氧调节蛋白150在人肝细胞癌中的过度表达及其对凋亡的作用

在前期研究中发现,氧调节蛋白150(ORP150)是与肝细胞癌相关的糖蛋白．进一步研究了ORP150的表达水平与肝细胞癌的相关性．免疫印迹、细胞免疫化学和定量PCR分别在蛋白质水平和mRNA水平检测了ORP150的表达．运用RNA干扰技术检测了其对凋亡和肝细胞癌侵袭性的影响．发现：无论是蛋白质水平还是mRNA水平,与正常肝细胞相比,ORP150在肝细胞癌中表达明显上调;经RNA干扰后,肝细胞癌的凋亡明显增加,但肿瘤细胞的侵袭性无改变．肝细胞癌中,ORP150表达上调,它可能抑制肿瘤细胞的凋亡而促进其生长．ORP150有可能成为肝细胞癌的治疗靶点．     

Reciprocal regulation of miR-23a and lysophosphatidic acid receptor signaling in cardiomyocyte hypertrophy

Earlier, our study demonstrated that lysophosphatidic acid (LPA) receptor mediated cardiomyocyte hypertrophy. However, the subtype-specific functions for LPA1 and LPA3 receptors in LPA-induced hypertrophy have not been distinguished. Growing evidence indicates that microRNAs (miRNAs) are involved in the pathogenesis of cardiac hypertrophy by down-regulating target molecules. The present work therefore aimed at elucidating the functions mediated by different subtypes of LPA receptors and investigating the modulatory role of miRNAs during LPA induced hypertrophy. Experiments were done with cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and we showed that knockdown of LPA1 by small interfering RNA (siRNA) enhanced LPA-induced cardiomyocyte hypertrophy, whereas LPA3 silencing repressed hypertrophy. miR-23a, a pro-hypertrophic miRNA, was up-regulated by LPA in cardiomyocytes and its down-regulation reduced LPA-induced cardiomyocyte hypertrophy. Importantly, luciferase reporter assay confirmed LPA1 to be a target of miR-23a, indicating that miR-23a is involved in mediating the LPA-induced cardiomyocyte hypertrophy by targeting LPA1. In addition, knockdown of LPA3, but not LPA1, eliminated miR-23a elevation induced by LPA. And PI3K inhibitor, LY294002, effectively prevented LPA-induced miR-23a expression in cardiomyocytes, suggesting that LPA might induce miR-23a elevation by activating LPA3 and PI3K/AKT pathway. These findings identified opposite subtype-specific functions for LPA1 and LPA3 in mediating cardiomyocyte hypertrophy and indicated LPA1 to be a target of miR-23a, which discloses a link between miR-23a and the LPA receptor signaling in cardiomyocyte hypertrophy.     

LPA1/3 signaling mediates tumor lymphangiogenesis through promoting CRT expression in prostate cancer

Lysophosphatidic acid (LPA) is a bioactive lipid growth factor which is present in high levels in serum and platelets. LPA binds to its specific G-protein-coupled receptors, including LPA₁ to LPA₆, thereby regulating various physiological functions, including cancer growth, angiogenesis, and lymphangiogenesis. Our previous study showed that LPA promotes the expression of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C in prostate cancer (PCa) cells. Interestingly, LPA has been shown to regulate the expression of calreticulin (CRT), a multifunctional chaperone protein, but the roles of CRT in PCa progression remain unclear. Here we investigated the involvement of CRT in LPA-mediated VEGF-C expression and lymphangiogenesis in PCa. Knockdown of CRT significantly reduced LPA-induced VEGF-C expression in PC-3 cells. Moreover, LPA promoted CRT expression through LPA receptors LPA₁ and LPA₃, reactive oxygen species (ROS) production, and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Tumor-xenografted mouse experiments further showed that CRT knockdown suppressed tumor growth and lymphangiogenesis. Notably, clinical evidence indicated that the LPA-producing enzyme autotaxin (ATX) is related to CRT and that CRT level is highly associated with lymphatic vessel density and VEGF-C expression. Interestingly, the pharmacological antagonist of LPA receptors significantly reduced the lymphatic vessel density in tumor and lymph node metastasis in tumor-bearing nude mice. Together, our results demonstrated that CRT is critical in PCa progression through the mediation of LPA-induced VEGF-C expression, implying that targeting the LPA signaling axis is a potential therapeutic strategy for PCa.     

