Excess Lipin enzyme activity contributes to TOR1A recessive disease and DYT-TOR1A dystonia.

TOR1A/TorsinA mutations cause two incurable diseases: a recessive congenital syndrome that can be lethal, and a dominantly-inherited childhood-onset dystonia (DYT-TOR1A). TorsinA has been linked to phosphatidic acid lipid metabolism in Drosophila melanogaster. Here we evaluate the role of phosphatidic acid phosphatase (PAP) enzymes in TOR1A diseases using induced pluripotent stem cell-derived neurons from patients, and mouse models of recessive Tor1a disease. We find that Lipin PAP enzyme activity is abnormally elevated in human DYT-TOR1A dystonia patient cells and in the brains of four different Tor1a mouse models. Its severity also correlated with the dosage of Tor1a/TOR1A mutation. We assessed the role of excess Lipin activity in the neurological dysfunction of Tor1a disease mouse models by interbreeding these with Lpin1 knock-out mice. Genetic reduction of Lpin1 improved the survival of recessive Tor1a disease-model mice, alongside suppressing neurodegeneration, motor dysfunction, and nuclear membrane pathology. These data establish that TOR1A disease mutations cause abnormal phosphatidic acid metabolism, and suggest that approaches that suppress Lipin PAP enzyme activity could be therapeutically useful for TOR1A diseases.

Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1.

Nuclear lipid droplets (nLDs) form on the inner nuclear membrane by a mechanism involving promyelocytic leukemia (PML), the protein scaffold of PML nuclear bodies. We report that PML structures on nLDs in oleate-treated U2OS cells, referred to as lipid-associated PML structures (LAPS), differ from canonical PML nuclear bodies by the relative absence of SUMO1, SP100, and DAXX. These nLDs were also enriched in CTP:phosphocholine cytidylyltransferase α (CCTα), the phosphatidic acid phosphatase Lipin1, and DAG. Translocation of CCTα onto nLDs was mediated by its α-helical M-domain but was not correlated with its activator DAG. High-resolution imaging revealed that CCTα and LAPS occupied distinct polarized regions on nLDs. PML knockout U2OS (PML KO) cells lacking LAPS had a 40-50% reduction in nLDs with associated CCTα, and residual nLDs were almost devoid of Lipin1 and DAG. As a result, phosphatidylcholine and triacylglycerol synthesis was inhibited in PML KO cells. We conclude that in response to excess exogenous fatty acids, LAPS are required to assemble nLDs that are competent to recruit CCTα and Lipin1.

Lipin1 mediates cognitive impairment in fld mice via PKD-ERK pathway.

Lipin1 is important in lipid synthesis because of its phosphatidate phosphatase activity, and it also functions as transcriptional coactivators to regulate the expression of genes involved in lipid metabolism. We found that fld mice exhibit cognitive impairment, and it is related to the DAG-PKD-ERK pathway. We used fld mice to explore the relationship between lipin1 and cognitive function. Our results confirmed the presence of cognitive impairment in the hippocampus of lipin1-deficient mice. As shown in behavioral test, the spatial learning and memory ability of fld mice was much worse than that of wild-type mice. Electron microscopy results showed that the number of synapses in hippocampus of fld mice was significantly reduced. BDNF,SYP, PSD95 were significantly reduced. These results suggest that lipin1 impairs synaptic plasticity. Hence,a deficiency of lipin1 leads to decreased DAG levels and inhibits PKD activation, thereby affecting the phosphorylation of ERK and the CREB.

Phosphatidate phosphatase Pah1 has a role in the hyphal growth and virulence of Candida albicans.

Phosphatidate phosphatases play essential roles in lipid metabolism by converting phosphatidic acid to diacylglycerol. Here, we have investigated the roles of a phosphatidate phosphatase, Pah1, in the fungal pathogen Candida albicans. Deleting PAH1 causes multiple phenotypes, especially severe hyphal defects, increased sensitivity to cell wall stress, and reduced virulence in mice. By qPCR, we detected a significant downregulation of hyphal-specific genes including two key hyphal-promoting genes UME6 and HGC1. Overexpression of UME6 in pah1Δ/Δ cells largely restored the hyphal growth, indicating that the reduced expression of UME6 is primarily responsible for the hyphal defects. We also detected decreased expression of three hyphal-promoting transcription factors EFG1, FLO8, and CPH1 in pah1 mutants, consistent with the reduced expression of UME6. Furthermore, the pah1Δ/Δ mutant exhibited increased sensitivity to cell wall stress. During systemic infection of mice, the mutant showed significantly impaired ability to colonize the kidney and to kill the host. Together, C. albicans PAH1 plays an important role in hyphal growth, adaptability to environmental stresses, and virulence. Thus, Pah1 could be targeted for the development of new antifungal drugs.

Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Lipin 1 regulates glycerolipid homeostasis by acting as a phosphatidic acid phosphohydrolase (PAP) enzyme in the triglyceride-synthesis pathway and by regulating transcription factor activity. Mutations in human lipin 1 are a common cause of recurrent rhabdomyolysis in children. Mice with constitutive whole-body lipin 1 deficiency have been used to examine mechanisms connecting lipin 1 deficiency to myocyte injury. However, that mouse model is confounded by lipodystrophy not phenocopied in people. Herein, 2 muscle-specific mouse models were studied: 1) Lpin1 exon 3 and 4 deletion, resulting in a hypomorphic protein without PAP activity, but which preserved transcriptional coregulatory function; and 2) Lpin1 exon 7 deletion, resulting in total protein loss. In both models, skeletal muscles exhibited a chronic myopathy with ongoing muscle fiber necrosis and regeneration and accumulation of phosphatidic acid and, paradoxically, diacylglycerol. Additionally, lipin 1-deficient mice had abundant, but abnormal, mitochondria likely because of impaired autophagy. Finally, these mice exhibited increased plasma creatine kinase following exhaustive exercise when unfed. These data suggest that mice lacking lipin 1-mediated PAP activity in skeletal muscle may serve as a model for determining the mechanisms by which lipin 1 deficiency leads to myocyte injury and for testing potential therapeutic approaches.-Schweitzer, G. G., Collier, S. L., Chen, Z., McCommis, K. S., Pittman, S. K., Yoshino, J., Matkovich, S. J., Hsu, F.-F., Chrast, R., Eaton, J. M., Harris, T. E., Weihl, C. C., Finck, B. N. Loss of lipin 1-mediated phosphatidic acid phosphohydrolase activity in muscle leads to skeletal myopathy in mice.

Mining the Plasma Cell Transcriptome for Novel Cell Surface Proteins.

Antibody Secreting Cells (ASCs) are a fundamental component of humoral immunity, however, deregulated or excessive antibody production contributes to the pathology of autoimmune diseases, while transformation of ASCs results in the malignancy Multiple Myeloma (MM). Despite substantial recent improvements in treating these conditions, there is as yet no widely used ASC-specific therapeutic approach, highlighting a critical need to identify novel methods of targeting normal and malignant ASCs. Surface molecules specifically expressed by the target cell population represent ideal candidates for a monoclonal antibody-based therapy. By interrogating the ASC gene signature that we previously defined we identified three surface proteins, Plpp5, Clptm1l and Itm2c, which represent potential targets for novel MM treatments. Plpp5, Clptm1l and Itm2c are highly and selectively expressed by mouse and human ASCs as well as MM cells. To investigate the function of these proteins within the humoral immune system we have generated three novel mouse strains, each carrying a loss-of-function mutation in either Plpp5, Clptm1l or Itm2c. Through analysis of these novel strains, we have shown that Plpp5, Clptm1l and Itm2c are dispensable for the development, maturation and differentiation of B-lymphocytes, and for the production of antibodies by ASCs. As adult mice lacking either protein showed no apparent disease phenotypes, it is likely that targeting these molecules on ASCs will have minimal on-target adverse effects.

LPP3 mediates self-generation of chemotactic LPA gradients by melanoma cells.

Melanoma cells steer out of tumours using self-generated lysophosphatidic acid (LPA) gradients. The cells break down LPA, which is present at high levels around the tumours, creating a dynamic gradient that is low in the tumour and high outside. They then migrate up this gradient, creating a complex and evolving outward chemotactic stimulus. Here, we introduce a new assay for self-generated chemotaxis, and show that raising LPA levels causes a delay in migration rather than loss of chemotactic efficiency. Knockdown of the lipid phosphatase LPP3 - but not of its homologues LPP1 or LPP2 - diminishes the cell's ability to break down LPA. This is specific for chemotactically active LPAs, such as the 18:1 and 20:4 species. Inhibition of autotaxin-mediated LPA production does not diminish outward chemotaxis, but loss of LPP3-mediated LPA breakdown blocks it. Similarly, in both 2D and 3D invasion assays, knockdown of LPP3 diminishes the ability of melanoma cells to invade. Our results demonstrate that LPP3 is the key enzyme in the breakdown of LPA by melanoma cells, and confirm the importance of attractant breakdown in LPA-mediated cell steering.This article has an associated First Person interview with the first author of the paper.

