LPAR2-mediated action promotes human renal cell carcinoma via MAPK/NF-κB signaling to regulate cytokine network.

PURPOSE: Lysophosphatidic acid (LPA) exerts various physiological and pathological effects by activating its distinct G-protein-coupled LPA receptors. We demonstrated that LPA can increase the migration and proliferation of renal carcinoma cells. Meanwhile, LPAR1 and LPAR2 were preferentially expressed in renal cancer (RC) cell lines. So, the study aimed to determine the LPA receptor subtypes involved in LPA-induced actions and whether they could be used as a precision therapeutic target for renal cancer. METHODS: Biological approaches combined with big data analysis were used to demonstrate the role of LPAR2 in the progression of renal cancer. RESULTS: We found that the proliferation, clone formation, and migration in response to LPA were enhanced in LPAR2-overexpressing renal cancer cells, whereas, the actions were suppressed by LPAR2 antagonist in the cells. LPAR2 has also shown clinical diagnostic and prognostic value in renal carcinoma based on bioinformatics analysis and clinical tissue microarray analysis. In vivo study shown that tumor growth and metastasis were significantly increased in the LPAR2-overexpressing cells-derived solid tumors. LPA stimulated MAPK and NF-κB activation, and LPA-induced actions were inhibited by MAPKs and NF-κB inhibitors, respectively. Subsequently, the transcriptomic results revealed that LPAR2 strongly affected the cytokines production, and the increased IL6, CXCL8, and TNF were confirmed again using Kit assay. CONCLUSIONS: We have identified that LPAR2 is critical for LPA-promoted renal cancer progression, and the actions mainly dependent the MAPK and NF-κB activation mechanism. Then, the expression of inflammatory factors activated by NF-κB is also suspected to be involved in LPAR2-mediated carcinogenesis. Thus, LPAR2 may be a promising therapeutic target for renal cancer.

Lpar1-mediated Effects in Endothelial Progenitor Cells Are Crucial for Lung Repair in Acute Respiratory Distress Syndrome/Acute Lung Injury.

Acute respiratory distress syndrome/acute lung injury (ARDS/ALI) involves acute respiratory failure characterized by vascular endothelial and lung alveolar epithelial injury. Endothelial progenitor cells (EPCs) can mediate vasculogenesis. However, the limitations of EPCs, such as low survival and differentiation, are believed to inhibit the effectiveness of autologous cell therapies. This study demonstrated that lysophosphatidic acid (LPA), a bioactive small molecule without immunogenicity, is involved in the survival and antiapoptotic effects in human umbilical cord mesenchymal stem cells. This study aimed to explore whether LPA improves the survival of EPCs, enhancing the cellular therapeutic efficacy in ARDS, and these results will expand the application of LPA in stem cells and regenerative medicine. LPA promoted the colony formation, proliferation, and migration of EPCs and upregulated the expression of vascular endothelial-derived growth factor (VEGF) in EPCs. LPA pretreatment of transplanted EPCs improved the therapeutic effect by increasing EPC numbers in the rat lungs. LPA enhanced EPC proliferation and migration through Lpar1 coupled to Gi/o and Gq/11, respectively. Activation of extracellular signal-related kinase 1/2, or ERK1/2, was related to LPA-induced EPC proliferation but not migration. LPA/Lpar1-mediated Gi/o protein was also shown to be involved in promoting VEGF expression and inhibiting IL-1α expression in EPCs. Low LPA concentrations are present after lung injury; thus, the restoration of LPA may promote endothelial cell homeostasis and lung repair in ARDS. Inhalation of LPA significantly promoted the homing of endogenous EPCs to the lung and reduced lung injury in both rats with LPS-induced ALI and Streptococcus pneumoniae-infected mice. Taken together, these data indicated that LPA/Lpar1-mediated effects in EPCs are involved in maintaining endothelial cell homeostasis and lung tissue repair under physiological conditions.

NF-κB and EGFR participate in S1PR3-mediated human renal cell carcinomas progression.

