonary morphology in the hyperoxia-induced BPD model in male and female mice with females showing more resistance to injury and better recovery of alveolar chord length, septal thickness, and radial alveolar count. Additionally, we reveal miR-195-dependent signaling pathways involved in BPD and identify PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) as a novel specific target protein of miR-195. Conclusions: Our data demonstrate that high levels of miR-195 in neonatal lungs cause the exacerbation of hyperoxia-induced experimental BPD while its inhibition results in amelioration. This finding suggests a therapeutic potential of miR-195 inhibition in preventing BPD.

PHLPP1 inhibits the growth and aerobic glycolysis activity of human ovarian granular cells through inactivating AKT pathway

BackgroundPolycystic ovary syndrome (PCOS) is a disorder characterized by hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphologic features, and PCOS is associated with infertility. PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) has been shown to regulate AKT. The aim of present study is to investigate the role of PHLPP1 in PCOS.MethodsThe expression levels of PHLPP1 in dihydrotestosterone (DHT)-treated human ovarian granular KGN cells were determined by qRT-PCR and Western blot. PHLPP1 was silenced or overexpressed using lentivirus. Cell proliferation was detected by CCK-8. Apoptosis and ROS generation were analyzed by flow cytometry. Glycolysis was analyzed by measuring extracellular acidification rate (ECAR).ResultsDHT treatment suppressed proliferation, promoted apoptosis, enhanced ROS, and inhibited glycolysis in KGN cells. PHLPP1 silencing alleviated the DHT-induced suppression of proliferation and glycolysis, and promotion of apoptosis and ROS in KGN cells. PHLPP1 regulated cell proliferation and glycolysis in human KGN cells via the AKT signaling pathway.ConclusionsOur results showed that PHLPP1 mediates the proliferation and aerobic glycolysis activity of human ovarian granular cells through regulating AKT signaling.

EXPLORING THE POTENTIAL OF PHLPP INHIBITORS FOR OSTEOARTHRITIS TREATMENT

Abstract Osteoarthritis (OA) is a degenerative joint disease characterized by pain, stiffness, and limited joint motion affecting 7% of the world’s population. PH domain leucine-rich repeat protein phosphates (PHLPPs) have been identified as modifiable targets for cartilage regeneration and joint pain. PHLPP1 and PHLPP2 are abnormally expressed in osteoarthritic cartilage and sensory neurons. PHLPP1-/- mice are protected from cartilage degradation and pain-related behaviors (allodynia and reduced mobility) after surgery that destabilizes the medial meniscus (DMM). Here, we investigate the effects of PHLPP inhibitors (i) on human chondrocytes and the effect of Phlpp1 deletion on cell populations within lumbar dorsal root ganglia (DRGs). Immunohistochemistry was performed on mouse DRGs by cryosectioning fixed L3-5 DRGs and using primary antibodies against Fabp7, Anti tubulin beta 3, and PGP9.5/UCHL1. Human chondrocytes were subjected to Cycloheximide chase experiments and treated with PHLPPi (NSC117079 or NSC45586), followed by Western blot analysis to assess protein levels including Phlpp1, Phlpp2, Sox9, p-AKT (Ser473), total AKT, and B-actin, at various time points. We found that PHLPPi prolongs the phosphorylated AKT response when compared to controls and does not affect degradation of Phlpp1 or Phlpp2; additionally, NSC117079 increased production of cartilage extracellular matrix components (glycosaminoglycans and aggrecan) in over 90% of human articular cartilage explants from OA patients. These data advance our understanding of how PHLPPi function within chondrocytes and provide a basis toward understanding their activity and safety in vivo. Our results indicate that PHLPPi NSC117079 is a novel osteoarthritis disease-modifying drug candidate that may have palliative effects.

