Phospholipase C-related Catalytically Inactive Protein Research Articles

Phospholipase C-related catalytically inactive protein enhances cisplatin-induced apoptotic cell death

Cisplatin is one of the most widely used chemotherapeutic agents and induces caspase-9-mediated apoptosis. Here, we examined whether phospholipase C-related catalytically inactive protein (PRIP) enhances cisplatin-induced apoptosis of breast cancer cells. PRIP depletion increased expression of X-linked inhibitor of apoptosis protein (XIAP) by inhibiting protein degradation, which is downstream of the phosphatidylinositol 3-kinase/AKT pathway and inhibits apoptotic signaling by blocking caspase-9 activation. Conversely, the viability of MCF-7 cells transfected with Prip1 was significantly lower than that of control cells in the presence of cisplatin. The number of apoptotic nuclei and expression levels of cleaved caspase-9 and downstream cleaved caspase-7 and poly-ADP ribose polymerase were greater in PRIP1-expressing MCF-7 cells treated with cisplatin than in control cells. XIAP was decreased by expression of pleckstrin homology domain of PRIP1 (PRIP1-PH domain) that blocked phosphatidylinositol 4,5 bisphosphate metabolism. In an orthotopic transplantation model, combined administration of PRIP1-PH domain-containing liposomes and cisplatin reduced the size of MCF-7 tumors compared with cisplatin alone. Our findings demonstrate that PRIP promotes XIAP degradation by inhibiting PI(3,4,5)P3/AKT signaling and enhances cisplatin-induced apoptotic cell death. Therefore, we propose that PRIP1-PH liposomes are a novel agent to avoid cisplatin resistance.

Expression of PRIP, a phosphatidylinositol 4,5-bisphosphate binding protein, attenuates PI3K/AKT signaling and suppresses tumor growth in a xenograft mouse model

Cancer is characterized by uncontrolled proliferation resulting from aberrant cell cycle progression. The activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling, a regulatory pathway for the cell cycle, stabilizes cyclin D1 in the G1 phase by inhibiting the activity of glycogen synthase kinase 3β (GSK3β) via phosphorylation. We previously reported that phospholipase C-related catalytically inactive protein (PRIP), a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding protein, regulates PI3K/AKT signaling by competitively inhibiting substrate recognition by PI3K. Therefore, in this study, we investigated whether PRIP is involved in cell cycle progression.PRIP silencing in MCF-7 cells, a human breast cancer cell line, demonstrated PI(3,4,5)P3 signals accumulated at the cell periphery compared to that of the control. This suggests that PRIP reduction enhances PI(3,4,5)P3-mediated signaling. Consistently, PRIP silencing in MCF-7 cells exhibited increased phosphorylation of AKT and GSK3β which resulted in cyclin D1 accumulation. In contrast, the exogenous expression of PRIP in MCF-7 cells evidenced stronger downregulation of AKT and GSK3β phosphorylation, reduced accumulation of cyclin D1, and diminished cell proliferation in comparison to control cells. Flow cytometry analysis indicated that MCF-7 cells stably expressing PRIP attenuate cell cycle progression. Importantly, tumor growth of MCF-7 cells stably expressing PRIP was considerably prevented in an in vivo xenograft mouse model. In conclusion, PRIP expression downregulates PI3K/AKT/GSK3β-mediated cell cycle progression and suppresses tumor growth. Therefore, we propose that PRIP is a new therapeutic target for anticancer therapy.

Phospholipase C-related catalytically inactive protein regulates lipopolysaccharide-induced hypothalamic inflammation-mediated anorexia in mice