Leptin Regulates KATP Channel Trafficking in Pancreatic β-Cells by a Signaling Mechanism Involving AMP-activated Protein Kinase (AMPK) and cAMP-dependent Protein Kinase (PKA)

Pancreatic β-cells secrete insulin in response to metabolic and hormonal signals to maintain glucose homeostasis. Insulin secretion is under the control of ATP-sensitive potassium (K_ATP) channels that play key roles in setting β-cell membrane potential. Leptin, a hormone secreted by adipocytes, inhibits insulin secretion by increasing K_ATP channel conductance in β-cells. We investigated the mechanism by which leptin increases K_ATP channel conductance. We show that leptin causes a transient increase in surface expression of K_ATP channels without affecting channel gating properties. This increase results primarily from increased channel trafficking to the plasma membrane rather than reduced endocytosis of surface channels. The effect of leptin on K_ATP channels is dependent on the protein kinases AMP-activated protein kinase (AMPK) and PKA. Activation of AMPK or PKA mimics and inhibition of AMPK or PKA abrogates the effect of leptin. Leptin activates AMPK directly by increasing AMPK phosphorylation at threonine 172. Activation of PKA leads to increased channel surface expression even in the presence of AMPK inhibitors, suggesting AMPK lies upstream of PKA in the leptin signaling pathway. Leptin signaling also leads to F-actin depolymerization. Stabilization of F-actin pharmacologically occludes, whereas destabilization of F-actin simulates, the effect of leptin on K_ATP channel trafficking, indicating that leptin-induced actin reorganization underlies enhanced channel trafficking to the plasma membrane. Our study uncovers the signaling and cellular mechanism by which leptin regulates K_ATP channel trafficking to modulate β-cell function and insulin secretion.     

Taurine supplementation increases KATP channel protein content,improving Ca2+ handling and insulin secretion in islets from malnourished mice fed on a high-fat diet

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and K_ATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5 % Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced K_ATP inhibition and Cav activity. RH islets secreted less insulin at high K⁺ concentration and showed enhanced K_ATP activity. Tau supplementation normalized K⁺-induced secretion and enhanced glucose-induced Ca²⁺ influx in RHT islets. R islets presented lower Ca²⁺ influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the K_ATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the K_ATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the K_ATP channels and enhancing Synt-1 islet content.     

Matricellular Protein Cyr61 Bridges Lysophosphatidic Acid and Integrin Pathways Leading to Cell Migration

Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA₁) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA₁ is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6β1 and ανβ3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.     

RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells

In the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small-interfering RNA (siRNA)-mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound-induced migration and cytoskeletal organization in the non-transformed intestinal epithelial cells IEC-6 and IEC-18 stimulated with the G protein-coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline-rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA-induced migration of both IEC-18 and IEC-6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA-transfected IEC-18 cells. Interestingly, in contrast to the FAK-/- cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC-18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA-induced migration or cytoskeletal reorganization in IEC-18 cells. In conclusion, our study shows that FAK plays a critical role in LPA-induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA.     

Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic β-Cells

In pancreatic β-cells, voltage-gated potassium 2.1 (Kv2.1) channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β-cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase (AMPK). Activation of AMPK mimics whereas inhibition of AMPK occludes the effect of leptin. Inhibition of Ca²⁺/calmodulin-dependent protein kinase kinase β, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase (PKA) is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, whereas stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization, and pharmacologically induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (K_ATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface K_ATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. This study supports a model in which leptin exerts concerted trafficking regulation of K_ATP and Kv2.1 channels to coordinately inhibit insulin secretion.     