Lipin proteins and glycerolipid metabolism: Roles at the ER membrane and beyond.

The regulation of glycerolipid biosynthesis is critical for homeostasis of cellular lipid stores and membranes. Here we review the role of lipin phosphatidic acid phosphatase enzymes in glycerolipid synthesis. Lipin proteins are unique among glycerolipid biosynthetic enzymes in their ability to transit among cellular membranes, rather than remain membrane tethered. We focus on the mechanisms that underlie lipin protein interactions with membranes and the versatile roles of lipins in several organelles, including the endoplasmic reticulum, mitochondria, endolysosomes, lipid droplets, and nucleus. We also review the corresponding physiological roles of lipins, which have been uncovered by the study of genetic lipin deficiencies. We propose that the growing body of knowledge concerning the biochemical and cellular activities of lipin proteins will be valuable for understanding the physiological functions of lipin proteins in health and disease. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Lipin-2 regulates NLRP3 inflammasome by affecting P2X7 receptor activation.

Mutations in human LPIN2 produce a disease known as Majeed syndrome, the clinical manifestations of which are ameliorated by strategies that block IL-1ß or its receptor. However the role of lipin-2 during IL-1ß production remains elusive. We show here that lipin-2 controls excessive IL-1ß formation in primary human and mouse macrophages by several mechanisms, including activation of the inflammasome NLRP3. Lipin-2 regulates MAPK activation, which mediates synthesis of pro-IL-1ß during inflammasome priming. Lipin-2 also inhibits the activation and sensitization of the purinergic receptor P2X7 and K+ efflux, apoptosis-associated speck-like protein with a CARD domain oligomerization, and caspase-1 processing, key events during inflammasome activation. Reduced levels of lipin-2 in macrophages lead to a decrease in cellular cholesterol levels. In fact, restoration of cholesterol concentrations in cells lacking lipin-2 decreases ion currents through the P2X7 receptor, and downstream events that drive IL-1ß production. Furthermore, lipin-2-deficient mice exhibit increased sensitivity to high lipopolysaccharide doses. Collectively, our results unveil lipin-2 as a critical player in the negative regulation of NLRP3 inflammasome.

Novel insights into the composition and function of the Toxoplasma IMC sutures.

The Toxoplasma inner membrane complex (IMC) is a specialized organelle underlying the parasite's plasma membrane that consists of flattened rectangular membrane sacs that are sutured together and positioned atop a supportive cytoskeleton. We have previously identified a novel class of proteins localizing to the transverse and longitudinal sutures of the IMC, which we named IMC sutures components (ISCs). Here, we have used proximity-dependent biotin identification at the sutures to better define the composition of this IMC subcompartment. Using ISC4 as bait, we demonstrate biotin-dependent labeling of the sutures and have uncovered two new ISCs. We also identified five new proteins that exclusively localize to the transverse sutures that we named transverse sutures components (TSCs), demonstrating that components of the IMC sutures consist of two groups: those that localize to the transverse and longitudinal sutures (ISCs) and those residing only in the transverse sutures (TSCs). In addition, we functionally analyze the ISC protein ISC3 and demonstrate that ISC3-null parasites have morphological defects and reduced fitness in vitro. Most importantly, Δisc3 parasites exhibit a complete loss of virulence in vivo. These studies expand the known composition of the IMC sutures and highlight the contribution of ISCs to the ability of the parasite to proliferate and cause disease.

The Role of Lipin-1 in the Regulation of Fibrogenesis and TGF-ß Signaling in Hepatic Stellate Cells.

