Identifying lysophosphatidic acid receptor subtype 1 (LPA₁) as a novel factor to modulate microglial activation and their TNF-α production by activating ERK1/2

Kwon, Jin Hyun,Gaire, Bhakta Prasad,Park, Se Jin,Shin, Dong-Yoon,Choi, Ji Woong Elsevier 2018 Biochimica et biophysica acta, Molecular and cell Vol.1863 No.10

<P><B>Abstract</B></P> <P>Microglia regulate immune responses in the brain, and their activation is key to the pathogenesis of diverse neurological diseases. Receptor-mediated lysophosphatidic acid (LPA) signaling has been known to regulate microglial biology, but it is still unclear which receptor subtypes guide the biology, particularly, microglial activation. Here, we investigated the pathogenic aspects of LPA receptor subtype 1 (LPA<SUB>1</SUB>) in microglial activation using a systemic lipopolysaccharide (LPS) administration-induced septic mouse model in vivo and LPS-stimulated rat primary microglia in vitro. LPA<SUB>1</SUB> knockdown in the brain with its specific shRNA lentivirus attenuated the sepsis-induced microglia activation, morphological transformation, and proliferation. LPA<SUB>1</SUB> knockdown also resulted in the downregulation of TNF-α, at both mRNA and protein levels in septic brains, but not IL-1β or IL-6. In rat primary microglia, genetic or pharmacological blockade of LPA<SUB>1</SUB> attenuated gene upregulation and secretion of TNF-α in LPS-stimulated cells. In particular, the latter was associated with the suppressed TNF-α converting enzyme (TACE) activity. We reaffirmed these biological aspects using a BV2 microglial cell line in which LPA<SUB>1</SUB> expression was negligible. LPA<SUB>1</SUB> overexpression in BV2 cells led to significant increments in TNF-α production upon stimulation with LPS, whereas inhibiting LPA<SUB>1</SUB> reversed the production. We further identified ERK1/2, but not p38 MAPK or Akt, as the underlying effector pathway after LPA<SUB>1</SUB> activation in both septic brains and stimulated microglia. The current findings of the novel role of LPA<SUB>1</SUB> in microglial activation along with its mechanistic aspects could be applied to understanding the pathogenesis of diverse neurological diseases that involve microglial activation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> LPA<SUB>1</SUB> signaling triggers microglial activation in LPS-induced septic mouse brain and LPS-stimulated rat primary microglia. </LI> <LI> LPA<SUB>1</SUB> signaling upregulates TNF-α production through the activation of TNF-α converting enzyme. </LI> <LI> ERK1/2 is identified as the underlying effector pathway responsible for the TNF-α production by LPA<SUB>1</SUB> in activated microglia. </LI> </UL> </P>

LPA-induced migration of ovarian cancer cells requires activation of ERM proteins via LPA₁ and LPA₂

Park, Jeongrak,Jang, Jin-Hyeok,Oh, Seojin,Kim, Minhye,Shin, Changhoon,Jeong, Minseok,Heo, Kyun,Park, Jong Bae,Kim, Sang Ryong,Oh, Yong-Seok Elsevier 2018 Cellular signalling Vol.44 No.-

<P><B>Abstract</B></P> <P>Lysophosphatidic acid (LPA) has been implicated in the pathology of human ovarian cancer. This phospholipid elicits a wide range of cancer cell responses, such as proliferation, trans-differentiation, migration, and invasion, via various G-protein-coupled LPA receptors (LPARs). Here, we explored the cellular signaling pathway via which LPA induces migration of ovarian cancer cells. LPA induced robust phosphorylation of ezrin/radixin/moesin (ERM) proteins, which are membrane-cytoskeleton linkers, in the ovarian cancer cell line OVCAR-3. Among the LPAR subtypes expressed in these cells, LPA<SUB>1</SUB> and LPA<SUB>2</SUB>, but not LPA<SUB>3</SUB>, induced phosphorylation of ERM proteins at their C-termini. This phosphorylation was dependent on the Gα<SUB>12/13</SUB>/RhoA pathway, but not on the Gα<SUB>q</SUB>/Ca<SUP>2+</SUP>/PKC or Gα<SUB>s</SUB>/adenylate cyclase/PKA pathway. The activated ERM proteins mediated cytoskeletal reorganization and formation of membrane protrusions in OVCAR-3 cells. Importantly, LPA-induced migration of OVCAR-3 cells was completely abolished not only by gene silencing of <I>LPA</I> <SUB> <I>1</I> </SUB> or <I>LPA</I> <SUB> <I>2</I> </SUB>, but also by overexpression of a dominant negative ezrin mutant (ezrin-T567A). Taken together, this study demonstrates that the LPA<SUB>1</SUB>/LPA<SUB>2</SUB>/ERM pathway mediates LPA-induced migration of ovarian cancer cells. These findings may provide a potential therapeutic target to prevent metastatic progression of ovarian cancer.</P> <P><B>Highlights</B></P> <P> <UL> <LI> LPA induces phosphorylation and activation of ERM proteins in ovarian cancer cells. </LI> <LI> The LPA<SUB>1</SUB>&LPA<SUB>2</SUB>/Gα<SUB>12/13</SUB>/RhoA pathway mediates activation of ERM proteins. </LI> <LI> The LPA<SUB>1</SUB>&LPA<SUB>2</SUB>/ERM pathway mediates LPA-induced migration of ovarian cancer cells. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Gintonin, Newly Identified Compounds from Ginseng, Is Novel Lysophosphatidic Acids-Protein Complexes and Activates G Protein-Coupled Lysophosphatidic Acid Receptors with High Affinity