Lysophosphatidic acid regulates inflammation-related genes in human endothelial cells through LPA1 and LPA3

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid (LPL), which regulates endothelial cells participating in inflammation processes via interactions with endothelial differentiation gene (Edg) family G protein-coupled receptors. In this study, we attempted to determine which LPA receptors mediate the inflammatory response in human endothelial cells. Introduction of siRNA against LPA₁ significantly suppressed LPA-induced ICAM-1 mRNA, total protein, and cell surface expressions, and subsequent U937 monocyte adhesion to LPA-treated human umbilical endothelial cells (HUVECs). By knock down of LPA₁ and LPA₃ in HUVECs, LPA-enhanced IL-1β mRNA expression was significantly attenuated. Moreover, LPA₁ and LPA₃ siRNA also inhibited LPA-enhanced IL-1-dependent long-term IL-8 and MCP-1 mRNA expression, and subsequent THP-1 cell chemotaxis toward LPA-treated HUVEC-conditioned media. These results suggest that the expression of LPA-induced inflammatory response genes is mediated by LPA₁ and LPA₃. Our findings suggest the possible utilization of LPA₁ or LPA₃ as drug targets to treat severe inflammation.     

The effect of hypoxia on the expression of 150 kDa oxygen-regulated protein (ORP 150) in HeLa cells.

Correct protein folding is an important factor, for the translocation of newly synthesised proteins to specific subcellular compartments, extracellular matrix or to biological fluids. This process is regulated by a group of specific proteins, referred to as chaperones. Many stress conditions, such as oxygen or glucose deprivation, slow down the folding process and cause accumulation of unfolded/misfolded proteins in the cell. Molecular chaperones are induced in these conditions; with some named as oxygen-regulated proteins (ORPs). These bind to unfolded / misfolded proteins to facilitate correct assembly. ORP 150 is the subject of this study. Hypoxia results in an enhancement of ORP 150 expression in several tumour cell lines cultured in vitro. HeLa cells grown in hypoxic conditions (despite an intensive expression of ORP 150) demonstrate higher rates of apoptosis in comparison to those cultured in normoxic conditions. Furthermore, the inhibition of ORP 150 synthesis by transfection of these cells with a specific siRNA resulted in an intensification of apoptosis, as indicated by specific markers of this process; the enhancement of poly ADP-ribose protein cleavage and the increase in Bim protein expression. We conclude from our study that the increase in ORP 150 synthesis protects the cells against the proapoptotic effect of hypoxia.     

The role of lysophosphatidic acid receptors in phenotypic modulation of vascular smooth muscle cells

Lysophosphatidic acid (LPA) is a bioactive lipid with diverse physiological effects via activation of G protein-coupled receptors (GPCRs). It has been implicated as a specific dedifferentiation factors that can promote phenotypic modulation of cultured vascular smooth muscle cells (VSMCs) which is critically involved in various vascular disease. However, the role of LPA receptors and details of their signaling in LPA induced phenotypic modulation are largely unexplored. In this study we detect the expression of LPA1 and LPA3 in rat aortic smooth muscle cells (RASMCs). LPA promoted RASMCs phenotypic modulation in a dose-dependent manner and coordinated induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell phenotypic modulation was significantly inhibited by specific LPA1/LPA3-receptor antagonist dioctyl-glycerol pyrophosphate (DGPP8:0) at concentration, but this inhibitive effect was lost when the antagonist was coadministered with a highly selective LPA3 agonist,1-oleoyl-2-Omethyl-rac-glycero-phosphothionate (OMPT). In addition, pertussis toxin (PTX), a Gi protein inhibitor had little affect on the LPA-induced phenotypic modulation in RASMC. These data suggest that LPA-induced phenotypic modulation is mediated through the PTX-insensitive G-protein(s), possibly Gq-coupled LPA3 receptor.     