The adipogenic transcriptional regulation was reported to inhibit transdifferentiation of hepatic stellate cells (HSCs), which constitute the main fibrogenic cell type in the liver. Lipin-1 exhibits a dual function: an enzyme that catalyzes the conversion of phosphatidate to diacylglycerol and a transcriptional regulator. However, the involvement of Lipin-1 in the regulation of transforming growth factor-ß (TGF-ß) signaling and fibrogenesis in HSCs is not fully understood. Here, we showed that Lipin-1 was downregulated in activated primary HSCs and TGF-ß-treated LX-2 cells, immortalized human HSC cell lines. The downregulation of Lipin-1 by TGF-ß was not dependent on altered mRNA stability but rather on protein stability. Treatment of LX-2 cells with the proteasome inhibitor led to the accumulation of Lipin-1. Moreover, we observed a significant increase in Lipin-1 polyubiquitination. Overexpression of Lipin-1 attenuated TGF-ß-induced fibrogenic gene expression. In addition, Lipin-1 inhibited TGF-ß-mediated activation of Sma and Mad-related family (SMAD), a major transcription factor that transduces intracellular signals from TGF-ß. Resveratrol, a well-known natural polyphenolic antioxidant, is known to inhibit liver fibrosis, although its mechanism of action remains unknown. Our data showed that resveratrol significantly increased the levels of Lipin-1 protein and mRNA in HSCs. Further investigation revealed that resveratrol blocked the polyubiquitination of Lipin-1. Resveratrol inhibited TGF-ß-induced fibrogenic gene expression. TGF-ß-induced SMAD binding element-luciferase reporter activity was significantly diminished by resveratrol with a simultaneous decrease in SMAD3 phosphorylation. Consistently, knockdown of the Lipin-1 gene using siRNA abolished the inhibitory effect of resveratrol. We conclude that Lipin-1 can antagonize HSC activation through the inhibition of TGF-ß/SMAD signaling and that resveratrol may affect Lipin-1 gene induction and contribute to the inhibition of TGF-ß-mediated hepatic fibrogenesis.

Lipin1-Mediated Repression of Adipogenesis by Rutin.

Rutin, also called rutoside or quercetin-3-O-rutinoside and sophorin, is a glycoside between the flavonol quercetin and the disaccharide rutinose. Although many effects of rutin have been reported in vitro and in vivo, the anti-adipogenic effects of rutin have not been fully reported. The aim of this study was to confirm how rutin regulates adipocyte related factors. In this study, rutin decreased the expressions of adipogenesis-related genes, including peroxisome proliferators, activated receptor [Formula: see text] (PPAR[Formula: see text], CCAAT/enhancer-binding protein [Formula: see text] (C/EBP[Formula: see text], fatty acid synthase, adipocyte fatty acid-binding protein, and lipoprotein lipase in 3T3-L1 cells. Rutin also repressed the expression of lipin1, which is an upstream regulator that controls PPAR[Formula: see text] and C/EBP[Formula: see text]. In addition, when 3T3-L1 was transfected with lipin1 siRNA to block lipin1 function, rutin did not affect the expressions of PPAR[Formula: see text] and C/EBP[Formula: see text]. These results suggest that rutin has an anti-adipogenic effect that acts through the suppression of lipin1, as well as PPAR[Formula: see text] and C/EBP[Formula: see text].

Lipin1 Regulates Skeletal Muscle Differentiation through Extracellular Signal-regulated Kinase (ERK) Activation and Cyclin D Complex-regulated Cell Cycle Withdrawal.

Lipin1, an intracellular protein, plays critical roles in controlling lipid synthesis and energy metabolism through its enzymatic activity and nuclear transcriptional functions. Several mouse models of skeletal muscle wasting are associated with lipin1 mutation or altered expression. Recent human studies have suggested that children with homozygous null mutations in the LPIN1 gene suffer from rhabdomyolysis. However, the underlying pathophysiologic mechanism is still poorly understood. In the present study we examined whether lipin1 contributes to regulating muscle regeneration. We characterized the time course of skeletal muscle regeneration in lipin1-deficient fld mice after injury. We found that fld mice exhibited smaller regenerated muscle fiber cross-sectional areas compared with wild-type mice in response to injury. Our results from a series of in vitro experiments suggest that lipin1 is up-regulated and translocated to the nucleus during myoblast differentiation and plays a key role in myogenesis by regulating the cytosolic activation of ERK1/2 to form a complex and a downstream effector cyclin D3-mediated cell cycle withdrawal. Overall, our study reveals a previously unknown role of lipin1 in skeletal muscle regeneration and expands our understanding of the cellular and molecular mechanisms underlying skeletal muscle regeneration.

Lipid phosphate phosphatases and their roles in mammalian physiology and pathology.

