Elevation of Sphingoid Base 1-Phosphate as a Potential Contributor to Hepatotoxicity in Fumonisin $B_1-Exposed$ Mice

Kim, Dong-Hyun,Lee, Youn-Sun,Lee, Yong-Moon,Oh, Sei-Kwan,Yun, Yeo-Pyo,Yoo, Hwan-Soo 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.8

Fumonisins are causative agents of diseases in mice and rats, including liver and renal toxicities, as well as cancer, and are specific inhibitors of ceramide synthase in the metabolism of sphingolipid. The purpose of this study was to determine whether an elevated level of sphingoid base 1-phosphate was related to the expressions of metabolism enzymes in the liver of fumonisin $B_1$ ($FB_1$)-treated mice and acted as a contributing factor to hepatotoxicity. In our previous study, $FB_1$ was confirmed to be toxic to both liver and kidneys, coupled with simultaneous elevation of sphinganine 1-phosphate (Kim et al., 2006). ICR mice were treated intraperitoneally with 10 mg/kg/day $FB_1$ for 5 days, with the concentrations of sphingolipid metabolites in the serum and liver measured using HPLC following Bligh-Dyer extraction. The levels of sphingoid bases and their 1-phosphates in the serum and liver were markedly elevated in response to treatment with $FB_1$. In the liver, $FB_1$ increased the expression of sphingosine kinase and inhibited the expression of sphingosine 1-phosphate lyase. The cleaved form of caspase-3 was detected in the liver of $FB_1$-treated mice, indicating the occurrence of apoptosis in the liver following exposure to FB1. The expressions of proapoptotic signaling molecules, such as phosphorylated forms of c-Jun N-terminus kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), were increased in the liver of $FB_1$-treated mice. In conclusion, these results suggest the elevation of sphingoid base 1-phosphate, as a result of the activation of sphingosine kinase and the inhibition of sphingosine 1-phosphate lyase, may be a major target for $FB_1$-induced hepatotoxicity via the activation of an apoptotic signaling pathway.

Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P2) Attenuates Bleomycin-Induced Pulmonary Fibrosis

박수진,임동순 한국응용약물학회 2019 Biomolecules & Therapeutics(구 응용약물학회지) Vol.27 No.3

Sphingosine 1-phosphate (S1P) levels are often found to be elevated in serum, bronchoalveolar lavage, and lung tissue of idiopathic pulmonary fibrosis patients and experimental mouse models. Although the roles of sphingosine kinase 1 and S1P receptors have been implicated in fibrosis, the underlying mechanism of fibrosis via Sphingosine 1-phosphate receptor 2 (S1P2) has not been fully investigated. Therefore, in this study, the roles of S1P2 in lung inflammation and fibrosis was investigated by means of a bleomycin-induced lung fibrosis model and lung epithelial cells. Bleomycin was found to induce lung inflammation on day 7 and fibrosis on day 28 of treatment. On the 7th day after bleomycin administration, S1P2 deficient mice exhibited significantly less pulmonary inflammation, including cell infiltration and pro-inflammatory cytokine induction, than the wild type mice. On the 28th day after bleomycin treatment, severe inflammation and fibrosis were observed in lung tissues from wild type mice, while lung tissues from S1P2 deficient mice showed less inflammation and fibrosis. Increase in TGF-β1-induced extracellular matrix accumulation and epithelial-mesenchymal transition were inhibited by JTE-013, a S1P2 antagonist, in A549 lung epithelial cells. Taken together, pro-inflammatory and pro-fibrotic functions of S1P2 were elucidated using a bleomycin-induced fibrosis model. Notably, S1P2 was found to mediate epithelial-mesenchymal transition in fibrotic responses. Therefore, the results of this study indicate that S1P2 could be a promising therapeutic target for the treatment of pulmonary fibrosis.

