JP4972745B2 - G protein-coupled receptor G2A agonist, and G2A activity modulator screening method - Google Patents

Description

  The present invention relates to G2A agonists containing oxidized fatty acids such as 9-hydroxyoctadecadienoic acid (9-HODE). The present invention also relates to a screening method for G2A activity modulators (agonists, antagonists and inverse agonists) using oxidized fatty acids such as 9-HODE.

  In 1998, one of the G protein-coupled receptors (GPCRs) expressed in lymphocytes was reported, induced by various DNA damage stimuli, and the nature of G2A (G2 accumulation) It was named. At this point, G2A was an orphan receptor (orphan receptor) whose ligand was unknown (Non-patent Document 1). Thereafter, it was reported that the ligands were lysophospholipids (lysophosphatidylcholine (LPC) and sphingosylphosphorylcholine (SPC)), and the relationship with inflammation, immune disease, and arteriosclerosis was pointed out (Non-patent Document 2). However, since the binding experiment of the radioligand could not be reproduced, this paper was subsequently withdrawn (Non-patent Document 3), and the ligand was still unknown.

Regarding the function of G2A, since a deficient (knockout) mouse developed autoimmune disease (Non-patent Document 4; Patent Document 1), involvement in immune function is suggested. In addition, since it is expressed abundantly in arteriosclerotic lesions (Non-patent Document 5), it is suggested that it is involved in arteriosclerosis. In addition, a report (Non-patent Document 6) that it works as a proton sensor (pH sensor) was also made.
Furthermore, G2A is expressed in hematopoietic cells and reports that hematopoietic cell proliferation is controlled (Patent Document 2), G2A is expressed in lymphocytes and is induced to induce expression by growth stimulation or genotoxic stimulation, Reports that various cellular signals are transmitted by affecting the cytoskeleton (Patent Document 3), reports that G2A is highly expressed in human malignant tumors such as prostate cancer, ovarian cancer, lung cancer, breast cancer, and colon cancer (Patent Document 4).
US Patent Publication No. 2002/0051980 WO99 / 25830 pamphlet WO 01/81918 pamphlet International Publication No. 02/090925 Pamphlet Proc. Natl. Acad. Sci. USA, vol 95: p12334-9, 1998 Science, vol293: p702-5, 2001 Science, vol307: p206, 2005 Immunity, vol14: p561-71, 2001 Arterioscler Thromb Vasc Biol, vol22: p2049-53, 2002 J Biol Chem, vol279: p42484-91, 2004

  G protein-coupled receptor (GPCR) is a receptor that exists in the cell membrane and has the role of transmitting external signals into the cell. GPCR activity modulators (agonists, antagonists and inverse agonists) are widely used as therapeutics, and 8 of the top 20 sales of drugs currently used worldwide target GPCRs There is a report. Typical examples include cold medicine, stomach medicine, antiemetics, and psychotropic drugs. Thus, GPCR is important as a target molecule for drug discovery. Genome analysis is almost complete, and many orphan GPCRs with unknown ligands have been found and ligands are being searched for. Under such circumstances, identifying a ligand for G2A, a GPCR with remarkable biological activity, and analyzing its biological significance leads to the development of new diagnostics, therapeutics, and therapeutics. there is a possibility. That is, an object of the present invention is to provide a novel screening method for a novel G2A agonist and a G2A activity modulator.

  The present inventor has intensively studied to solve the above problems. As a result, the inventors discovered that oxidized fatty acids such as 9-HODE are ligands of G2A and act as G2A agonists, and that these oxidized fatty acids can be used to screen for novel G2A activity regulators, thereby completing the present invention. It came to do.