Lipid phosphate phosphatases (LPPs) are a group of enzymes that belong to a phosphatase/phosphotransferase family. Mammalian LPPs consist of three isoforms: LPP1, LPP2, and LPP3. They share highly conserved catalytic domains and catalyze the dephosphorylation of a variety of lipid phosphates, including phosphatidate, lysophosphatidate (LPA), sphingosine 1-phosphate (S1P), ceramide 1-phosphate, and diacylglycerol pyrophosphate. LPPs are integral membrane proteins, which are localized on plasma membranes with the active site on the outer leaflet. This enables the LPPs to degrade extracellular LPA and S1P, thereby attenuating their effects on the activation of surface receptors. LPP3 also exhibits noncatalytic effects at the cell surface. LPP expression on internal membranes, such as endoplasmic reticulum and Golgi, facilitates the metabolism of internal lipid phosphates, presumably on the luminal surface of these organelles. This action probably explains the signaling effects of the LPPs, which occur downstream of receptor activation. The three isoforms of LPPs show distinct and nonredundant effects in several physiological and pathological processes including embryo development, vascular function, and tumor progression. This review is intended to present an up-to-date understanding of the physiological and pathological consequences of changing the activities of the different LPPs, especially in relation to cell signaling by LPA and S1P.

Arguing the case for the autotaxin-lysophosphatidic acid-lipid phosphate phosphatase 3-signaling nexus in the development and complications of atherosclerosis.

The structurally simple glycero- and sphingo-phospholipids, lysophosphatidic acid (LPA) and sphingosine-1-phosphate, serve as important receptor-active mediators that influence blood and vascular cell function and are positioned to influence the events that contribute to the progression and complications of atherosclerosis. Growing evidence from preclinical animal models has implicated LPA, LPA receptors, and key enzymes involved in LPA metabolism in pathophysiologic events that may underlie atherosclerotic vascular disease. These observations are supported by genetic analysis in humans implicating a lipid phosphate phosphatase as a novel risk factor for coronary artery disease. In this review, we summarize current understanding of LPA production, metabolism, and signaling as may be relevant for atherosclerotic and other vascular disease.

Lipin1 regulates PPARγ transcriptional activity.

PPARγ (peroxisome-proliferator-activated receptor γ) is a master transcription factor involved in adipogenesis through regulating adipocyte-specific gene expression. Recently, lipin1 was found to act as a key factor for adipocyte maturation and maintenance by modulating the C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ network; however, the precise mechanism by which lipin1 affects the transcriptional activity of PPARγ is largely unknown. The results of the present study show that lipin1 activates PPARγ by releasing co-repressors, NCoR1 (nuclear receptor co-repressor 1) and SMRT (silencing mediator of retinoid and thyroid hormone receptor), from PPARγ in the absence of the ligand rosiglitazone. We also identified a novel lipin1 TAD (transcriptional activation domain), between residues 217 and 399, which is critical for the activation of PPARγ, but not PPARα. Furthermore, this TAD is unique to lipin1 since this region does not show any homology with the other lipin isoforms, lipin2 and lipin3. The activity of the lipin1 TAD is enhanced by p300 and SRC-1 (steroid receptor co-activator 1), but not by PCAF (p300/CBP-associated factor) and PGC-1α (PPAR co-activator 1α). The physical interaction between lipin1 and PPARγ occurs at the lipin1 C-terminal region from residues 825 to 926, and the VXXLL motif at residue 885 is critical for binding with and the activation of PPARγ. The action of lipin1 as a co-activator of PPARγ enhanced adipocyte differentiation; the TAD and VXXLL motif played critical roles, but the catalytic activity of lipin1 was not directly involved. Collectively, these data suggest that lipin1 functions as a key regulator of PPARγ activity through its ability to release co-repressors and recruit co-activators via a mechanism other than PPARα activation.

Phosphorylation of lipin 1 and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association.

The lipin gene family encodes a class of Mg(2+)-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Unlike other enzymes in the Kennedy pathway, lipins are not integral membrane proteins, and they need to translocate from the cytosol to intracellular membranes to participate in glycerolipid synthesis. The movement of lipin 1 within the cell is closely associated with its phosphorylation status. Although cellular analyses have demonstrated that highly phosphorylated lipin 1 is enriched in the cytosol and dephosphorylated lipin 1 is found on membranes, the effects of phosphorylation on lipin 1 activity and binding to membranes has not been recapitulated in vitro. Herein we describe a new biochemical assay for lipin 1 using mixtures of phosphatidic acid (PA) and phosphatidylethanolamine that reflects its physiological activity and membrane interaction. This depends on our observation that lipin 1 binding to PA in membranes is highly responsive to the electrostatic charge of PA. The studies presented here demonstrate that phosphorylation regulates the ability of the polybasic domain of lipin 1 to recognize di-anionic PA and identify mTOR as a crucial upstream signaling component regulating lipin 1 phosphorylation. These results demonstrate how phosphorylation of lipin 1 together with pH and membrane phospholipid composition play important roles in the membrane association of lipin 1 and thus the regulation of its enzymatic activity.