황성희,나승열,Tae-Joon Shin,최선혜,Hee-Jung Cho,이병환,Mi Kyung Pyo,Jun-Ho Lee,Jiyeon Kang,Hyeon-Joong Kim,Chan-Woo Park,신호철 한국분자세포생물학회 2012 Molecules and cells Vol.33 No.2

Recently, we isolated a subset of glycolipoproteins from Panax ginseng, that we designated gintonin, and demon-strated that it induced [Ca2+]i transients in cells via G-protein-coupled receptor (GPCR) signaling pathway(s). However, active components responsible for Ca2+ mobili-zation and the corresponding receptor(s) were unknown. Active component(s) for [Ca2+]i transients of gintonin were analyzed by liquid chromatography-electrospray ioniza-tion-tandem mass spectrometry and ion-mobility mass spectrometry, respectively. The corresponding receptor(s) were investigated through gene expression assays. We found that gintonin contains LPA C18:2 and other LPAs. Proteomic analysis showed that ginseng major latex-like protein and ribonuclease-like storage proteins are protein components of gintonin. Gintonin induced [Ca2+]i tran-sients in B103 rat neuroblastoma cells transfected with human LPA receptors with high affinity in order of LPA2 > LPA5 > LPA1 > LPA3 > LPA4. The LPA1/LPA3 receptor antagonist Ki16425 blocked gintonin action in cells ex-pressing LPA1 or LPA3. Mutations of binding sites in the LPA3 receptor attenuated gintonin action. Gintonin acted via pertussis toxin (PTX)-sensitive and -insensitive G pro-tein-phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3)- Ca2+ pathways. However, gintonin had no effects on other receptors examined. In human umbilical vein endothelial cells (HUVECs) gintonin stimulated cell proliferation and migration. Gintonin stimulated ERK1/2 phosphorylation. PTX blocked gintonin-mediated migration and ERK1/2 phosphorylation. In PC12 cells gintonin induced morpho-logical changes, which were blocked by Rho kinase inhibi-tor Y-27632. Gintonin contains GPCR ligand LPAs in com-plexes with ginseng proteins and could be useful in the development of drugs targeting LPA receptors.

Reactive Oxygen Species Mediates Lysophosphatidic Acid-induced Migration of SKOV-3 Ovarian Cancer Cells

Eun Kyoung Kim(김은경),Hye Sun Lee(이혜선),Hong Koo Ha(하홍구),Sung Ji Yun(윤성지),Jung Min Ha(하정민),Young Whan Kim(김영환),In Hye Jin(진인혜),Hwa Kyoung Shin(신화경),Sun Sik Bae(배순식) 한국생명과학회 2012 생명과학회지 Vol.22 No.12