NAP1L5 facilitates pancreatic ductal adenocarcinoma progression via TRIM29-mediated ubiquitination of PHLPP1

Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most aggressive solid tumours in humans. Despite its high mortality rate, effective targeted therapeutic strategies remain limited due to incomplete understanding of the underlying biological mechanisms. The NAP1L gene family has been implicated in the development and progression of various human tumours. However, the specific function and role of NAP1L5 (nucleosome assembly protein-like 5) in PDAC have not been fully elucidated. Therefore, in this study, we aimed to investigate the role of NAP1L5 in PDAC and explore the regulatory relationship between NAP1L5 and its potential downstream molecule PHLPP1 (PH domain Leucine-rich repeat Protein Phosphatase 1) in PDAC. Our study revealed that NAP1L5 is notably upregulated in PDAC. Moreover, both in vivo and in vitro experiments demonstrated that knockdown of NAP1L5 suppressed the proliferation of PDAC cells. Mechanistically, NAP1L5 was found to promote PDAC progression by activating the AKT/mTOR signalling pathway in a PHLPP1-dependent manner. Specifically, NAP1L5 binds to PHLPP1 and facilitates the ubiquitination-mediated degradation of PHLPP1, ultimately resulting in reduced PHLPP1 expression. Notably, TRIM29, recruited by NAP1L5, was found to be involved in facilitating K48-linked ubiquitination of PHLPP1. Our findings indicate that NAP1L5 overexpression promotes the proliferation of PDAC cells by inhibiting PHLPP1 expression. These novel insights suggest that NAP1L5 may serve as a potential therapeutic target for PDAC.

SKP2 Contributes to AKT Activation by Ubiquitination Degradation of PHLPP1, Impedes Autophagy, and Facilitates the Survival of Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid carcinoma. Despite a good prognosis, approximately a quarter of PTC patients are likely to relapse. Previous reports suggest an association between S-phase kinase-associated protein 2 (SKP2) and the prognosis of thyroid cancer. SKP1 is related to apoptosis of PTC cells; however, its role in PTC remains largely elusive. This study aimed to understand the expression and molecular mechanism of SKP2 in PTC. SKP2 expression was upregulated in PTC tissues and closely associated with clinical diagnosis. In vitro and in vivo knockdown of SKP2 expression in PTC cells suppressed cell growth and proliferation and induced apoptosis. SKP2 depletion promoted cell autophagy under glucose deprivation. SKP2 interacted with PH domain leucine-rich repeat protein phosphatase-1 (PHLPP1), triggering its degradation by ubiquitination. Furthermore, SKP2 activates the AKT-related pathways via PHLPP1, which leads to the cytoplasmic translocation of SKP2, indicating a reciprocal regulation between SKP2 and AKT. In conclusion, the upregulation of SKP2 leads to PTC proliferation and survival, and the regulatory network among SKP2, PHLPP1, and AKT provides novel insight into the molecular basis of SKP2 in tumor progression.

Abstract P1077: Nicotine Compromises Metabolic Adaptation Of The Heart To Injury By Altering PHLPP Isoforms