The bioactive lipid sphingosine 1-phosphate (S1P) is implicated in many pivotal processes for the physiological and pathological actions via activating five types of G-protein-coupled S1P receptors (S1PR1-5). The role of S1P in renal cell carcinoma (RCC) and its receptor subtype specific mediating mechanism are poorly studied. So we focus on the regulatory role of S1P in RCC progression and the receptor subtypes involved in S1P-induced actions, intending to further clarify a novel therapeutic target for RCC. Analysis of The Cancer Genome Atlas (TCGA) databases showed that the patients with high expression of S1PR3 had significantly worse overall than with low expression. We further demonstrated that S1P could promote proliferation, migration, and epithelial-mesenchymal transition (EMT) of renal cancer cells in vitro, and the actions were enhanced with the increase of S1PR3 expression. Meanwhile, the results in animal experiments also showed that S1PR3 could accelerate tumorigenesis and metastasis of RCC. Our study also clarified the mechanism for S1P induced cell proliferation is mediated by S1PR3/Gi/p38/Akt/p65/cyclin D1-CDK4 pathway and the main pathway for migration is S1PR3/Gi/q/ERK/p38/p65. In addition, S1PR3 was involved in epidermal growth factor (EGF)-induced actions by enhancing protein expression, not by transactivation of epidermal growth factor receptor (EGFR). These results also further supported our conclusion that the carcinogenic role of S1P/S1PR3 axis. Thus, our findings provide that S1PR3 may be a promising small molecular therapeutic target for S1PR3 expressed cancers.

LPA3 is a precise therapeutic target and potential biomarker for ovarian cancer.

Current studies have demonstrated that significant increased LPA levels to be observed in ascites in patients with ovarian cancer. Although several studies have shown that Lysophosphatidic acid (LPA) related to the progression of ovarian cancer, which LPA receptors (LPARs) and G-coupled protein subtypes mediated in LPA actions have not been clearly elucidated. This study aimed to clarify the roles of LPA and it is subtype-specific LPARs mediating mechanisms in ovarian cancer integrated using bioinformatic analysis and biological experimental approaches. The big data analysis shown that LPA3 was the only differentially expressed LPA receptor among the six LPARs in ovarian cancer and further verified in immunohistochemistry of tissue microarrays. Also found that LPA3 was also highly expressed in ovarian cancer tissue and ovarian cancer cells. Importantly, LPA significantly promoted the proliferation and migration of LPA3-overexpressing ovarian cancer cells, while the LPA-induced actions blocked by Ki16425, a LPAR1/3 antagonist treated, and LPA3-shRNA transfected. In vivo study indicated that the LPA3-overexpressing cell-derived tumors metastasis, tumors volume, and tumors mass were apparently increased in xenografted nude mice. In addition, we also observed that LPA3 was differential high expression in ovarian cancer tissue of the patients. Our studies further confirmed the LPA3/Gi/MAPKs/NF-κB signals were involved in LPA-induced oncogenic actions in ovarian cancer cells. Our findings indicated that the LPA3 might be a novel precise therapeutic target and potential biomarker for ovarian cancer.

Outstanding Drug-Loading/Release Capacity of Hollow Fe-Metal-Organic Framework-Based Microcapsules: A Potential Multifunctional Drug-Delivery Platform.

Owing to their characteristic structures, metal-organic frameworks (MOFs) are considered as the leading candidate for drug-delivery materials. However, controlling the synthesis of MOFs with uniform morphology and high drug-loading/release efficiencies is still challenging, which greatly limits their applications and promotion. Herein, a multifunctional MOF-based drug-delivery system (DDS) with a controlled pore size of 100-200 nm for both therapeutic and bioimaging purposes was successfully synthesized in one step. Fe-MOF-based microcapsules were synthesized through a competitive coordination method, which was profited from the intrinsic coordination characteristics of the Fe element and the host-guest supramolecular interactions between Fe3+ and polyoxometalates anions. This as-synthesized macroporous DDS could greatly increase the drug-loading/release rate (77%; 83%) and serve as a magnetic resonance (MR) contrast agent. Because an Fe-containing macroporous DDS presents ultrahigh drug loading/release, the obtained 5-FU/Fe-MOF-based microcapsules displayed good biocompatibility, extremely powerful inhibition of tumor growth, and satisfactory MR imaging capability. Given all these advantages, this study integrates high therapeutic effect and diagnostic capability via a simple and effective morphology-controlling strategy, aiming at further facilitating the applications of MOFs in multifunctional drug delivery.