세포의 이동은 성장, 면역 작용, 그리고 혈관 신생 등 많은 생리현상에 중요한 역할을 한다. 또한 염증 및 종양세포 침윤 등의 다양한 병리적 현상과도 밀접한 연관이 있다. 본 연구에서는 lysophosphatidic acid (LPA)는 활성산소의 생성을 통해 SKOV-3 난소암세포의 이동을 조절한다는 것을 관찰하였다. 먼저, 난소 암세포인 SKOV-3에서 LPA에 의한 세포의 이동이 강하게 일어남을 확인하였다. LPA에 의한 SKOV-3 세포의 이동은 phosphatidylinositol 3-kinase (PI3K)/Akt 신호전달체계를 저해시키는 약물에 의해서 완벽히 억제됨을 확인하였으나 ERK 신호전달체계를 저해시키는 약물에 의해서는 전혀 영향을 받지 않았다. 그리고 SKOV-3 세포에서 LPA에 의한 활성산소 형성이 시간에 따라 강하게 일어남을 확인하였다. 더욱이 LPA에 의한 활성산소 형성도 PI3K 또는 Akt의 저해제에 의해서 완벽히 억제됨을 확인하였으나 ERK 신호전달을 억제하였을 때는 거의 영향을 받지 않았다. SKOV-3 세포에서 LPA에 의해 생성된 활성산소는 diphenylene idonium (DPI, 10 μM), apocyanin (Apo, 10 μM)과 같은 NADPH oxidase 억제제를 전 처리하였을 때 활성산소가 형성되지 못함을 관찰하였다. 그러나 xanthine oxidase (allopurinol, Allo, 10 μM), cyclooxygenase (indomethacin, Indo, 10 μM), 또는 mitochondrial respiratory chain complex I (rotenone, Rot, 10 μM)를 억제하였을 때는 LPA에 의한 활성산소 형성에 영향을 주지 못함을 확인하였다. 마지막으로 활성산소 억제제인 N-acetylcysteine (NAC, 10 μM)에 의해서 LPA에 의한 암세포의 이동이 억제됨을 관찰하였다. 이와 더불어 LPA에 의한 SKOV-3 세포의 이동도 NADPH oxidase 억제에 의해 저해가 됨을 확인하였다. 이러한 연구결과로 보아 LPA에 의한 활성산소의 형성에는 3K/Akt/NADPH oxidase 신호전달체계가 중추적인 역할을 하며 이를 통해 암세포의 이동을 조절한다는 것을 알 수 있었다. Cell motility plays an essential role in many physiological responses, such as development, immune reaction, and angiogenesis. In the present study, we showed that lysophosphatidic acid (LPA) modulates cancer cell migration by regulation of generation of reactive oxygen species (ROS). Stimulation of SKOV-3 ovarian cancer cells with LPA strongly promoted migration. but this migration was completely blocked by pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Inhibition of the ERK pathway had no effect on migration. Stimulation of SKOV-3 ovarian cancer cells with LPA significantly induced the generation of ROS in a time-dependent manner. LPA-induced generation of ROS was significantly blocked by pharmacological inhibition of PI3K or Akt, but inhibition of the ERK signaling pathway had little effect. LPA-induced generation of ROS was blocked by pretreatment of SKOV-3 ovarian cancer cells with an NADPH oxidase inhibitor, whereas inhibition of xanthine oxidase, cyclooxygenase, or mitochondrial respiratory chain complex I had no effect. Scavenging of ROS by N-acetylcysteine completely blocked LPA-induced migration of SKOV-3 ovarian cancer cells. Inhibition of NADPH oxidase blocked LPA-induced migration whereas inhibition of xanthine oxidase, cyclooxygenase, or mitochondrial respiratory chain complex I did not affect LPA-induced migration of SKOV-3 ovarian cancer cells. Given these results, we suggest that LPA induces ROS generation through the PI3K/Akt/NADPH oxidase signaling axis, thereby regulating cancer cell migration.

Activation of Lysophosphatidic Acid Receptor Is Coupled to Enhancement of $Ca^{2+}$ -Activated Potassium Channel Currents

Choi, Sun-Hye,Lee, Byung-Hwan,Kim, Hyeon-Joong,Hwang, Sung-Hee,Lee, Sang-Mok,Nah, Seung-Yeol The Korean Society of Pharmacology 2013 The Korean Journal of Physiology & Pharmacology Vol.17 No.3

The calcium-activated $K^+$ ($BK_{Ca}$) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. $Ca^{2+}$ is the main regulator of $BK_{Ca}$ channel activation. The $BK_{Ca}$ channel contains two high affinity $Ca^{2+}$ binding sites, namely, regulators of $K^+$ conductance, RCK1 and the $Ca^{2+}$ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular $Ca^{2+}$ levels through diverse G proteins such as $G{\alpha}_{q/11}$, $G{\alpha}_i$, $G{\alpha}_{12/13}$, and $G{\alpha}s$ and the related signal transduction pathway. In the present study, we examined LPA effects on $BK_{Ca}$ channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated $BK_{Ca}$ channel activation was also attenuated by the PLC inhibitor U-73122, $IP_3$ inhibitor 2-APB, $Ca^{2+}$ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated $BK_{Ca}$ channel activation. The present study indicates that LPA-mediated activation of the $BK_{Ca}$ channel is achieved through the PLC, $IP_3$, $Ca^{2+}$, and PKC pathway and that LPA-mediated activation of the $BK_{Ca}$ channel could be one of the biological effects of LPA in the nervous and vascular systems.