Peripheral lipopolysaccharide (LPS) injection induces systemic inflammation through the activation of the inhibitor of nuclear factor kappa B (NF-κB) kinase (IKK)/NF-κB signaling pathway, which promotes brain dysfunction resulting in conditions including anorexia. LPS-mediated reduction of food intake is associated with activation of NF-κB signaling and phosphorylation of the transcription factor signal transducer and activator of transcription 3 (STAT3) in the hypothalamus. We recently reported phospholipase C-related catalytically inactive protein (PRIP) as a new negative regulator of phosphatidylinositol 3-kinase/AKT signaling. AKT regulates the IKK/NF-κB signaling pathway; therefore, this study aimed to investigate the role of PRIP/AKT signaling in LPS-mediated neuroinflammation-induced anorexia.PRIP gene (Prip1 and Prip2) knockout (Prip-KO) mice intraperitoneally (ip) administered with LPS exhibited increased anorexia responses compared with wild-type (WT) controls. Although few differences were observed between WT and Prip-KO mice in LPS-elicited plasma pro-inflammatory cytokine elevation, hypothalamic pro-inflammatory cytokines were significantly upregulated in Prip-KO rather than WT mice. Hypothalamic AKT and IKK phosphorylation and IκB degradation were significantly increased in Prip-KO rather than WT mice, indicating further promotion of AKT-mediated NF-κB signaling. Consistently, hypothalamic STAT3 was further phosphorylated in Prip-KO rather than WT mice. Furthermore, suppressor of cytokine signaling 3 (Socs3), a negative feedback regulator for STAT3 signaling, and cyclooxogenase-2 (Cox2), a candidate molecule in LPS-induced anorexigenic responses, were upregulated in the hypothalamus in Prip-KO rather than WT mice. Pro-inflammatory cytokines were upregulated in hypothalamic microglia isolated from Prip-KO rather than WT mice.Together, these findings indicate that PRIP negatively regulates LPS-induced anorexia caused by pro-inflammatory cytokine expression in the hypothalamus, which is mediated by AKT-activated NF-κB signaling. Importantly, hypothalamic microglia participate in this PRIP-mediated process. Elucidation of PRIP-mediated neuroinflammatory responses may provide novel insights into the pathophysiology of many brain dysfunctions.

Phospholipase C-related catalytically inactive protein regulates cytokinesis by protecting phosphatidylinositol 4,5-bisphosphate from metabolism in the cleavage furrow

Cytokinesis is initiated by the formation and ingression of the cleavage furrow. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] accumulation followed by RhoA translocation to the cleavage furrow are prerequisites for cytokinesis progression. Here, we investigated whether phospholipase C (PLC)-related catalytically inactive protein (PRIP), a metabolic modulator of PI(4,5)P2, regulates PI(4,5)P2-mediated cytokinesis. We found that PRIP localised to the cleavage furrow during cytokinesis. Moreover, HeLa cells with silenced PRIP displayed abnormal cytokinesis. Importantly, PI(4,5)P2 accumulation at the cleavage furrow, as well as the localisation of RhoA and phospho-myosin II regulatory light chain to the cleavage furrow, were reduced in PRIP-silenced cells. The overexpression of oculocerebrorenal syndrome of Lowe-1 (OCRL1), a phosphatidylinositol-5-phosphatase, in cells decreased PI(4,5)P2 levels during early cytokinesis and resulted in cytokinesis abnormalities. However, these abnormal cytokinesis phenotypes were ameliorated by the co-expression of PRIP but not by co-expression of a PI(4,5)P2-unbound PRIP mutant. Collectively, our results indicate that PRIP is a component at the cleavage furrow that maintains PI(4,5)P2 metabolism and regulates RhoA-dependent progression of cytokinesis. Thus, we propose that PRIP regulates phosphoinositide metabolism correctively and mediates normal cytokinesis progression.

Suppression of cell migration by phospholipase C-related catalytically inactive protein-dependent modulation of PI3K signalling

The metabolic processes of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] into PI(3,4,5)P3 and the subsequent PI(3,4,5)P3 signalling are involved in cell migration. Dysfunctions in the control of this pathway can cause human cancer cell migration and metastatic growth. Here we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a PI(4,5)P2-binding protein, regulates cancer cell migration. PRIP overexpression in MCF-7 and BT-549 human breast cancer cells inhibited cell migration in vitro and metastasis development in vivo. Overexpression of the PRIP pleckstrin homology domain, a PI(4,5)P2 binding motif, in MCF-7 cells caused significant suppression of cell migration. Consistent with these results, in comparison with wild-type cells, Prip-deficient mouse embryonic fibroblasts exhibited increased cell migration, and this was significantly attenuated upon transfection with a siRNA targeting p110α, a catalytic subunit of class I phosphoinositide 3-kinases (PI3Ks). PI(3,4,5)P3 production was decreased in Prip-overexpressing MCF-7 and BT-549 cells. PI3K binding to PI(4,5)P2 was significantly inhibited by recombinant PRIP in vitro, and thus the activity of PI3K was downregulated. Collectively, PRIP regulates the production of PI(3,4,5)P3 from PI(4,5)P2 by PI3K, and the suppressor activity of PRIP in PI(4,5)P2 metabolism regulates the tumour migration, suggesting PRIP as a promising target for protection against metastatic progression.