Rationale: Cardiovascular (CV) disease remains the leading cause of death and disability in the US and worldwide and tobacco use is a major risk factor for increasing the incidence of disease. Consequently, there is a need for understanding the cardiovascular impact of systemic nicotine on cardiac physiology and heart failure progression following injury. We have demonstrated that removal of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) increases Akt activity and protects the heart from oxidative damage, however downregulation of PHLPP2 causes damage. Preliminary data suggest that exposure to nicotine increases PHLPP1 and decreases PHLPP2 levels in the heart. Therefore, linking the direct pathogenic effects of nicotine on these cell-survival intracellular pathways such as those that utilize PHLPP1 activity needs to be rigorously examined for therapeutic efficacy and intervention. Hypothesis: We hypothesize that exposure to high levels of nicotine will compromise the heart and increase susceptibility to ischemic injury by altering PHLPP isoform expression. Methods: Using in vivo systems we investigated the effect of nicotine exposure on PHLPP expression and cardiomyocyte function and injury. Male and female wild-type WT (n=4) and PHLPP1 KO (n=4) mice were implanted with osmotic minipumps containing nicotine (5mg/kg/day) or saline for 7days and changes in protein and gene expression were examined. A second cohort of WT animals was exposed to nicotine (7 days) and subjected to 1 hour ischemia and 24 hours reperfusion (n=6). Results: Following 7 days of nicotine exposure, we found PHLPP1 protein expression was significantly increased (1.7 ± 0.25 fold) and PHLPP2 significantly decreased (0.8 ± 0.04 fold) in the hearts of WT mice. However, in PHLPP1 KO mice, nicotine exposure significant increased PHLPP2 levels in the heart (1.5 ± 0.044fold). Also, we found that exposure to nicotine suppressed genes important for mitochondrial biogenesis and decreased antioxidant protein expression in the hearts of WT mice while antioxidant signaling was significantly increased in KO hearts. Following 7 days of nicotine exposure WT mice have increased infarct injury (39% ± 3.2, n=6) following 24-hour reperfusion compared to saline treated WT (31% ± 0.55, n=4) with the same area at risk. Preliminary studies found that PHLPP1 KO significantly protected the heart from ischemic injury (26% ± 0.38, n=3), however the effect in the presence of nicotine is under investigation. Conclusions: Our data suggests that nicotine exposure has detrimental effects on the heart by altering PHLPP isoform expression which compromises oxidative signaling and increases injury. Further studies will investigate the mechanism of nicotine induced PHLPP isoform expression both in vitro and in vivo and determine whether targeting PHLPP1 could significantly impact cardiac outcome associated with nicotine use.

Hyperglycemia-Induced Overexpression of PH Domain Leucine-Rich Repeat Protein Phosphatase 1 (PHLPP1) Compromises the Cardioprotective Effect of Ischemic Postconditioning Via Modulation of the Akt/Mst1 Pathway Signaling.

Ischemic postconditioning (IPostC) alleviates myocardial ischemia/reperfusion (IR) injury, but the protective effect is lost during diabetes. PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) is able to inactivate Akt. Our previous study found that PHLPP1 expression was upregulated in diabetic hearts. We presumed that the attenuation of myocardial injury by IPostC might be hindered by PHLPP1 overexpression in diabetic animals. Nondiabetic and diabetic mice were subjected to 45 min of ischemia followed by 2 h of reperfusion with or without IPostC. H9c2 cells were exposed to normal or high glucose and were subjected to 4 h of hypoxia followed by 4 h of reoxygenation with or without hypoxic postconditioning (HPostC). IPostC attenuated postischemic infarction, apoptosis, creatine kinase-MB, and oxidative stress, which were accompanied by increased p-Akt and decreased PHLPP1 expression and p-Mst1 in nondiabetic but not in diabetic mice. PHLPP1 knockdown or an Mst1 inhibitor reduced hypoxia/reoxygenation (HR)-induced cardiomyocyte damage in H9c2 cells exposed to normal glucose, but the effect was abolished by a PI3K/Akt inhibitor. HPostC attenuated HR-induced cardiomyocyte injury and oxidative stress accompanied by increased p-Akt as well as decreased PHLPP1 expression and p-Mst1 in H9c2 cells exposed to normal glucose but not high glucose. In addition, HPostC in combination with PHLPP1 knockdown or PHLPP1 knockdown alone reduced cell death and oxidative stress in H9c2 cells exposed to high glucose, which was hindered by PI3K/Akt inhibitor. IPostC prevented myocardial IR injury partly through PHLPP1/Akt/Mst1 signaling, and abnormalities in this pathway may be responsible for the loss of IPostC cardioprotection in diabetes.