초고성능액체크로마토그래피-텐덤질량분석법에 의한 혈청 중스핑고신과 스핑고신-1-포스페이트의 동시 분석

나승훈(Seonghoon Na),윤혜란(Hye-Ran Yoon) 대한약학회 2021 약학회지 Vol.65 No.1

Sphingosine (SPH) and sphingosine-1-phosphate (S1P) are emerging as key players in asthma metabolism and in numerous cellular inflammation processes. To identify potential biomarkers of asthma and inflammatory therapeutics, it is essential to determine their levels. Herein, we developed a rapid and sensitive UHPLC-MS/MS method to simultaneously quantify SPH and S1P in human serum using C17-SPH and C17-S1P as internal standards. After methanol precipitation of serum proteins, the supernatants were analyzed by MS/MS performed in the positive ion mode by multiple reaction monitoring. UHPLC analysis (C18 column) was performed using two mobile phase systems (water containing 0.1% formic acid, and 85% acetonitrile containing 0.1% formic acid) within 5 min of the short run. The calibration curves were linear in the range of 0.002-1.5 µg/mL for S1P and SPH with an R 2 greater than 0.9999. The LOD and LOQ were 0.0002 and 0.0004 µg/mL for S1P, and 0.0005 and 0.001 µg/mL for SPH, respectively. The accuracy and precision of the method were in the range of 89.8-100.7% (RSD, 1.5-2.8%) for both SPH and S1P species. We were able to quantify both molecules in serum from healthy and asthmatic patients. These results suggest that SPH and S1P are promising potential biomarkers, and also contribute to the basic data for the construction of an omics-based platform for preventive index prior to asthma diagnosis.

Effects of Sphingosine-1-phosphate on Vestibular Nuclear Neurons

Jae-Hyuk Lee,Sujeong Jang,Song-Hee Kim,Han-Seong Jeong,Jong-Seong Park(박종성) 대한의생명과학회 2010 Biomedical Science Letters Vol.16 No.1

This study was designed to investigate the effects of sphingosine-1-phosphate on the neuronal activity of rat medial vestibular nuclear neurons. Sprague-Dawley rats aged 14 to 16 days were decapitated under ether anesthesia. After treatment with pronase and thermolysin, the dissociated medial vestibular nuclear neurons were transferred into a chamber on an inverted microscope. Spontaneous action potentials and potassium currents were recorded by standard patch-clamp techniques under current and voltage-clamp modes respectively. 15 medial vestibular nuclear neurons revealed excitatory responses to 1 and 5 μM of sphingosine-1-phosphate. The spike frequency and resting membrane potential of these cells were increased by sphingosine-1-phosphate. The amplitude of afterhyperpolarization was decreased by sphingosine-1-phosphate. Whole potassium currents of medial vestibular nuclear neurons were decreased by sphingosine-1-phosphate (n=12). Sphingosine-1-phosphate did not affect the charybdotoxin-treated potassium currents. These experimental results suggest that sphingosine-1-phosphate increases the neuronal activity of the medial vestibular nuclear neurons by altering the resting membrane potential and afterhyperpolarization.

Elevation of Sphingoid Base 1-Phosphate as a Potential Contributor to Hepatotoxicity in Fumonisin B1-Exposed Mice

Dong-Hyun Kim,이윤선,Yong-Moon Lee,Seikwan Oh,Yeo-Pyo Yun,유환수 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.8

Fumonisins are causative agents of diseases in mice and rats, including liver and renal toxicities, as well as cancer, and are specific inhibitors of ceramide synthase in the metabolism of sphingolipid. The purpose of this study was to determine whether an elevated level of sphingoid base 1-phosphate was related to the expressions of metabolism enzymes in the liver of fumonisin B1 (FB1)-treated mice and acted as a contributing factor to hepatotoxicity. In our previous study, FB1 was confirmed to be toxic to both liver and kidneys, coupled with simultaneous elevation of sphinganine 1-phosphate (Kim et al., 2006). ICR mice were treated intraperitoneally with 10 mg/kg/day FB1 for 5 days, with the concentrations of sphingolipid metabolites in the serum and liver measured using HPLC following Bligh-Dyer extraction. The levels of sphingoid bases and their 1-phosphates in the serum and liver were markedly elevated in response to treatment with FB1. In the liver, FB1 increased the expression of sphingosine kinase and inhibited the expression of sphingosine 1-phosphate lyase. The cleaved form of caspase-3 was detected in the liver of FB1-treated mice, indicating the occurrence of apoptosis in the liver following exposure to FB1. The expressions of proapoptotic signaling molecules, such as phosphorylated forms of c-Jun N-terminus kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), were increased in the liver of FB1-treated mice. In conclusion, these results suggest the elevation of sphingoid base 1-phosphate, as a result of the activation of sphingosine kinase and the inhibition of sphingosine 1-phosphate lyase, may be a major target for FB1-induced hepatotoxicity via the activation of an apoptotic signaling pathway.