Glucose effect on the expression of 150 kDa oxygen-regulated protein in HeLa cells

Chaperones assist in the correct folding of newly synthesised proteins in the endoplasmic reticulum (ER) of cells, this being essential for the translocation of protein molecules to specific subcellular compartments, extracellular matrix or to biological fluids. The biosynthesis of some ER chaperones is regulated by glucose. They are named "glucose-regulated proteins" (GRPs). The function of some GRPs depends on oxygen, a subgroup named "oxygen-regulated proteins" (ORPs). The biosynthesis of ORPs is induced by deprivation of glucose or oxygen. Exposure of HeLa cells to glucose starvation induces the biosynthesis of various GRPs including ORP 150. The expression of ORP 150 is regulated by the concentration of glucose in the culture medium, being induced by a shortage and repressed by a presence of glucose. We have shown that both glucose starvation and transfection of cells with siRNA (specific to ORP 150 mRNA) evoke similar, but quantitatively different, effects. The cells grown for 72 h in a 4.5 mg/ml glucose-containing medium demonstrated low apoptosis (3.7%) whereas in a 0.5 mg/ml glucose-containing medium the apoptosis was increased to 10%. The effect of transfection on apoptosis was distinctly higher with almost 22% of apoptotic cells detected in 72 h cultures. One may conclude that ORP 150 reduces the pro-apoptotic effects of glucose starvation. Such a hypothesis is supported by the observation that the transfection procedure makes HeLa cells resistant to the regulatory effect of glucose on ORP 150 production. The transfected cells do not respond to glucose starvation with an overexpression of ORP 150. It is apparent from our experiments that ORP 150 plays an important role in adaptation of cells to the shortage of glucose and reduces the pro-apoptotic effect of glucose starvation.     

Role of Hsp90 in Biogenesis of the β-Cell ATP-sensitive Potassium Channel Complex

The pancreatic β-cell ATP-sensitive potassium (K_ATP) channel is a multimeric protein complex composed of four inwardly rectifying potassium channel (Kir6.2) and four sulfonylurea receptor 1 (SUR1) subunits. K_ATP channels play a key role in glucose-stimulated insulin secretion by linking glucose metabolism to membrane excitability. Many SUR1 and Kir6.2 mutations reduce channel function by disrupting channel biogenesis and processing, resulting in insulin secretion disease. To better understand the mechanisms governing K_ATP channel biogenesis, a proteomics approach was used to identify chaperone proteins associated with K_ATP channels. We report that chaperone proteins heat-shock protein (Hsp)90, heat-shock cognate protein (Hsc)70, and Hsp40 are associated with β-cell K_ATP channels. Pharmacologic inhibition of Hsp90 function by geldanamycin reduces, whereas overexpression of Hsp90 increases surface expression of wild-type K_ATP channels. Coimmunoprecipitation data indicate that channel association with the Hsp90 complex is mediated through SUR1. Accordingly, manipulation of Hsp90 protein expression or function has significant effects on the biogenesis efficiency of SUR1, but not Kir6.2, expressed alone. Interestingly, overexpression of Hsp90 selectively improved surface expression of mutant channels harboring a subset of disease-causing SUR1 processing mutations. Our study demonstrates that Hsp90 regulates biogenesis efficiency of heteromeric K_ATP channels via SUR1, thereby affecting functional expression of the channel in β-cell membrane.     

Lysophosphatidic Acid Enhanced the Angiogenic Capability of Human Chondrocytes by Regulating Gi/NF-kB-Dependent Angiogenic Factor Expression

Lysophosphatidic acid (LPA) has been found to mediate myeloid differentiation, stimulate osteogenesis, alter cell proliferation and migration, and inhibit apoptosis in chondrocytes. The effect of LPA on the angiogenic capability of chondrocytes is not clear. This study aimed to investigate its effect on the angiogenic capability of human chondrocytes and the underlying mechanism of these effects. Human chondrocyte cell line, CHON-001, commercialized human chondrocytes (HC) derived from normal human articular cartilage, and human vascular endothelial cells (HUVECs) were used as cell models in this study. The angiogenic capability of chondrocytes was determined by capillary tube formation, monolayer permeability, cell migration, and cell proliferation. An angiogenesis protein array kit was used to evaluate the secretion of angiogenic factors in conditioned medium. Angiogenin, insulin-like growth factor-binding protein 1 (IGFBP-1), interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase (MMP)-9, and vascular endothelial growth factor (VEGF) mRNA and protein expressions were evaluated by Q-RT-PCR and EIA, respectively. LPA receptor (LPAR) expression was determined by RT-PCR. Signaling pathways were clarified using inhibitors, Western blot analysis, and reporter assays. The LPA treatment promoted the angiogenic capability of CHON-001 cells and HC, resulting in enhanced HUVEC capillary tube formation, monolayer permeability, migration, and cell growth. Angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF mRNA and protein expressions were significantly enhanced in LPA-treated chondrocytes. LPA2, 3, 4 and 6 were expressed in CHON-001 and HC cells. Pretreatment with the Gi/o type G protein inhibitor, pertussis toxin (PTX), and the NF-kB inhibitor, PDTC, significantly inhibited LPA-induced angiogenin, IGFBP-1, IL-8, MCP-1, MMP-9, and VEGF expressions in chondrocytes. The PTX pretreatment also inhibited LPA-mediated NF-kB activation, suggesting the presence of active Gi/NF-kB signaling in CHON-001 and HC cells. The effect of LPA on the angiogenesis-inducing capacity of chondrocytes may be due to the increased angiogenesis factor expression via the Gi/NF-kB signaling pathway.     