Peptides derived from sea cucumber accelerate cells proliferation and migration for wound healing by promoting energy metabolism and upregulating the ERK/AKT pathway

Numerous bioactive peptides have high potential in accelerating skin repair. This study aimed to explore wound healing capacity of sea cucumber-derived tetrapeptides with amino acid sequence of Val-Thr-Pro-Tyr (VTPY) and Val-Leu-Leu-Tyr (VLLY) by human skin fibroblast (HSF) and human umbilical vein endothelial cells (HUVEC) in vitro. The results showed that VTPY and VLLY possessed excellent capacity to induce the proliferation and migration of HSF cells and HUVEC cells. Among them, VTPY possessed a higher cell migration-promotion capacity than VLLY. Meanwhile, VTPY and VLLY increased mitochondrial respiratory capacity of cells, leading to the significant increase in adenosine triphosphate (ATP) production. Additionally, VTPY and VLLY significantly increased the percentage of S phase in cell cycle of HUVEC via upregulation of extracellular regulated protein kinases (ERK) and protein kinase B (AKT) signaling pathway. Molecular docking further indicated the interaction of peptides with mitogen-activated protein kinase phosphatase (MKP) and PH domain leucine-rich repeat protein phosphatase (PHLPP) binding site in the ERK2 and AKT, respectively, leading to subsequent increased phosphorylation of ERK1/2 and AKT. Overall, peptides of VTPY and VLLY possessed outstanding capacity to induce the proliferation and migration of HSF cells and HUVEC cells in vitro and the mechanism was mainly related to improving mitochondrial respiratory capacity to produce more ATP for biological energy, blocking the binding of MKP to ERK2 and PHLPP to AKT and thus upregulating the ERK/AKT pathway.

PHLPP1 deficiency ameliorates cardiomyocyte death and cardiac dysfunction through inhibiting Mcl-1 degradation

Myocardial infarction (MI), ischemia-reperfusion injury or chemotherapy can trigger excessive loss of terminally differentiated cardiomyocytes, leading to the development of heart failure. Whereas apoptosis has been considered to be the major form of cell death in various myocardial damage, the means by which to reduce cardiomyocyte loss are limited, and the mechanism that underlies cardiomyocyte apoptosis need to be further investigated. PH domain leucine-rich repeat protein phosphatase1 (PHLPP1) belongs to a novel family of Ser/Thr protein phosphatases that functions as a tumor suppressor. Here, we identified PHLPP1 as an important pro-apoptosis factor of cardiomyocytes in response to pathogenic stresses. The conditional PHLPP1 deficiency in cardiomyocytes alleviated myocardial ischemic injury, improved cardiac function and inhibited myocardial fibrosis, in turn preventing adverse cardiac remodeling and heart failure after MI. The conditional PHLPP1 deficiency in cardiomyocytes also attenuated doxorubicin (Dox)-induced myocardial injury, suppressed the inflammation and fibrosis in cardiac tissues, and protected from cardiac dysfunction. Mechanically, PHLPP1 bound the anti-apoptosis protein myeloid cell leukemia sequence 1 (Mcl-1) in cardiomyocytes. Thr163 phosphorylation of Mcl-1 was reported to slow Mcl-1 protein turnover. We further found that PHLPP1 deficiency enhanced Thr163 phosphorylation of Mcl-1, inhibited Mcl-1 degradation and maintained Mcl-1 protein expression level in myocardium and cardiomyocytes upon MI or Dox treatment. PHLPP1 could directly dephosphorylate Thr163 of Mcl-1. Thus, PHLPP1 promotes cardiomyocyte death and cardiac dysfunction through binding and enhancing Mcl-1 degradation under ischemic or toxic injury conditions, which sheds new light on the development of potential therapies to control cardiomyocyte loss.

LncRNA LOC729178 acts as a sponge of miR-144-3p to mitigate cigarette smoke extract-induced inflammatory injury via regulating PHLPP2 in 16HBE cells.