That is, the present invention is as follows.
(1) G2A containing one or more selected from 9-hydroxyoctadecadienoic acid, 9-hydroperoxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid and ricinoleic acid Agonist.
(2) A medicament comprising the G2A agonist of (1).
(3) A food containing the G2A agonist of (1).
(4) Test using one or more compounds selected from 9-hydroxyoctadecadienoic acid, 9-hydroperoxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid and ricinoleic acid A method for screening for a G2A activity modulator, comprising evaluating the G2A binding ability of a compound.
(5) A method for evaluating whether or not a test compound has G2A binding ability, comprising 9-hydroxyoctadecadienoic acid, 9-hydroperoxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, hydroxyeicosa A method comprising evaluating one or more compounds selected from tetraenoic acid and ricinoleic acid as a control compound to evaluate the G2A binding ability of a test compound.
(6) One or more compounds selected from 9-hydroxyoctadecadienoic acid, 9-hydroperoxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid and ricinoleic acid are used as control compounds. A method for screening a G2A activity modulator, comprising evaluating a test compound's G2A activation ability or G2A activation inhibition ability.
(7) A method for evaluating whether a test compound has G2A activation ability or G2A activation inhibition ability, comprising 9-hydroxyoctadecadienoic acid, 9-hydroperoxyoctadecadienoic acid, To evaluate G2A activation ability or G2A activation inhibition ability of a test compound using at least one compound selected from 13-hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid and ricinoleic acid as a control compound A method characterized by.

The figure which shows the increase in intracellular calcium concentration when 9 (S) -HODE is added. Add Fura-2 to CHO-G2A cells (◆), CHO-G2A-Gqi (●), and CHO-Gqi (□), and then add 9 (S) -HODE, and adjust the intracellular calcium concentration using FLEXstation. Measured. Data represent the mean ± standard deviation of 4 independent experiments. Measurements were also made on parental CHO-K1 cells (◯). The figure which shows the ligand specificity of G2A. (A) 1 μM each of oxidized derivatives of linoleic acid or arachidonic acid was added to CHO-Gqi (□) and CHO-G2A-Gqi (■), and the increase in intracellular calcium concentration was measured using a FLEXstation. Data represent the mean ± standard deviation of 4 independent experiments. The structures of 9-HODE and 11-HETE are shown in parentheses. * Indicates p <0.01 (Student ’s t test) compared to CHO-Gqi. (B) CHO-G2A-Gqi cells were treated with 9 (S) -HODE (●) and 9 (R) -HODE (◯) HODE, and the increase in intracellular calcium concentration was measured using a FLEXstation. Data represent the mean ± standard deviation of 4 independent experiments. * Indicates p <0.01 (Student ’s t test) compared to 9 (R) -HODE. The figure which shows the binding of [ ³⁵ S] GTPγS dependent on 9 (S) -HODE to the membrane fraction of G2A-expressing cells. Membrane fractions were prepared from CHO-K1 cells and CHO-G2A stably expressing cells (A), or HEK293 cells (B) transiently expressing G2A and / or Gi, and 20 μM of non-binding buffer in binding buffer. 20 μg membrane fraction was incubated with 0.5 nM [ ³⁵ S] GTPγS and 9 (S) -HODE for 30 minutes at 30 ° C in the presence (non-specific binding) or absence (total binding) of labeled GTPγS . The specific binding amount was calculated by subtracting the non-specific binding amount from the total binding amount. Data represent the mean ± standard deviation of 4 independent experiments. The figure which shows the influence of linoleic acid addition in the CHO-K1 cell in which G2A was expressed. Add Fura-2 to CHO-K1 (○) and CHO-G2A (●) cells, add 1 μM 9 (S) -HODE, 10 μM ricinoleic acid, or 100 μM ATP, and adjust the intracellular calcium concentration to FLEXstation. Measured by. The figure (photograph) which shows the influence on foaming of G2A overexpression in J774 cell or G2A expression suppression. Mouse macrophage-derived J774 cells were seeded on a 24 well plate at 5 × 10 ⁴ and cultured overnight. Lentiviral supernatant containing pLenti6-mG2A or pBlock-iT-mG2A was added to the cells. After 48 hours of incubation, cells were incubated with 50 μg / ml LDL or oxidized LDL (OxLDL) in medium containing 5% fetal calf serum in the presence or absence of 10 μM 9 (S) -HODE. Treated for 24 hours. Subsequently, the cells were fixed with 4% formaldehyde in PBS (−), and lipid droplets accumulated in the cells were stained with Oil-Red O. The figure which shows the expression of G2A in human skin (photograph). (A) shows the result of fluorescent staining using anti-G2A antibody. Sections from shoulder skin were incubated with control rabbit IgG or anti-G2A antibody, then incubated with fluorescently labeled anti-rabbit IgG and observed with a fluorescence microscope. The magnification is 400 times and the bar represents 20 μm. (B) shows the detection results of mRNA in human cultured keratinocytes NHEK. G2A mRNA was detected by PCR with or without RT. The pCXN2.1-G2A vector was used as a positive control. Figure 5 shows intracellular calcium mobilization triggered by 9 (S) -HODE in NHEK cells. Fura-2 / AM was added to the cells, stimulated with 9 (S) -HODE, and analyzed using an RF5300PC spectrofluorometer. The figure which shows the secretion of the cytokine induced by 9 (S) -HODE in NHEK cells. After incubating NHEK cells with 9 (S) -HODE for 0-24 hours, the culture supernatant was collected, and the concentration of cytokine was measured using Bio-Plex ELISA system. (A) IL-6; (B) IL-8; (C) GM-CSF. Data are mean ± SD (n = 3), * indicates p <0.05 (Student ’s t-test) relative to control.