LPA receptor1 antagonists as anticancer agents suppress human lung tumours.

Lysophosphatidic acid (LPA), as a bioactive lipid, plays a variety of physiological and pathological roles via activating six types of G-protein-coupled LPA receptors (LPA1-6). Our preliminary study found that LPA1 is highly expressed in lung cancer tissues compared with paracancerous tissues, but the role of LPA1 in lung carcinoma is unclear. This study aimed to elucidate the association between LPA1 and lung tumour behaviour at the cellular and animal model levels. We found that LPA promoted the migration, proliferation and colony formation of a lung cancer cell line (A549). LPA1 and LPA3 are preferentially expressed in A549 cells, and both Ki16425 (LPA1 and LPA3 antagonist) and ono7300243 (LPA1 antagonist) completely blocked the LPA-induced actions. These results were further verified by experiments of the LPA1/3 overexpression and LPA1 knockdown A549 cells. Furthermore, LPA1 overexpression and knockdown A549 cells were used to assess the in vivo tumour-bearing animal model and the mechanism underlying LPA-induced actions. In the animal model, A549 cell-derived tumour volume was significantly increased by LPA1 overexpression and significantly decreased by LPA1 knockdown respectively, suggesting that LPA1 is a regulator of in vivo tumour formation. Our results also indicated that the LPA1/Gi/MAP kinase/NF-κB pathway is involved in LPA-induced oncogenic actions in A549 cells. Thus, targeting LPA1 may be a novel strategy for treating lung carcinoma.

The atherogenic actions of LPC on vascular smooth muscle cells and its LPA receptor mediated mechanism.

Lysophosphatidylcholine (LPC) is a bioactive lipid constituent of oxidized low density lipoprotein (ox-LDL). It regulates various cellular functions, including migration of circulating monocytes, expression of endothelial adhesion molecules, proliferation and migration of vascular smooth muscle cells (VSMCs). LPC can also be hydrolyzed into lysophosphatidic acid (LPA) by autotaxin (ATX) which possesses lysophospholipase D (lyso-PLD) activity. The aim of this study was to explore the effects of LPC on proliferation and migration of human artery smooth muscle cells (HASMCs) and the involvement of LPC-ATX-LPA pathway in these processes. In vitro, we found that LPC and LPA stimulated HASMCs proliferation and migration. Knockdown of LPA1 by siRNA and inhibit Gi protein with pertussis toxin (PTX) showed the contrary results. Silencing of LPC receptor genes did not significantly affect the LPC induced proliferation and migration. We detected the higher expressed mRNA and protein of ATX in HASMCs, and measured lyso-PLD activity. In atherosclerotic rabbit model, we observed high LPC level and high lyso-D activity in blood, and high expression of LPA1 in aorta walls. We also found that neointima appeared to be thickened and mRNA expressions of LPA1 appeared to be increased. These results revealed that LPC was converted into LPA by ATX to induce the proliferation and migration in HASMCs through LPA1/Gi/o/MAP Kinase signaling pathway. Our research suggested that LPC-ATX-LPA system contributed to the atherogenic action induced by ox-LDL. LPA1 antagonist may be considered as a potential therapeutic and preventative drug for cardiovascular disease.

Lysophosphatidic acid enhances human umbilical cord mesenchymal stem cell viability without differentiation via LPA receptor mediating manner.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are potential stromal cells which are regarded as the most feasible stem cell group in cell therapy. The maintenance of cell survival without differentiation is important in cell transplantation and stem cell therapy. However, negative factors exist in cell transplantation. Lysophosphatidic acid (LPA) is a non-antigenic small molecule phospholipid which induced several fundamental cellular responses, such as cell proliferation, apoptosis and migration. In this study we aimed to explore the effects of LPA on the survival and differentiation of MSCs and its availability in cell therapy. We found that LPA stimulated hUC-MSC proliferation and protected hUC-MSCs from lipopolysaccharide (LPS) induced apoptosis. We also observed that CD29, CD44, CD73, CD90 and CD105 were expressed, whereas CD34 and CD45 were not expressed in hUC-MSCs, and these makers have no change in LPA containing medium, which indicated that LPA accelerated the survival of hUC-MSCs in an undifferentiating status. We also demonstrated that higher expressed LPAR1 involved in LPA stimulated cell survival action. LPA stimulated cell proliferation was associated with LPAR1 mediated Gi/o-proteins/ERK1/2 pathway. On the other hand, LPA protected hUC-MSCs from LPS-induced apoptosis through suppressing caspase-3 activation by LPAR1 coupled with a G protein, but not Gi/o or Gq/11 in hUC-MSC. Collectively, this study demonstrated that LPA increased the proliferation and survival of hUC-MSCs without differentiation through LPAR1 mediated manner. Our findings provide that LPA as a anti-apoptotic agent having potential application prospect in cell transplantation and stem cell therapy.