Chronic obstructive pulmonary disease (COPD) is an inflammatory respiratory disease. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of COPD. In the present study, we set to investigate the role and mechanism of LOC729178 on cigarette smoke extract (CSE)-induced inflammatory damage in 16HBE cells. The expression levels of LOC729178, miR-144-3p, and PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell viability and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) and flow cytometry, respectively. Enzyme-linked immunosorbent assay (ELISA) assay was performed to evaluate the levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α), and IL-8. Targeted relationships among LOC729178, miR-144-3p, and PHLPP2 were verified by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Our data indicated that LOC729178 was underexpressed in COPD tissues and CSE-treated 16HBE cells. Exogenous expression of LOC729178 alleviated CSE-induced inflammatory injury in 16HBE cells. LOC729178 targeted miR-144-3p by directly binding to miR-144-3p. miR-144-3p was a downstream effector of LOC729178 function. PHLPP2 was identified as a direct and functional target of miR-144-3p. Furthermore, LOC729178 operated as a post-transcriptional regulator of PHLPP2 expression through miR-144-3p. Our current study suggested that LOC729178 overexpression alleviated CSE-induced inflammatory injury in 16HBE cells at least in part by up-regulating PHLPP2 via sponging miR-144-3p, providing a rationale for developing LOC729178 as a potential therapeutic agent against COPD.

The PHLPP1 N-Terminal Extension Is a Mitotic Cdk1 Substrate and Controls an Interactome Switch.

PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) is a tumor suppressor that directly dephosphorylates a wide array of substrates, most notably the prosurvival kinase Akt. However, little is known about the molecular mechanisms governing PHLPP1 itself. Here, we report that PHLPP1 is dynamically regulated in a cell cycle-dependent manner and deletion of PHLPP1 results in mitotic delays and increased rates of chromosomal segregation errors. We show that PHLPP1 is hyperphosphorylated during mitosis by Cdk1 in a functionally uncharacterized region known as the PHLPP1 N-terminal extension (NTE). A proximity-dependent biotin identification (BioID) interaction screen revealed that during mitosis, PHLPP1 dissociates from plasma membrane scaffolds, such as Scribble, by a mechanism that depends on its NTE and gains proximity to kinetochore and mitotic spindle proteins such as KNL1 and TPX2. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows accurate progression through mitosis. The finding that PHLPP1 binds mitotic proteins in a cell cycle- and phosphorylation-dependent manner may have relevance to its tumor-suppressive function.

PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2.

IntroductionTreatment of bortezomib (BTZ) improves the clinical outcomes of patients with multiple myeloma (MM). However, primary resistance and acquired resistance to BTZ frequently develop in patients with MM. PH domain leucine-rich repeat protein phosphatase (PHLPP) plays an important role in chemoresistance in a number of cancers. However, the role of PHLPP on MM remains unclear. In this study, we investigated the role of PHLPP in BTZ-resistant MM cells.MethodsBrdU assays, immunoprecipitation, flow cytometry analyses, and immunofluorescence assays were performed.ResultsPHLPP and lysosome-associated membrane protein 2 (LAMP2) levels were downregulated in BTZ-resistant MM cells compared with BTZ-sensitive MM cells, accompanied by inactivation of autophagy pathway evaluated by a reduction in Beclin1, Atg5 and LC3B and increase in p62. Gain- and loss-of-function experiments revealed that PHLPP partially re-sensitized MM cells to BTZ. In addition, PHLPP overexpression increased whereas PHLPP knockdown reduced LAMP2 expression, subsequently regulating the autophagy pathway in MM cells. Further findings demonstrated that LAMP2 knockdown reversed PHLPP-mediated cell apoptosis and autophagy activation in MM cells.ConclusionThis study demonstrated that PHLPP is a potential strategy for overcoming BTZ resistance in patients with MM.

Phlpp1 is associated with human intervertebral disc degeneration and its deficiency promotes healing after needle puncture injury in mice