Activation of Lysophosphatidic Acid Receptor Is Coupled to Enhancement of Ca2+-Activated Potassium Channel Currents

최선혜,이병환,황성희,이상목,나승열,김현중 대한약리학회 2013 The Korean Journal of Physiology & Pharmacology Vol.17 No.3

The calcium-activated K+ (BKCa) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. Ca2+ is the main regulator of BKCa channel activation. The BKCa channel contains two high affinity Ca2+ binding sites, namely, regulators of K+ conductance,RCK1 and the Ca2+ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G proteincoupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular Ca2+ levels through diverse G proteins such as Gαq/11, Gαi, Gα12/13, and Gαs and the related signal transduction pathway. In the present study, we examined LPA effects on BKCa channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated BKCa channel activation was also attenuated by the PLC inhibitor U-73122, IP3 inhibitor 2-APB, Ca2+ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated BKCa channel activation. The present study indicates that LPA-mediated activation of the BKCa channel is achieved through the PLC, IP3, Ca2+, and PKC pathway and that LPA-mediated activation of the BKCa channel could be one of the biological effects of LPA in the nervous and vascular systems.

Activation of Lysophosphatidic Acid Receptor Is Coupled to Enhancement of Ca<SUP>2+</SUP>-Activated Potassium Channel Currents

Sun-Hye Choi,Byung-Hwan Lee,Hyeon-Joong Kim,Sung-Hee Hwang,Sang-Mok Lee,Seung-Yeol Nah 대한생리학회-대한약리학회 2013 The Korean Journal of Physiology & Pharmacology Vol.17 No.3

The calcium-activated K⁺ (BK_Ca) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. Ca²⁺ is the main regulator of BK_Ca channel activation. The BK_Cachannel contains two high affinity Ca²⁺ binding sites, namely, regulators of K⁺ conductance, RCK1 and the Ca²⁺ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein- coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular Ca²⁺ levels through diverse G proteins such as GՁ_q/11, GՁ_i, GՁ_12/13, and GՁs and the related signal transduction pathway. In the present study, we examined LPA effects on BK_Cachannel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated BK_Ca channel activation was also attenuated by the PLC inhibitor U-73122, IP₃inhibitor 2-APB, Ca²⁺ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated BK_Ca channel activation. The present study indicates that LPA-mediated activation of the BK_Ca channel is achieved through the PLC, IP₃, Ca²⁺, and PKC pathway and that LPA-mediated activation of the BK_Ca channel could be one of the biological effects of LPA in the nervous and vascular systems.

Action and Signaling of Lysophosphatidylethanolamine in MDA-MB-231 Breast Cancer Cells

( Dong Soon Im ),( Soo Jin Park ),( Kyoung Pil Lee ) 한국응용약물학회 2014 Biomolecules & Therapeutics(구 응용약물학회지) Vol.22 No.2

Previously, we reported that lysophosphatidylethanolamine (LPE), a lyso-type metabolite of phosphatidylethanolamine, can increaseintracellular Ca2+ ([Ca2+]i) via type 1 lysophosphatidic acid (LPA) receptor (LPA1) and CD97, an adhesion G-protein-coupledreceptor (GPCR), in MDA-MB-231 breast cancer cells. Furthermore, LPE signaling was suggested as like LPA1/CD97-Gi/oproteins-phospholipase C-IP3-Ca2+ increase in these cells. In the present study, we further investigated actions of LPE not onlyin the [Ca2+]i increasing effect but also in cell proliferation and migration in MDA-MB-231 breast cancer cells. We utilized chemicallydifferent LPEs and a specific inhibitor of LPA1, AM-095 in comparison with responses in SK-OV3 ovarian cancer cells. It wasfound that LPE-induced Ca2+ response in MDA-MB-231 cells was evoked in a different manner to that in SK-OV3 cells in terms ofstructural requirements. AM-095 inhibited LPE-induced Ca2+ response and cell proliferation in MDA-MB-231 cells, but not in SKOV3cells, supporting LPA1 involvement only in MDA-MB-231 cells. LPA had significant effects on cell proliferation and migrationin MDA-MB-231 cells, whereas LPE had less or no significant effect. However, LPE modulations of MAPKs (ERK1/2, JNK and p38MAPK) was not different to those by LPA in the cells. These data support the involvement of LPA1 in LPE-induced Ca2+ responseand cell proliferation in breast MDA-MB-231 cells but unknown GPCRs (not LPA1) in LPE-induced responses in SK-OV3 cells. Furthermore, although LPE and LPA utilized LPA1, LPA utilized more signaling cascades than LPE, resulting in stronger responsesby LPA in proliferation and migration than LPE in MDA-MB-231 cells.