Distinct tissue expression profiles of chicken Lpin1-α/ß isoforms and the effect of the variation on muscle fiber traits.

Here we cloned chicken Lpin1-ß, conducted the temporal and spatial expression pattern analysis of chicken Lpin1 isoforms by real-time PCR, and studied the 5' flanking region variation and the potential effect. It was found that chicken Lpin1-α and Lpin1-ß exhibited distinct tissue expression profiling, with prominent expression in the ovary and muscle tissues respectively. Chicken Lpin1 presented a tissue-specific temporal expression pattern in postnatal development (0-16 weeks). Energy restriction significantly elevated the mRNA level of total Lpin1 by increasing the expression of Lpin1-α and Lpin1-ß in a nearly same magnitude. Eight variants/four haplotypes among six breeds were detected from the 5' flanking region of chicken Lpin1, one multiple-nucleotide length polymorphism (g.258M>N) was found and predicted causing the change of 31 transcription factor binding sites including MyoD et al. Both g.258M>N and g. 65C>T variants showed significant association with muscle fiber traits, which suggested one novel role of Lpin1 on muscle fiber development.

Compartmentalizing the embryo: lipids and septate junction mediated barrier function.

Lipid phosphate phosphatases (LPPs) are a class of enzymes that can dephosphorylate a number of lysophopholipids in vitro. Analysis of knockouts of LPP family members has demonstrated striking but diverse developmental roles for these enzymes. LPP3 is required for mouse vascular development while the Drosophila LPPs Wunen (Wun) and Wunen2 (Wun2) are required during embryogenesis for germ cell migration and survival. In a recent publication we examined if these fly LPPs have further developmental roles and found that Wun is required for proper tracheal formation. In particular we highlight a role for Wun in septate junction mediated barrier function in the tracheal system. In this paper we discuss further the possible mechanisms by which LPPs may influence barrier activity.

Suppression of intestinal calcium entry channel TRPV6 by OCRL, a lipid phosphatase associated with Lowe syndrome and Dent disease.

Oculocerebrorenal syndrome of Lowe (OCRL) gene product is a phosphatidyl inositol 4,5-bisphosphate [PI(4,5)P(2)] 5-phosphatase, and mutations of OCRL cause Lowe syndrome and Dent disease, both of which are frequently associated with hypercalciuria. Transient receptor potential, vanilloid subfamily, subtype 6 (TRPV6) is an intestinal epithelial Ca(2+) channel mediating active Ca(2+) absorption. Hyperabsorption of Ca(2+) was found in patients of Dent disease with increased Ca(2+) excretion. In this study, we tested whether TRPV6 is regulated by OCRL and, if so, to what extent it is altered by Dent-causing OCRL mutations using Xenopus laevis oocyte expression system. Exogenous OCRL decreased TRPV6-mediated Ca(2+) uptake by regulating the function and trafficking of TRPV6 through different domains of OCRL. The PI(4,5)P(2) 5-phosphatase domain suppressed the TRPV6-mediated Ca(2+) transport likely through regulating the PI(4,5)P(2) level needed for TRPV6 function without affecting TRPV6 protein abundance of TRPV6 at the cell surface. The forward trafficking of TRPV6 was decreased by OCRL. The Rab binding domain in OCRL was involved in regulating the trafficking of TRPV6. Knocking down endogenous X. laevis OCRL by antisense approach increased TRPV6-mediated Ca(2+) transport and TRPV6 forward trafficking. All seven Dent-causing OCRL mutations examined exhibited alleviation of the inhibitory effect on TRPV6-mediated Ca(2+) transport together with decreased overall PI(4,5)P(2) 5-phosphatase activity. In conclusion, OCRL suppresses TRPV6 via two separate mechanisms. The disruption of PI(4,5)P(2) 5-phosphatase activity by Dent-causing mutations of OCRL may lead to increased intestinal Ca(2+) absorption and, in turn, hypercalciuria.