Controllable Synthesis of a Smart Multifunctional Nanoscale Metal-Organic Framework for Magnetic Resonance/Optical Imaging and Targeted Drug Delivery.

As a result of their extraordinarily large surfaces and well-defined pores, the design of a multifunctional metal-organic framework (MOF) is crucial for drug delivery but has rarely been reported. In this paper, a novel drug delivery system (DDS) based on nanoscale MOF was developed for use in cancer diagnosis and therapy. This MOF-based tumor targeting DDS was fabricated by a simple postsynthetic surface modification process. First, magnetic mesoporous nanomaterial Fe-MIL-53-NH2 was used for encapsulating the drug and served as a magnetic resonance contrast agent. Moreover, the Fe-MIL-53-NH2 nanomaterial exhibited a high loading capacity for the model anticancer drug 5-fluorouracil (5-FU). Subsequently, the fluorescence imaging agent 5-carboxyfluorescein (5-FAM) and the targeting reagent folic acid (FA) were conjugated to the 5-FU-loaded Fe-MIL-53-NH2, resulting in the advanced DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU. Owing to the multifunctional surface modification, the obtained DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU shows good biocompatibility, tumor enhanced cellular uptake, strong cancer cell growth inhibitory effect, excellent fluorescence imaging, and outstanding magnetic resonance imaging capability. Taken together, this study integrates diagnostic and treatment aspects into a single platform by a simple and efficient strategy, aiming for facilitating new possibilities for MOF use for multifunctional drug delivery.

Overexpression of p58ipk protects neuroblastoma against paraquat-induced toxicity.

BACKGROUND: Paraquat (PQ) is a powerful pathologic pesticide that contribute to the neurotoxicity, however, the pathogenic mechanism between them was unclear. The aims of this study were to explore the underlying mechanism of PQ-induced toxicity and then make potential contribute to such neuronal diseases therapy. METHODS: Human cell line SH-SY5Y was pretreated with a set concentrations of PQ to detect the cell apoptosis and the expression of related genes and proteins. Next, pcDNA 3.1-p58ipk or si-p58ipk was transfected the PQ-induced cells to detect the cytotoxicity. RESULTS: PQ significantly increased the cell apoptosis as well as the expression of p58ipk and CHOP, but decreased the expression of pAKT. p58ipk suppression resulted in an increase of cell apoptosis and CHOP expression, but the expression of pAKT was significantly decreased in PQ-induced SH-SY5Y cells. However, overexpressed p58ipk led to an opposite result. CONCLUSION: The results indicated that the expression of p58ipk was related to the toxicity level of PQ-induced cells and the mechanism between them was that p58ipk regulated the toxicity might through regulating the endoplasmic reticulum stress (ER-stress) and then regulating cell apoptosis. Further studies take emphasize on the effect of ER-stress on neuron system and explore ER-stress-related therapy are important on the treatment of neurodegenerative disease.

Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner.

The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF.

Migration of gastric cancer cells in response to lysophosphatidic acid is mediated by LPA receptor 2.