The present invention is described in detail below.
In the present invention, G2A is a G protein-coupled receptor (GPCR) that is mainly expressed in lymphocytes and macrophages, and is reported to be induced by various DNA damage stimuli and stop the cell cycle in the G2 phase. Receptor. The amino acid sequences of human and mouse G2A are shown in SEQ ID NOs: 2 and 4, respectively, and the nucleotide sequences of the genes encoding them are shown in SEQ ID NOs: 1 and 3, respectively. Since the amino acid sequence of G2A varies depending on the species and the like, G2A used in the evaluation of the G2A agonist of the present invention and the screening method of the present invention is not limited to the above sequence, and 9-HODE, 13 As long as it has the property of increasing intracellular calcium concentration in response to ligands such as HODE, HETE, and ricinoleic acid, an amino acid in which one or several amino acids are substituted, deleted, inserted or added in the above sequence It may be a protein having a sequence. Here, the number is preferably 2 to 50, more preferably 2 to 20, and particularly preferably 2 to 10.
G2A activity modulators include G2A agonists, G2A antagonists and G2A inverse agonists. An inverse agonist refers to a substance that attenuates constitutive receptor activity that is not affected by endogenous ligands or drugs. For example, if a G2A mutation always activates the receptor signal without stimulating the endogenous ligand, causing abnormal biological function, an inverse agonist that suppresses the activity of the mutant receptor is beneficial for these diseases. Can be expected.

The G2A agonist of the present invention includes 9-hydroxyoctadecadienoic acid (9-HODE), 9-hydroperoxyoctadecadienoic acid (9-HPODE), 13-hydroxyoctadecadienoic acid (13-HODE), hydroxyeico. It contains 1 or 2 or more oxidized fatty acids selected from the group consisting of satetraenoic acid (HETE) (particularly, HETE having a hydroxyl group at any of positions 5, 8, 9, 11, 12, 15), and ricinoleic acid. These oxidized fatty acids are sometimes called G2A ligands.
9-HODE is octadecadienoic acid in which the 9th carbon is hydroxylated (structure shown in FIG. 2), and 13-HODE is octadecadienoic acid in which the 13th carbon is hydroxylated. . 9-HOODE is octadecadienoic acid in which the 9-position carbon is peroxidized and hydroxylated.
HETE is eicosatetraenoic acid in which carbons such as positions 5, 8, 9, 11, 12, 15 are hydroxylated. The structure of 11-HETE is shown in FIG.
Ricinoleic acid is an oxidized fatty acid having the structure of the following formula (I), is contained in a large amount in plant-derived castor oil, and is released from the glycerol skeleton of castor oil by various lipases in animals.
The oxidized fatty acids such as 9-HODE, 13-HODE and HETE have optical isomers of (S) and (R), but the oxidized fatty acid contained in the G2A agonist of the present invention is ( S) body, (R) body, or a mixture of both.