Rho-mediated cytoskeletal rearrangement in response to LPA is functionally antagonized by Rac1 and PIP2

G-protein-coupled receptors signal through Rho to induce actin cytoskeletal rearrangement. We previously demonstrated that thrombin stimulates Rho-dependent process retraction and rounding of 1321N1 astrocytoma cells. Surprisingly, while lysophosphatidic acid (LPA) activated RhoA in 1321N1 cells, it failed to produce cell rounding. Thrombin, unlike LPA, decreased Rac1 activity, and activated (GTPase-deficient) Rac1 inhibited thrombin-stimulated cell rounding, while expression of dominant-negative Rac1 promoted LPA-induced rounding. LPA and thrombin receptors appear to differ in coupling to Gi, as LPA but not thrombin-stimulated 1321N1 cell proliferation was pertussis toxin-sensitive. Blocking Gi with pertussis toxin enabled LPA to induce cell rounding and to decrease activated Rac1. These data support the hypothesis that Rac1 and Gi activation antagonize cell rounding. Thrombin and LPA receptors also differentially activated Gq pathways as thrombin but not LPA increased InsP3 formation and reduced phosphatidylinositol 4,5-bisphosphate (PIP2) levels. Microinjection of the plekstrin homology domain of phospholipase C (PLC)delta1, which binds PIP2, enabled LPA to elicit cell rounding, consistent with a requirement for PIP2 reduction. We suggest that Rho-mediated cytoskeletal responses are enhanced by concomitant reductions in cellular levels of PIP2 and Rac1 activation and thus effected only by G-protein-coupled receptors with appropriate subsets of G protein activation.     

Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins

Lysophosphatidate (LPA), the simplest natural phospholipid, is highly mitogenic for quiescent fibroblasts. LPA-induced cell proliferation is not dependent on other mitogens and is blocked by pertussis toxin. LPA initiates at least three separate signaling cascades: activation of a pertussis toxin-insensitive G protein mediating phosphoinositide hydrolysis with subsequent Ca2+ mobilization and stimulation of protein kinase C; release of arachidonic acid in a GTP-dependent manner, but independent of prior phosphoinositide hydrolysis; and activation of a pertussis toxin-sensitive Gi protein mediating inhibition of adenylate cyclase. The peptide bradykinin mimics LPA in inducing the first two responses but fails to activate Gi and to stimulate DNA synthesis. Our data suggest that the mitogenic action of LPA occurs through Gi or a related pertussis toxin substrate and that the phosphoinositide hydrolysis-protein kinase C pathway is neither required nor sufficient, by itself, for mitogenesis. The results further suggest that LPA or LPA-like phospholipids may have a novel role in G protein-mediated signal transduction.     

Identification and Characterization of a Novel Lysophosphatidic Acid Receptor, p2y5/LPA6

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA₄. Here we report that p2y5 is a novel LPA receptor coupling to the G₁₃-Rho signaling pathway. “LPA receptor-null” RH7777 and B103 cells exogenously expressing p2y5 showed [³H]LPA binding, LPA-induced [³⁵S]guanosine 5′-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and G_s/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA₆.