Lysophosphatidic acid (LPA), a natural phospholipid, is able to modulate diverse cellular responses through LPA receptors (LPARs). Several studies have reported that LPAR2 gene expression is increased in a variety of cancer cells, suggesting that LPAR2 is involved in gastric cancer. The present study investigated the expression profiles of the LPAR and involvement of the receptor subtypes in the LPA-induced migration of gastric cancer cells using cell migration assays, RNA interference, quantitative real-time PCR and western blotting. LPAR2 was observed to be highly expressed in SGC-7901 cells, a human gastric cancer cell line, while LPAR1 and LPAR3 were not. Transient transfection with LPAR2 siRNA was observed to reduce LPAR2 mRNA in SGC-7901 cells and eliminate the LPA-induced cell migration. It was also observed that LPA-induced SGC-7901 cell migration was inhibited by the inhibitor for Gq/11 protein and p38. The results suggest that the LPAR2/Gq/11/p38 pathway regulates LPA-induced SGC-7901 cell migration. The present findings suggest that LPAR2 may be a potential target for the clinical treatment of gastric cancer.

Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice.

AIM: To improve the outcome of orthotopic transplantation in a mouse model, we used an absorbable gelatin sponge (AGS) in nude mice to establish an orthotopic implantation tumor model. METHODS: MHCC-97L hepatocellular carcinoma (HCC) cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice. One week later, the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice. The AGS was used to establish the nude mouse orthotopic implantation tumor model. The tumor suppressor gene, paired box gene 5 (PAX5), which is a tumor suppressor in HCC, was transfected into HCC cells to validate the model. Tumor growth was measured by bioluminescence imaging technology. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line. RESULTS: We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS. The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS. The detection of fluorescent signals showed that tumors grew in all live nude mice. The mice were divided into 3 groups: AGS-, AGS+/PAX5- and AGS+/PAX5+. Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice (P < 0.0001). These fluorescent signal results were consistent with observations made during surgery. Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC. Results from RT-PCR proved that the HCC originated from MHCC-97L cells. CONCLUSION: Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

Ovarian cancer G protein-coupled receptor 1-dependent and -independent vascular actions to acidic pH in human aortic smooth muscle cells.

Atherosclerosis is a chronic inflammation disease characterized by acidic micromilieu and the accumulation of numerous bioactive lipid mediators, such as lysophosphatidic acid (LPA) and prostaglandins, in the atherosclerotic lesion. Chronic acidification induced various effects on vascular smooth muscle cells, but the molecular mechanisms underlying these effects remain unknown. In this study, we examine the role of proton-sensing ovarian cancer G protein-coupled receptor 1 (OGR1) in extracellular acidification-induced regulation of cyclooxygenase (COX)-2 induction, PGI(2) production, MAPK phosphatase (MKP)-1 expression, and plasminogen activator inhibitor (PAI)-1 expression and proliferation in human aortic smooth muscle cells (AoSMCs). Experiments with knockdown with small interfering RNA specific to OGR1 and specific inhibitors for G proteins showed that acidification-induced COX-2 expression, PGI(2) production, and MKP-1 expression, but not PAI-1 expression and inhibition of proliferation, were dependent on OGR1 and mainly mediated by G(q/11) protein. LPA remarkably enhanced, through the LPA(1) receptor/G(i) protein, the OGR1-mediated vascular actions to acidic pH. In conclusion, acidic pH-induced vascular actions of AoSMCs can be dissected to OGR1-dependent and -independent pathways: COX-2 expression, PGI(2) production, and MKP-1 expression are mediated by OGR1, but PAI-1 expression and inhibition of proliferation are not. LPA, which is usually thought to be a proatherogenic lipid mediator, may exert antiatherogenic actions under acidic micromilieu through cross-talk between LPA(1)/G(i) protein and OGR1/G(q/11) protein.

Each one of certain histidine residues in G-protein-coupled receptor GPR4 is critical for extracellular proton-induced stimulation of multiple G-protein-signaling pathways.

GPR4, previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways, including the G(s)-protein/cAMP, G(12/13)-protein/Rho, and G(q)-protein/phospholipase C pathways. In the present study, we examined whether extracellularly located histidine residues of GPR4 sense extracellular protons and, if so, whether a certain histidine residue is critical for coupling to the single or multiple signaling pathway(s). We found that the mutation of histidine residue at 79, 165, or 269 from the N-terminal of GPR4 to phenylalanine shifted the half-maximal effective concentration (EC(50)) of proton-induced signaling activities to the right, including cAMP accumulation, SRE promoter activity reflecting Rho activity, and NFAT promoter activity reflecting phospholipase C signaling activity, without an appreciable change in the maximal activities. These results suggest that the protonation of each one of histidine residues at 79, 165, and 269 in GPR4 may be critical for conformational change of the receptor for coupling to multiple intracellular signaling pathways through G-proteins.