Effects of Gintonin-enriched fraction on the gene expression of six lysophosphatidic receptor subtypes

Rami Lee,Byung-Hwan Lee,Sun-Hye Choi,Yeon-Jin Cho,Han-Sung Cho,Hyoung-Chun Kim,Hyewhon Rhim,Ik-Hyun Cho,Man Hee Rhee,Seung-Yeol Nah 고려인삼학회 2021 Journal of Ginseng Research Vol.45 No.5

Background: Gintonin, isolated from ginseng, acts as a ginseng-derived lysophosphatidic acid (LPA) receptor ligand and elicits the [Ca<SUP>2+</SUP>]i transient through six LPA receptor subtypes (LPARSs). However, the long-term effects of gintonin-enriched fraction (GEF) on the gene expression of six LPARSs remain unknown. We examined changes in the gene expression of six LPA receptors in the mouse whole brain, heart, lungs, liver, kidneys, spleen, small intestine, colon, and testis after long-term oral GEF administration. Methods: C57BL/6 mice were divided into two groups: control vehicle and GEF (100 mg/kg, p.o.). After 21-day saline or GEF treatment, total RNA was extracted from nine mouse organs. Quantitative-real-time PCR (qRT-PCR) and western blot were performed to quantify changes in the gene and protein expression of the six LPARSs, respectively. Results: qRT-PCR analysis before GEF treatment revealed that the LPA6 RS was predominant in all organs except the small intestine. The LPA2 RS was most abundant in the small intestine. Long-term GEF administration differentially regulated the six LPARSs. Upon GEF treatment, the LPA6 RS significantly increased in the liver, small intestine, colon, and testis but decreased in the whole brain, heart, lungs, and kidneys. Western blot analysis of the LPA6 RS confirmed the differential effects of GEF on LPA6 receptor protein levels in the whole brain, liver, small intestine, and testis. Conclusion: The LPA6 receptor was predominantly expressed in all nine organs examined; long-term oral GEF administration differentially regulated LPA3, LPA4, and LPA6 receptors in the whole brain, heart, lungs, liver, kidneys, small intestine, and testis.

Signaling Pathway of Lysophosphatidic Acid-Induced Contraction in Feline Esophageal Smooth Muscle Cells

남윤성,서정숙,송현주,손의동 대한약리학회 2013 The Korean Journal of Physiology & Pharmacology Vol.17 No.2

Lysolipids such as LPA, S1P and SPC have diverse biological activities including cell proliferation,differentiation, and migration. We investigated signaling pathways of LPA-induced contraction in feline esophageal smooth muscle cells. We used freshly isolated smooth muscle cells and permeabilized cells from cat esophagus to measure the length of cells. Maximal contraction occurred at 10−6 M and the response peaked at 30s. To identify LPA receptor subtypes in cells, western blot analysis was performed with antibodies to LPA receptor subtypes. LPA1 and LPA3 receptor were detected at 50 kDa and 44kDa. LPA-induced contraction was almost completely blocked by LPA receptor (1/3) antagonist KI16425. Pertussis toxin (PTX) inhibited the contraction induced by LPA, suggesting that the contraction is mediated by a PTX-sensitive G protein. Phospholipase C (PLC) inhibitors U73122 and neomycin, and protein kinase C (PKC) inhibitor GF109203X also reduced the contraction. The PKC-mediated contraction may be isozyme-specific since only PKCε antibody inhibited the contraction. MEK inhibitor PD98059 and JNK inhibitor SP600125 blocked the contraction. However, there is no synergistic effect of PKC and MAPK on the LPA-induced contraction. In addition, RhoA inhibitor C3exoenzyme and ROCK inhibitor Y27632 significantly, but not completely, reduced the contraction. The present study demonstrated that LPA-induced contraction seems to be mediated by LPA receptors (1/3),coupled to PTX-sensitive G protein, resulting in activation of PLC, PKC-ε pathway, which subsequently mediates activation of ERK and JNK. The data also suggest that RhoA/ROCK are involved in the LPA-induced contraction.