Back pain is a leading cause of global disability and is strongly associated with intervertebral disc (IVD) degeneration (IDD). Hallmarks of IDD include progressive cell loss and matrix degradation. The Akt signaling pathway regulates cellularity and matrix production in IVDs and its inactivation is known to contribute to a catabolic shift and increased cell loss via apoptosis. The PH domain leucine-rich repeat protein phosphatase (Phlpp1) directly regulates Akt signaling and therefore may play a role in regulating IDD, yet this has not been investigated. The aim of this study was to investigate if Phlpp1 has a role in Akt dysregulation during IDD. In human IVDs, Phlpp1 expression was positively correlated with IDD and the apoptosis marker cleaved Caspase-3, suggesting a key role of Phlpp1 in the progression of IDD. In mice, 3 days after IVD needle puncture injury, Phlpp1 knockout (KO) promoted Akt phosphorylation and cell proliferation, with less apoptosis. At 2 and 8 months after injury, Phlpp1 deficiency also had protective effects on IVD cellularity, matrix production, and collagen structure as measured with histological and immunohistochemical analyses. Specifically, Phlpp1-deletion resulted in enhanced nucleus pulposus matrix production and more chondrocytic cells at 2 months, and increased IVD height, nucleus pulposus cellularity, and extracellular matrix deposition 8 months after injury. In conclusion, Phlpp1 has a role in limiting cell survival and matrix degradation in IDD and research targeting its suppression could identify a potential therapeutic target for IDD.

PHLPP1 counter-regulates STAT1-mediated inflammatory signaling.

Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and live Escherichia coli infection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages.

Excessive miR-25-3p maturation via N6-methyladenosine stimulated by cigarette smoke promotes pancreatic cancer progression

N6-methyladenosine (m6A) modification is an important mechanism in miRNA processing and maturation, but the role of its aberrant regulation in human diseases remained unclear. Here, we demonstrate that oncogenic primary microRNA-25 (miR-25) in pancreatic duct epithelial cells can be excessively maturated by cigarette smoke condensate (CSC) via enhanced m6A modification that is mediated by NF-κB associated protein (NKAP). This modification is catalyzed by overexpressed methyltransferase-like 3 (METTL3) due to hypomethylation of the METTL3 promoter also caused by CSC. Mature miR-25, miR-25-3p, suppresses PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2), resulting in the activation of oncogenic AKT-p70S6K signaling, which provokes malignant phenotypes of pancreatic cancer cells. High levels of miR-25-3p are detected in smokers and in pancreatic cancers tissues that are correlated with poor prognosis of pancreatic cancer patients. These results collectively indicate that cigarette smoke-induced miR-25-3p excessive maturation via m6A modification promotes the development and progression of pancreatic cancer.

Pink1 regulates FKBP5 interaction with AKT/PHLPP and protects neurons from neurotoxin stress induced by MPP.

Loss of function mutations in the PTEN-induced putative kinase 1 (Pink1) gene have been linked with an autosomal recessive familial form of early onset Parkinson's disease (PD). However, the underlying mechanism(s) responsible for degeneration remains elusive. Presently, using co-immunoprecipitation in HEK (Human embryonic kidney) 293 cells, we show that Pink1 endogenously interacts with FK506-binding protein 51 (FKBP51 or FKBP5), FKBP5 and directly phosphorylates FKBP5 at Serine in an invitro kinase assay. Both FKBP5 and Pink1 have been previously associated with protein kinase B (AKT) regulation. We provide evidence using primary cortical cultured neurons from Pink1-deficient mice that Pink1 increases AKT phosphorylation at Serine 473 (Ser473) challenged by 1-methyl-4-phenylpyridinium (MPP+ ) and that over-expression of FKBP5 using an adeno-associated virus delivery system negatively regulates AKT phosphorylation at Ser473 in murine-cultured cortical neurons. Interestingly, FKBP5 over-expression promotes death in response to MPP+ in the absence of Pink1. Conversely, shRNA-mediated knockdown of FKBP5 in cultured cortical neurons is protective and this effect is reversed with inhibition of AKT signaling. In addition, shRNA down-regulation of PH domain leucine-rich repeat protein phosphatase (PHLPP) in Pink1 WT neurons increases neuronal survival, while down-regulation of PHLPP in Pink1 KO rescues neuronal death in response to MPP+ . Finally, using co-immunoprecipitation, we show that FKBP5 interacts with the kinase AKT and phosphatase PHLPP. This interaction is increased in the absence of Pink1, both in Mouse Embryonic Fibroblasts (MEF) and in mouse brain tissue. Expression of kinase dead Pink1 (K219M) enhances FKBP5 interaction with both AKT and PHLPP. Overall, our results suggest a testable model by which Pink1 could regulate AKT through phosphorylation of FKBP5 and interaction of AKT with PHLPP. Our results suggest a potential mechanism by which PINK1-FKBP5 pathway contributes to neuronal death in PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulates G-protein–coupled receptor kinase 5 (GRK5)-induced cardiac hypertrophy in vitro

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.