Signaling pathways involved in DNA synthesis and migration in response to lysophosphatidic acid and low-density lipoprotein in coronary artery smooth muscle cells.

Low-density lipoprotein (LDL) and lysophosphatidic acid (LPA), one of the lipid components of lipoprotein, induced the DNA synthesis of coronary artery smooth muscle cells (CASMCs). The LDL- and LPA-induced DNA synthesis was markedly inhibited by the LPA receptor antagonist Ki16425, pertussis toxin, small interfering RNAs targeted for LPA1 receptors, and a potent calcineurin inhibitor cyclosporine A. It has been reported that LDL and LPA induced a migration response in a manner sensitive to Ki16425, pertussis toxin, and a LPA1 receptor-specific small interfering RNA. However, cyclosporine A was ineffective in inhibiting the migration response. Instead, an epidermal growth factor (EGF) receptor tyrosine kinase inhibitor markedly suppressed the migration response to LDL and LPA without having any significant effect on DNA synthesis. Thus, the LDL-induced stimulation of DNA synthesis and migration in CASMCs is mediated by its component LPA through LPA1 receptors and G(i/o)-proteins. Ca2+/calcineurin pathways and transactivation of EGF receptors mediate LPA1-receptor-induced DNA synthesis and migration, respectively.

Cyclooxygenase-2 expression and prostaglandin E2 production in response to acidic pH through OGR1 in a human osteoblastic cell line.

Acidosis has been shown to induce depletion of bone calcium from the body. This calcium release process is thought to be partially cell mediated. In an organ culture of bone, acidic pH has been shown to induce cyclooxygenase-2 (COX-2) induction and prostaglandin E(2) (PGE(2)) production, resulting in stimulation of bone calcium release. However, the molecular mechanisms whereby osteoblasts sense acidic circumstances and thereby induce COX-2 induction and PGE(2) production remain unknown. In this study, we used a human osteoblastic cell line (NHOst) to characterize cellular activities, including inositol phosphate production, intracellular Ca(2+) concentration ([Ca(2+)](i)), PGE(2) production, and COX-2 mRNA and protein expression, in response to extracellular acidification. Small interfering RNA (siRNA) specific to the OGR1 receptor and specific inhibitors for intracellular signaling pathways were used to characterize acidification-induced cellular activities. We found that extracellular acidic pH induced a transient increase in [Ca(2+)](i) and inositol phosphate production in the cells. Acidification also induced COX-2 induction, resulting in PGE(2) production. These proton-induced actions were markedly inhibited by siRNA targeted for the OGR1 receptor and the inhibitors for G(q/11) protein, phospholipase C, and protein kinase C. We conclude that the OGR1/G(q/11)/phospholipase C/protein kinase C pathway regulates osteoblastic COX-2 induction and subsequent PGE(2) production in response to acidic circumstances.

Previously postulated "ligand-independent" signaling of GPR4 is mediated through proton-sensing mechanisms.

GPR4 was initially identified as a receptor for sphingosylphosphorylcholine and lysophosphatidylcholine; however, lipid actions have not always been confirmed. Instead, ligand-independent actions have sometimes been observed in GPR4- and other OGR1 family receptor-expressing cells. Here, we examined the possible involvement of extracellular protons, which have recently been proposed as another ligand for GPR4. At pH 7.4, the epidermal growth factor-induced extracellular signal-regulated kinase activity was lower in GPR4-transfected RH7777 cells, in association with increased cAMP accumulation, than in vector-transfected cells. The serum response element (SRE)-driven transcriptional activity was also clearly higher in GPR4-expressing HEK293 cells than in vector-transfected cells at pH 7.4. These apparent ligand-independent actions were very small at alkalinic 7.8. The SRE activity was further increased by extracellular acidification in a manner dependent on the G13 protein/Rho signaling pathway in HEK293 cells expressing GPR4 or other OGR1 receptor family members. GPR4-expressing cells also showed a calcineurin-dependent nuclear factor of activated T cell (NFAT) promoter activation at pH 7.4, and this activity was further increased by pH below 7.2 in association with inositol phosphate production. In contrast to the cAMP and SRE responses, however, alkalinization to pH 7.8 hardly affected the high basal activity. Finally, the expression of GPR4 hardly modulated the sphingosylphosphorylcholine- or lysophosphatidylcholine-induced action. These results suggest that an extracellular proton play a role as a ligand in some of previously postulated ligand-independent actions through GPR4 receptors. Moreover, GPR4 may be a multi-functional receptor coupling to Gs, G13, and Gq/11 proteins in response to extracellular acidification.