Phospholipase C-related catalytically inactive protein-knockout mice exhibit uncoupling protein 1 upregulation in adipose tissues following chronic cold exposure

ObjectivesWe have previously demonstrated that phospholipase C-related catalytically inactive protein (PRIP) is involved in fat metabolism and energy consumption. However, whether PRIP participates in body energy metabolism in vivo remains to be determined. Therefore, we examined whether PRIP deficiency affects whole-body energy homeostasis, which is modulated by non-shivering thermogenesis in brown adipose tissue, using a cold exposure animal model. MethodsFasting plasma triacylglycerol levels were measured to evaluate fat metabolism in wild-type and Prip-KO mice. In addition, a glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed. To determine changes in energy consumption, mice were exposed to a cold environment for 7 days, and expression of uncoupling protein 1 (UCP1) in brown adipose tissue was analyzed via western blotting. ResultsFasting plasma levels of triacylglycerols were significantly higher in Prip-KO mice than in wild-type mice. However, Prip-KO mice showed a healthy phenotype based on GTT and ITT. UCP1 expression was significantly upregulated in the brown and white adipose tissues of Prip-KO mice exposed to cold conditions. ConclusionPrip-KO mice exhibit greater ability to consume lipids as an energy source, indicating that PRIP modulates of systemic energy expenditure. Our findings provide increased understanding of PRIP-mediated non-shivering thermogenic mechanisms and offers important insights for the treatment and control of obesity.

General anesthetic actions on GABAA receptors in vivo are reduced in phospholipase C-related catalytically inactive protein knockout mice.

The aim of this study was to investigate the action of general anesthetics in phospholipase C-related catalytically inactive protein (PRIP)-knockout (KO) mice that alter GABAA receptor signaling. PRIP regulates the intracellular trafficking of β subunit-containing GABAA receptors in vitro. In this study, we examined the effects of intravenous anesthetics, propofol and etomidate that act via β subunit-containing GABAA receptors, in wild-type and Prip-KO mice. Mice were intraperitoneally injected with a drug, and a loss of righting reflex (LORR) assay and an electroencephalogram analysis were performed. The cell surface expression of GABAA receptor β3 subunit detected by immunoblotting was decreased in Prip-knockout brain compared with that in wild-type brain without changing the expression of other GABAA receptor subunits. Propofol-treated Prip-KO mice exhibited significantly shorter duration of LORR and had lower total anesthetic score than wild-type mice in the LORR assay. The average duration of sleep time in an electroencephalogram analysis was shorter in propofol-treated Prip-KO mice than in wild-type mice. The hypnotic action of etomidate was also reduced in Prip-KO mice. However, ketamine, an NMDA receptor antagonist, had similar effects in the two genotypes. PRIP regulates the cell surface expression of the GABAA receptor β3 subunit and modulates general anesthetic action in vivo. Elucidation of the involved regulatory mechanisms of GABAA receptor-dependent signaling would inform the development of safer anesthetic therapies for clinical applications.

Phospholipase C-related catalytically inactive protein can regulate obesity, a state of peripheral inflammation.

SummaryObesity is defined as abnormal or excessive fat accumulation. Chronic inflammation in fat influences the development of obesity-related diseases. Many reports state that obesity increases the risk of morbidity in many diseases, including hypertension, dyslipidemia, type 2 diabetes, coronary heart disease, stroke, sleep apnea, and breast, prostate and colon cancers, leading to increased mortality. Obesity is also associated with chronic neuropathologic conditions such as depression and Alzheimer's disease. However, there is strong evidence that weight loss reduces these risks, by limiting blood pressure and improving levels of serum triglycerides, total cholesterol, low-density lipoprotein (LDL)-cholesterol, and high-density lipoprotein (HDL)-cholesterol. Prevention and control of obesity is complex, and requires a multifaceted approach. The elucidation of molecular mechanisms driving fat metabolism (adipogenesis and lipolysis) aims at developing clinical treatments to control obesity. We recently reported a new regulatory mechanism in fat metabolism: a protein phosphatase binding protein, phospholipase C-related catalytically inactive protein (PRIP), regulates lipolysis in white adipocytes and heat production in brown adipocytes via phosphoregulation. Deficiency of PRIP in mice led to reduced fat accumulation and increased energy expenditure, resulting in a lean phenotype. Here, we evaluate PRIP as a new therapeutic target for the control of obesity.

Phospholipase C-related Catalytically Inactive Protein Is a New Modulator of Thermogenesis Promoted by β-Adrenergic Receptors in Brown Adipocytes

Phospholipase C-related catalytically inactive protein (PRIP) was first identified as an inositol 1,4,5-trisphosphate-binding protein, and was later found to be involved in a variety of cellular events, particularly those related to protein phosphatases. We previously reported that Prip knock-out (KO) mice exhibit a lean phenotype with a small amount of white adipose tissue. In the present study, we examined whether PRIP is involved in energy metabolism, which could explain the lean phenotype, using high-fat diet (HFD)-fed mice. Prip-KO mice showed resistance to HFD-induced obesity, resulting in protection from glucose metabolism dysfunction and insulin resistance. Energy expenditure and body temperature at night were significantly higher in Prip-KO mice than in wild-type mice. Gene and protein expression of uncoupling protein 1 (UCP1), a thermogenic protein, was up-regulated in Prip-KO brown adipocytes in thermoneutral or cold environments. These phenotypes were caused by the promotion of lipolysis in Prip-KO brown adipocytes, which is triggered by up-regulation of phosphorylation of the lipolysis-related proteins hormone-sensitive lipase and perilipin, followed by activation of UCP1 and/or up-regulation of thermogenesis-related genes (e.g. peroxisome proliferator-activated receptor-γ coactivator-1α). The results indicate that PRIP negatively regulates UCP1-mediated thermogenesis in brown adipocytes.