Synthesis and Characterization of an Amino-oxy-modified Sphingosine-1-Phosphate Derivative that Can Replace Thiolated-S1P in Competitive ELSIA

박지혜,Yeji Lee,Eunjin Kim,Yong-Tae Kim,홍인석 대한화학회 2020 Bulletin of the Korean Chemical Society Vol.41 No.4

Sphingosine-1-phosphate (S1P) is a signaling molecule that has various roles in vivo. Recently, S1P has emerged as a biomarker in the early diagnosis of osteoporosis and cancer. A competitive enzyme-linked immunosorbent assay (ELISA) assay kit for detecting S1P in blood or in various samples is now commercially available. Competitive ELISA requires an anti-S1P antibody and S1P-coated microtiter plates. The antigenic derivative used here is the thiolated-S1P in which a sulfur hydryl group is introduced at the terminal end of the S1P hydrophobic chain. However, the sulfur hydryl group is easily oxidized, and its solubility in water is considerably low, making it difficult to handle. In this study, amino-oxy-modified S1P derivatives that can replace thiolated-S1P were synthesized. Amino-oxy groups are easy to attach to proteins and other polymers without any linker, and are easy to handle because they are not easily oxidized, and soluble in water. The synthesized S1P derivative was directly immobilized on a microtiter plate, and an ELISA assay was performed which obtained comparable results with the conventional thiolated-S1P reference. It is expected that the anti-S1P antibody can be easily induced by attaching this amino-oxy-modified S1P to reduced carrier proteins.

Sphingosine-1-Phosphate-Induced Migration and Differentiation of Human Mesenchymal Stem Cells to Smooth Muscle Cells

Hae Young Song(송해영),Sang Hun Shin(신상훈),Min Young Kim(김민영),Jae Ho Kim(김재호) 한국생명과학회 2011 생명과학회지 Vol.21 No.2

중간엽 줄기세포의 이동과 분화는 손상된 조직의 재생을 위해 필수적이다. Sphingosine-1-phosphate (S1P)는 세포성장, 생존, 분화, 이동성 등 여러 가지 생명현상에 중요한 역할을 하는 생리활성 지질이다. 본 연구에서는 인체 골수유래 중간엽 줄기세포의 이동과 세포분화에 대한 S1P의 영향을 조사하였다. S1P는 중간엽 줄기세포의 이동을 증가시켰으며 pertussis toxin의 전처리는 S1P에 의한 세포이동을 억제하였다. 본 결과는 S1P에 의한 세포이동과정에 Gi에 연결된 수용체가 관여함을 제시한다. S1P₁과 S1P₃ 수용체에 대한 길항제인 VPC23019의 전처리나 siRNA를 이용한 S1P₁ 수용체의 발현억제는 S1P에 의한 세포 내 칼슘 증가와 중간엽 줄기세포의 이동을 저해하였다. 또한, S1P의 처리는 중간엽 줄기세포에서 평활근세포의 표지유전자인 α-smooth muscle actin (α-SMA)의 발현을 증가시켰으며 VPC23019의 전처리는 S1P에 의한 α-SMA의 발현증가를 저해하였다. S1P는 중간엽 줄기세포에서 p38 mitogen-activated protein kinase (p38 MAPK)의 인산화를 촉진하였으며 p38 MAPK의 저해제인 SB202190의 전처리 또는 p38 MAPK의 dominant negative mutant의 과발현은 S1P에 의한 중간엽 줄기세포의 이동 α-SMA 발현증가를 억제하였다. 본 연구결과는 S1P가 S1P1-p38 MAPK 신호전달기전을 통해 중간엽 줄기세포의 이동과 평활근세포로의 분화를 촉진함으로써 중간엽 줄기세포를 이용한 조직재생에의 활용 가능성을 제시한다. Migration and differentiation of mesenchymal stem cells are crucial for tissue regeneration in response to injury. Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a variety of biological processes, including proliferation, survival, differentiation and motility. In the present study, we determined the role of S1P in migration and differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs). S1P stimulated migration of BMSCs in a dose- and time-dependent manner, and pre-incubation of the cells with pertussis toxin completely abrogated S1P-induced migration, suggesting involvement of Gi-coupled receptors in S1P-induced cell migration. S1P elicited elevation of intracellular concentration of Ca<SUP>2+</SUP> ([Ca<SUP>2+</SUP>]i) and pretreatment with VPC23019, an antagonist of S1P₁/S1P₃, blocked S1P-induced migration and increase of [Ca<SUP>2+</SUP>]i. Small interfering RNA-mediated knockdown of endogenous S1P1 attenuated S1P-induced migration of BMSCs. Furthermore, S1P treatment induced expression of α-smooth muscle actin (α-SMA), a smooth muscle marker, and pretreatment with VPC23019 abrogated S1P-induced α-SMA expression. S1P induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), and pretreatment of cells with SB202190, an inhibitor of p38 MAPK, or adenoviral overexpression of a dominant-negative mutant of the p38 MAPK blocked S1P-induced cell migration and α-SMA expression. Taken together, these results suggest that S1P stimulates migration and smooth muscle differentiation of BMSCs through an S1P1-p38 MAPK-dependent mechanism.