Oxidized fatty acids such as 9-HODE bind to G2A and activate G2A to transmit signals such as an increase in intracellular calcium concentration. As a result, it exhibits various medicinal effects.
For example, the following medicinal effects are mentioned.
G2A is induced by various growth stimuli and DNA damage stimuli, and is known to stop the cell cycle at the G2 phase. In addition, G2A is expressed in hematopoietic cells and reported to regulate the growth of hematopoietic cells (WO 99/25830). It is expressed in lymphocytes and is induced by proliferation and genotoxic stimuli. Report that various cell signals are transmitted by affecting the skeleton (WO 01/81918), it is expressed abundantly in human malignant tumors such as prostate cancer, ovarian cancer, lung cancer, breast cancer, colon cancer, skin cancer There are reports (WO 02/090925). From these facts, the G2A agonist of the present invention can be used as an anticancer agent for various cancers such as lymphoma and leukemia.

  In addition, G2A-deficient (knockout) mice developed autoimmune disease (Immunity, 14: 561-71, 2001; US 2002/0051980), which strongly suggests that G2A is involved in immune functions. Furthermore, as shown in the Examples of the present application, it was revealed that G2A ligand induces the production of inflammatory cytokines in peripheral tissues such as skin. From these facts, it can be easily understood that G2A activity modulators are used as therapeutic agents for immune diseases and inflammatory diseases. Examples of immune diseases include rheumatoid arthritis and other autoimmune diseases, and examples of inflammatory diseases include psoriasis, sun dermatitis (sunburn), inflammatory bowel disease, and hepatitis. Since the liver is a central organ of lipid metabolism, it is exposed to stress caused by oxidized fat in the state of intake of oxidized fat from the diet, viral hepatitis, fatty liver, drug metabolism disorder, hyperlipidemia and the like. Therefore, G2A activity modulators are used as therapeutic agents for liver diseases such as viral hepatitis, drug-induced hepatitis, alcoholic hepatitis, fatty liver, cirrhosis. In addition, the G2A agonist of the present invention is also useful as an immunosuppressant used for bone marrow transplantation.

  In addition, G2A is highly expressed in arteriosclerotic lesions (Arterioscler Thromb Vasc Biol, 22: 2049-53, 2002), which strongly suggests that G2A is involved in arteriosclerosis. Actually, as shown in the examples below, when the G2A gene was forcibly expressed in J774 cells, which are macrophage cells, the effect of accumulating G2A activity was further increased. It is considered useful as a therapeutic agent for adult diseases (metabolic syndrome) such as obesity drugs and antidiabetic drugs.

  G2A agonists containing oxidized fatty acids such as 9-HODE, and G2A activity modulators obtained by screening methods described below (hereinafter sometimes collectively referred to as G2A activity modulators) are acceptable as is or pharmaceutically. In combination with such a pharmaceutical carrier, it can be used as a medicament for treating or preventing the above-mentioned diseases. The oxidized fatty acid such as 9-HODE can be converted into a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include salts of metals such as sodium and potassium.

  The pharmaceutical preparation form of the pharmaceutical of the present invention is not particularly limited and can be appropriately selected depending on the purpose of treatment. Specifically, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, syrups Suppositories, injections, ointments, patches, eye drops, nasal drops and the like. Additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, solvents for injections, etc. that are commonly used in ordinary pharmaceuticals as pharmaceutical carriers Can be used. Moreover, you may use together a G2A activity regulator and another pharmaceutical.