Phospholipase C-Related Catalytically Inactive Protein (PRIP) Regulates Lipolysis in Adipose Tissue by Modulating the Phosphorylation of Hormone-Sensitive Lipase

Phosphorylation of hormone-sensitive lipase (HSL) and perilipin by protein kinase A (PKA) promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A), is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO) mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP-KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis.

Phospholipase C-related catalytically inactive protein participates in the autophagic elimination of Staphylococcus aureus infecting mouse embryonic fibroblasts.

Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP) that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs). We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.

Phospholipase C-related catalytically inactive protein (PRIP) controls KIF5B-mediated insulin secretion.

ABSTRACTWe previously reported that phospholipase C-related catalytically inactive protein (PRIP)-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6) cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrin-labeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA-receptor-associated protein (GABARAP), a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms.

Phospholipase C-related catalytically inactive protein, a novel microtubule-associated protein 1 light chain 3-binding protein, negatively regulates autophagosome formation

Upon starvation, cells undergo autophagy, an intracellular bulk-degradation process, to provide the required nutrients. Here, we observed that phospholipase C-related catalytically inactive protein (PRIP) binds to microtubule-associated protein 1 light chain 3 (LC3), a mammalian autophagy-related initiator that regulates the autophagy pathway. Then, we examined the involvement of PRIP in the nutrient depletion-induced autophagy pathway. Enhanced colocalization of PRIP with LC3 was clearly seen in nutrient-starved mouse embryonic fibroblasts under a fluorescent microscope, and interaction of the proteins was revealed by immunoprecipitation experiments with an anti-LC3 antibody. Under starvation conditions, there were more green fluorescent protein fused-LC3 dots in mouse embryonic fibroblasts from PRIP-deficient mice than in fibroblasts from wild type cells. The formation of new dots in a single cell increased, as assessed by time-lapse microscopy. Furthermore, the increase in autophagosome formation in PRIP-deficient cells was notably inhibited by exogenously overexpressed PRIP. Taken together, PRIP is a novel LC3-binding protein that acts as a negative modulator of autophagosome formation.

Orofacial movements in phospholipase C‐related catalytically inactive protein‐1/2 double knockout mice: Effect of the GABAergic agent diazepam and the D1 dopamine receptor agonist SKF 83959

Orofacial movements are regulated by D(1)-like dopamine receptors interacting with additional mechanisms. Phospholipase C-related catalytically inactive protein (PRIP) regulates cell surface expression of GABA(A) receptors containing a gamma2 subunit. Mutant mice with double knockout of PRIP-1 and PRIP-2 were used to investigate aspects of GABAergic regulation of orofacial movements and interactions with D(1) mechanisms. Vertical jaw movements, tongue protrusions and movements of the head and vibrissae were reduced in PRIP-1/2 double knockouts. The GABA(A)ergic agent diazepam reduced movements of the head and vibrissae; these effects were unaltered in PRIP-1/2 double knockouts. The D(1)-like agonist SKF 83959 induced vertical jaw movements, incisor chattering, and movements of the head and vibrissae that were unaltered in PRIP-1/2 double knockouts. However, SKF 83959-induced tongue protrusions were reduced in PRIP-1/2 double knockouts. PRIP-mediated regulation of GABA(A)ergic receptor mechanisms influences topographically distinct aspects of orofacial movement and interacts with D(1) receptor systems.

Phospholipase C‐related inactive protein is implicated in the constitutive internalization of GABAAreceptors mediated by clathrin and AP2 adaptor complex

A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABA(A) receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABA(A) receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABA(A) receptors through association with receptor beta subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABA(A) receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABA(A) receptor alpha/beta/gamma subunits and PRIP. PRIP was internalized together with GABA(A) receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to beta subunits. The clathrin heavy chain, mu2 and beta2 subunits of AP2 and PRIP-1, were complexed with GABA(A) receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-beta2/3 GABA(A) receptor antibody or by pull-down assay using beta subunits of GABA(A) receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABA(A) receptor that requires clathrin and AP2 protein complex.