Sphingosine 1-Phosphate Receptor Modulators and Drug Discovery

Park, Soo-Jin,Im, Dong-Soon The Korean Society of Applied Pharmacology 2017 Biomolecules & Therapeutics(구 응용약물학회지) Vol.25 No.1

Initial discovery on sphingosine 1-phosphate (S1P) as an intracellular second messenger was faced unexpectedly with roles of S1P as a first messenger, which subsequently resulted in cloning of its G protein-coupled receptors, $S1P_{1-5}$. The molecular identification of S1P receptors opened up a new avenue for pathophysiological research on this lipid mediator. Cellular and molecular in vitro studies and in vivo studies on gene deficient mice have elucidated cellular signaling pathways and the pathophysiological meanings of S1P receptors. Another unexpected finding that fingolimod (FTY720) modulates S1P receptors accelerated drug discovery in this field. Fingolimod was approved as a first-in-class, orally active drug for relapsing multiple sclerosis in 2010, and its applications in other disease conditions are currently under clinical trials. In addition, more selective S1P receptor modulators with better pharmacokinetic profiles and fewer side effects are under development. Some of them are being clinically tested in the contexts of multiple sclerosis and other autoimmune and inflammatory disorders, such as, psoriasis, Crohn's disease, ulcerative colitis, polymyositis, dermatomyositis, liver failure, renal failure, acute stroke, and transplant rejection. In this review, the authors discuss the state of the art regarding the status of drug discovery efforts targeting S1P receptors and place emphasis on potential clinical applications.

Effect of Sphingosine-1-Phosphate on Intracellular Free Ca²⁺ in Cat Esophageal Smooth Muscle Cells

Lee, Dong Kyu,Min, Young Sil,Yoo, Seong Su,Shim, Hyun Sub,Park, Sun Young,Sohn, Uy Dong The Korean Society of Applied Pharmacology 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.6

A comprehensive collection of proteins senses local changes in intracellular $Ca^{2+}$ concentrations ($[Ca^{2+}]_i$) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular $Ca^{2+}$ concentrations in cat esophageal smooth muscle cells. To measure $[Ca^{2+}]_i$ levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in $[Ca^{2+}]_i$ in the cells. Pretreatment with EGTA, an extracellular $Ca^{2+}$ chelator, decreased the S1P-induced increase in $[Ca^{2+}]_i$, and an L-type $Ca^{2+}$-channel blocker, nimodipine, decreased the effect of S1P. This indicates that $Ca^{2+}$ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an $InsP_3$ receptor blocker, the S1P-evoked increase in $[Ca^{2+}]_i$ was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of $G_i$-protein, suppressed the increase in $[Ca^{2+}]_i$ evoked by S1P. These results suggest that the S1P-induced increase in $[Ca^{2+}]_i$ in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of $Ca^{2+}$ from the $InsP_3$-sensitive $Ca^{2+}$ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular $Ca^{2+}$ via the L type $Ca^{2+}$ channel, which was dependent on activation of the $S1P_4$ receptor coupled to PTX-sensitive $G_i$ protein, via phospholipase C-mediated $Ca^{2+}$ release from the $InsP_3$-sensitive $Ca^{2+}$ pool in cat esophageal smooth muscle cells.

Effect of Sphingosine-1-Phosphate on Intracellular Free Ca²⁺ in Cat Esophageal Smooth Muscle Cells

( Dong Kyu Lee ),( Young Sil Min ),( Seong Su Yoo ),( Hyun Sub Shim ),( Sun Young Park ),( Uy Dong Sohn ) 한국응용약물학회 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.6

A comprehensive collection of proteins senses local changes in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca²⁺ concentrations in cat esophageal smooth muscle cells. To measure [Ca²⁺]_i levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca²⁺]_i in the cells. Pretreatment with EGTA, an extracellular Ca²⁺ chelator, decreased the S1P-induced increase in [Ca²⁺]_i, and an L-type Ca²⁺-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca²⁺ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP₃ receptor blocker, the S1P-evoked increase in [Ca²⁺]_i was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of Gi-protein, suppressed the increase in [Ca²⁺]_i evoked by S1P. These results suggest that the S1P-induced increase in [Ca²⁺]_i in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca²⁺ from the InsP₃-sensitive Ca²⁺ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca²⁺ via the L type Ca²⁺ channel, which was dependent on activation of the S1P₄ receptor coupled to PTX-sensitive G_i protein, via phospholipase Cmediated Ca²⁺ release from the InsP₃-sensitive Ca²⁺ pool in cat esophageal smooth muscle cells.