MiR-214-Dependent Increase of PHLPP2 Levels Mediates the Impairment of Insulin-Stimulated Akt Activation in Mouse Aortic Endothelial Cells Exposed to Methylglyoxal.

Evidence has been provided linking microRNAs (miRNAs) and diabetic complications, by the regulation of molecular pathways, including insulin-signaling, involved in the pathophysiology of vascular dysfunction. Methylglyoxal (MGO) accumulates in diabetes and is associated with cardiovascular complications. This study aims to analyze the contribution of miRNAs in the MGO-induced damaging effect on insulin responsiveness in mouse aortic endothelial cells (MAECs). miRNA modulation was performed by transfection of specific miRNA mimics and inhibitors in MAECs, treated or not with MGO. miRNA-target protein levels were evaluated by Western blot. PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulation by miR-214 was tested by luciferase assays and by the use of a target protector specific for miR-214 on PHLPP2-3′UTR. This study reveals a 4-fold increase of PHLPP2 in MGO-treated MAECs. PHLPP2 levels inversely correlate with miR-214 modulation. Moreover, miR-214 overexpression is able to reduce PHLPP2 levels in MGO-treated MAECs. Interestingly, a direct regulation of PHLPP2 is proved to be dependent by miR-214. Finally, the inhibition of miR-214 impairs the insulin-dependent Akt activation, while its overexpression rescues the insulin effect on Akt activation in MGO-treated MAECs. In conclusion, this study shows that PHLPP2 is a target of miR-214 in MAECs, and identifies miR-214 downregulation as a contributing factor to MGO-induced endothelial insulin-resistance.

GYY4137 protects against myocardial ischemia/reperfusion injury via activation of the PHLPP-1/Akt/Nrf2 signaling pathway in diabetic mice

BackgroundThis study explores the protective effects of a hydrogen sulfide donor, morpholin-4-ium 4-methoxyphenyl-morpholino-phosphinodithioate (GYY4137), in the hearts of diabetic mice that had been subjected to myocardial ischemia/reperfusion injury. Diabetes impairs the Akt pathway, in which the Akt protein is dephosphorylated and inactivated by PH domain leucine-rich repeat protein phosphatase-1 (PHLPP-1). However, the function of PHLPP-1 and molecular mechanism that underlies the cardiac protection exerted by GYY4137 remains unknown. MethodsDiabetic or nondiabetic mice were subjected to 45 min of coronary artery occlusion followed by 2 h of reperfusion. H9c2 cells were cultured with normal or high glucose and then subjected to 3 h of hypoxia followed by 6 h of reoxygenation. Pretreatment with GYY4137 was performed in a randomized manner before ischemia/reperfusion or hypoxia/reoxygenation. The infarct size, cardiomyocyte apoptosis, and oxidative stress were measured. Western blotting was conducted to elucidate the protective mechanism. ResultsDiabetic mice or H9c2 cells exposed to high glucose displayed a larger infarct size, more severe cardiomyocyte apoptosis, lower cell viability, and increased oxidative stress, which were associated with increased levels of PHLPP-1 and reduced levels of p-Akt and nuclear factor-erythroid-2-related factor 2 (Nrf2) protein expression. These changes were prevented/reversed by GYYG4137 pretreatment. At the cellular level, PHLPP-1 siRNA attenuated cellular injury, and this was associated with increased p-Akt and nuclear Nrf2 protein, whereas the decrement of Akt phosphorylation induced by LY294002 augmented cellular injury and decreased nuclear Nrf2. ConclusionsGYY4137 activates the PHLPP-1/Akt/Nrf2 pathway to protect against diabetic myocardial ischemia/reperfusion injury.