The G2A activity regulator of the present invention can also be contained in food. Although the specific form of a foodstuff is not restrict | limited in particular, Edible oil, a seasoning, processed food, etc. are illustrated. The food can be produced by mixing a G2A activity regulator with a raw material usually used for food.
The amount of the G2A agonist contained in the food of the present invention is not particularly limited and may be appropriately selected. For example, the amount of the G2A activity regulator is 0.1 to 50% by mass, preferably 1 in the food. It is good to set it as -10 mass%.
In addition, the food of the present invention can be a health food or a food for specified health that has a preventive or therapeutic effect on the above-mentioned diseases. The food of the present invention can be sold with a label such as “a food containing an ingredient having an effect such as an anti-cancer action, an anti-inflammatory action, an immunosuppressive action, an anti-arteriosclerosis, or a liver protecting action”.

<2> Screening Method The screening method of the present invention is 1 selected from 9-hydroxyoctadecadienoic acid, 9-hydroperoxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid and ricinoleic acid. It is a screening method for a G2A activity modulator, characterized by using species or two or more species.

Examples of screening systems using G2A include systems for screening for compounds that bind to G2A and systems for screening for compounds that promote or inhibit G2A activation. For the former, 9-HODE, which can bind to G2A protein, is labeled with a radioisotope as a control compound, and the labeled control compound and G2A protein are mixed in advance, and a test compound is added thereto. A method of selecting a compound that can bind to the G2A protein by competing with the control compound from the test compounds can be mentioned. In this method, for example, a test compound is added to and reacted with a solution containing a G2A protein and a labeled control compound bound thereto, and the dissociated control compound and free test substance are removed by washing, and then remain in the G2A protein. Whether or not the test substance binds to the G2A protein can be determined by measuring the radiation dose to be measured. That is, when the labeled control compound dissociates from the G2A protein due to competition with the test substance and the radiation dose decreases, it can be determined that the test compound binds to the G2A protein. As a control compound, 9-HODE, 13-HODE, ricinoleic acid, HETE, and the like, which have been shown to bind to the G2A protein in the present invention, are used.
The compounds that bind to G2A obtained by screening are thought to include agonists, antagonists, and even inverse agonists of G2A, so investigate whether to increase intracellular calcium concentration as described below It is possible to determine which of these is true.

  The G2A protein used in this screening system may be a protein produced by gene recombination or may be purified from cells or the like. Further, it may be chemically synthesized. Further, it may be a partial protein containing a ligand binding site of G2A protein. When these proteins are produced by gene recombination, for example, DNA having the nucleotide sequence of SEQ ID NO: 1 or 3 is linked to an appropriate vector and introduced into a host such as E. coli or animal cells to express the protein. And purifying according to a conventional method. The DNA used in this case is stringent with the DNA having the nucleotide sequence of SEQ ID NO: 1 or 3 as long as it encodes a protein capable of increasing intracellular calcium concentration in response to a ligand such as 9-HODE. DNA that hybridizes under various conditions may be used. Here, as stringent conditions, for example, at a salt concentration corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably once. Conditions for washing 2 to 3 times are mentioned. Examples of vectors for genetic recombination in E. coli include pET vectors (Novagen) and pGEX vectors (Amersham Pharmacia). Vectors for genetic recombination in animal cells include pcDNA vectors (Invitrogen). ) And the like.