Role of lipoprotein-associated lysophospholipids in migratory activity of coronary artery smooth muscle cells.

The migration of vascular smooth muscle cells (SMCs) is a hallmark of the pathogenesis of atherosclerosis and restenosis after angioplasty. Plasma low-density lipoprotein (LDL), but not high-density lipoprotein (HDL), induced the migration of human coronary artery SMCs (CASMCs). Among bioactive lipids postulated to be present in LDL, lysophosphatidic acid (LPA) appreciably mimicked the LDL action. In fact, the LDL-induced migration was markedly inhibited by pertussis toxin, an LPA receptor antagonist Ki-16425, and a small interfering RNA (siRNA) targeted for LPA(1) receptors. Moreover, LDL contains a higher amount of LPA than HDL does. HDL markedly inhibited LPA- and platelet-derived growth factor (PDGF)-induced migration, and sphingosine 1-phosphate (S1P), the content of which is about fourfold higher in HDL than in LDL, mimicked the HDL action. The inhibitory actions of HDL and S1P were suppressed by S1P(2) receptor-specific siRNA. On the other hand, the degradation of the LPA component of LDL by monoglyceride lipase or the antagonism of LPA receptors by Ki-16425 allowed LDL to inhibit the PDGF-induced migration. The inhibitory effect of LDL was again suppressed by S1P(2) receptor-specific siRNA. In conclusion, LPA/LPA(1) receptors and S1P/S1P(2) receptors mediate the stimulatory and inhibitory migration response to LDL and HDL, respectively. The balance of not only the content of LPA and S1P in lipoproteins but also the signaling activity between LPA(1) and S1P(2) receptors in the cells may be critical in determining whether the lipoprotein is a positive or negative regulator of CASMC migration.

Role of scavenger receptor class B type I and sphingosine 1-phosphate receptors in high density lipoprotein-induced inhibition of adhesion molecule expression in endothelial cells.

We characterized the molecular mechanisms by which high density lipoprotein (HDL) inhibits the expression of adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, induced by sphingosine 1-phosphate (S1P) and tumor necrosis factor (TNF) alpha in endothelial cells. HDL inhibited S1P-induced nuclear factor kappaB activation and adhesion molecule expression in human umbilical vein endothelial cells. The inhibitory HDL actions were associated with nitric-oxide synthase (NOS) activation and were reversed by inhibitors for phosphatidylinositol 3-kinase and NOS. The HDL-induced inhibitory actions were also attenuated by the down-regulation of scavenger receptor class B type I (SR-BI) and its associated protein PDZK1. When TNFalpha was used as a stimulant, the HDL-induced NOS activation and the inhibitory action on adhesion molecule expression were, in part, attenuated by the down-regulation of the expression of S1P receptors, especially S1P(1), in addition to SR-BI. Reconstituted HDL composed mainly of apolipoprotein A-I and phosphatidylcholine mimicked the SR-BI-sensitive part of HDL-induced actions. Down-regulation of S1P(3) receptors severely suppressed the stimulatory actions of S1P. Although G(i/o) proteins may play roles in either stimulatory or inhibitory S1P actions, as judged from pertussis toxin sensitivity, the coupling of S1P(3) receptors to G(12/13) proteins may be critical to distinguish the stimulatory pathways from the inhibitory ones. In conclusion, even though S1P alone stimulates adhesion molecule expression, HDL overcomes S1P(3) receptor-mediated stimulatory actions through SR-BI/PDZK1-mediated signaling pathways involving phosphatidylinositol 3-kinase and NOS. In addition, the S1P component of HDL plays a role in the inhibition of TNFalpha-induced actions through S1P receptors, especially S1P(1).