In addition, a compound that activates G2A or a compound that inhibits activation of G2A in a cell line may be screened. Activation of G2A refers to the function of G2A in vivo, such as increasing intracellular calcium concentration in response to a ligand. Further, the intracellular cAMP concentration may be used as an index. For example, G2A protein can be expressed in cells such as CHO and HEK293 by introducing a gene encoding G2A, and the change in intracellular calcium concentration when a test substance is added to the cells can be examined. . Using a G2A ligand such as 9-HODE as a positive control, screening for a substance (G2A agonist) that activates G2A to the same extent or higher than these ligands may be performed, or the G2A ligand and the test substance may be screened simultaneously. In addition, a substance that inhibits G2A activation by a G2A ligand (G2A antagonist) may be screened.
The calcium concentration can be measured by a conventional method, and can be measured using, for example, Fura-2 (Dojindo). Moreover, cAMP density | concentration can be measured using AlphaScreen cAMP assay kit (PerkinElmer), for example.

In the screening method of the present invention, the compound to be screened is not particularly limited, and may be, for example, a low-molecular synthetic compound or a compound contained in a natural product. Moreover, a peptide may be sufficient. Although individual test substances may be used for screening, a compound library containing these substances may be used.
The above method should be used not only for screening for G2A agonists or antagonists from multiple compounds, but also for evaluating the G2A binding ability, G2A activation ability, and G2A activation inhibition ability of individual compounds. Can do.

The G2A agonist obtained by the screening method of the present invention can be used as a therapeutic or prophylactic agent for cancer, immune disease, inflammatory disease, arteriosclerosis, liver disease and the like as described above.
On the other hand, the G2A antagonist obtained by the screening method of the present invention can be used as a therapeutic agent or a prophylactic agent for many diseases and conditions associated with oxidative stress. That is, skin epithelial cells express G2A, but in response to oxidized fatty acids such as 9-HODE, cell growth stops and various cytokines are released. The skin is in contact with the outside air and is constantly exposed to constant oxidative stress. In particular, the oxidative stress is increased by sunburn (exposure to ultraviolet rays). At this time, it is considered that oxidized fatty acids are produced in the skin.
Therefore, G2A antagonists are considered useful as therapeutic agents for diseases associated with oxidative stress such as various inflammations, tumors, arteriosclerosis, smoking, autoimmune diseases, diabetes, and liver diseases. In addition, it can be applied to burn medicines, sun creams and the like.
Furthermore, it has been reported that G2A is induced by radiation (WO 01/81918 pamphlet). Therefore, G2A activity modulators are UV radiation, radiation, particle radiation treatment, radiation therapy and It is also considered useful as an action enhancer for particle beam therapy, a healthy tissue protective agent, and the like.

  The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.

Various HODE, HPODE, HETE and cholesteryl-9-HODE were purchased from Cayman Chemical. Linoleic acid, arachidonic acid, 1-palmitoyl lysophosphatidylcholine, and 1-palmitoyl-2-linoleoylphosphatidylcholine were purchased from Sigma. [ ³⁵ S] GTPγS was purchased from Perkin Elmer.

Synthesis of 9 (S) -HODE
5-lipoxygenase was purified from tubers of potato according to the method described in Proc Natl Acad Sci USA 81, 689-693 (1984). Linoleic acid was oxidized with 5-lipoxygenase in reaction buffer (20 mM Tris-HCl, pH 7.4, 0.1 mM diethylenetriaminepentaacetic acid) and then reduced with NaBH ₄ . The product was separated from unreacted substrate by silica column chromatography. LC-MS analysis confirmed that the product contained more than 95% 9 (S) -HODE.

Plasmid construction
A gene encoding human G2A with a FLAG epitope added to the N-terminus was amplified by Pyrobest (Takara Bio) using the primers of SEQ ID NOs: 5 and 6. The PCR product was digested with BamHI and EoRI and inserted into the mammalian cell expression vector pCXN2.1 (Gene, vol108: p193-200, 1991). The resulting plasmid was named pCXN2.1-G2A.
A plasmid containing the entire ORF of Gqi (in which the C-terminal 9 peptide of mouse Gq protein was replaced with the corresponding peptide of mouse Gi protein) was subcloned into pcDNA3.1 / Zeo vector (Invitrogen) and named pcDNA3.1 / Gqi It was. The C-terminal region of the G protein is an important site for determining the interaction with the G protein-coupled receptor, and this Gqi chimeric protein in which the C-terminal region is replaced with the corresponding region of the Gi protein is a receptor that couples to the Gi protein. It has been reported that it can interact with the body and transmit Gq-like signals (Nature vol363: p274-6, 1993). Therefore, it can be a useful tool for observing a receptor signal coupled to Gi as a calcium signal transmitted by Gq.

Cell culture, genetic gene transfer, flow cytometry Chinese hamster ovary cells (CHO cells) and human embryonic kidney cells 293 cells (HEK293 cells) are each Ham's F-12 medium (Sigma) containing 10% fetal bovine serum. And maintained in a humidified 5% CO ₂ incubator at 37 ° C. in Dulbecco's modified Eagle's medium (Sigma). Each plasmid DNA was transfected into the cells using Lipofectamine 2000 reagent (Invitrogen).
To observe membrane expression of FLAG-tagged G2A protein, add 10 μg / ml M5 anti-FLAG antibody (Sigma) at room temperature in PBS (-) containing 1% BSA without permeabilizing the cells. Incubated for 1 hour. FITC-conjugated anti-mouse IgG was added, incubated at room temperature for 30 minutes, and detected using an EPICS XL flow cytometer system (Beckman Coulter).

Stable expression of G2A gene in CHO cells
PCXN2.1-G2A was transfected into CHO cells using Lipofectamine 2000. Clones resistant to 1 mg / ml Geneticin (Invitrogen) were selected, and the expression level of G2A gene was confirmed by RT-PCR and flow cytometry.
G2A high expression clones (CHO-G2A cells) were further transfected with pcDNA3.1 / Gqi, and stable clones resistant to 1 mg / ml Zeocin (Invitrogen) were selected. The expression of Gqi was confirmed by RT-PCR, and CHO-G2A-Gqi cells were obtained. In addition, CHO cells that stably express Gqi were obtained (CHO-Gqi cells).

Measurement of intracellular calcium concentration
Hepes-Tyrode's-BSA buffer containing 1.25 mM probenecid and 0.02% pluronic F127 (BASF) (25 mM Hepes-NaOH, pH 7.4, 140 mM NaCl, 2.7 mM KCl, 1.0 mM CaCl ₂ , 12 mM NaHCO ₃ , 5.6 mM 5 μM Fura-2 AM (Dojin) was added to CHO cells in D-glucose, 0.37 mM NaH ₂ PO ₄ , 0.49 mM MgCl ₂ , 0.01% defatted BSA) and incubated at 37 ° C. for 1 hour. The cells were washed with Hepes-Tyrode's-BSA buffer, stimulated with the ligand, and changes in intracellular calcium concentration were measured using a scanning fluorometer system (FLEXstation, Molecular Devices) or RF5300PC spectrofluorometer (Shimazu).

GTPγS binding assay
CHO cells were sonicated in a homogenization buffer (20 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 10 mM MgCl ₂ , 1 mM EDTA, and Complete protease inhibitor cocktail (Roche)). The crushed material was centrifuged at 12,000 × g for 10 minutes, and the supernatant was centrifuged at 100,000 × g for 1 hour. The precipitate (membrane fraction) was resuspended in homogenization buffer and the protein concentration was determined by BCA Protein Assay Reagent (Pierce) with BSA as standard. Presence of 20 μM unlabeled GTPγS in 200 μl binding buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl ₂ , 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 5 μM GDP, and 0.1% BSA) In the absence or absence, membrane protein (20 μg) was incubated with 0.5 nM [ ³⁵ S] GTPγS and each concentration of 9 (S) -HODE at 30 ° C. for 30 minutes.
The reaction was terminated by filtration using GF / C glass-fiber filters (Whatman). The filter was carefully washed with PBS (−), dried at 50 ° C., and immersed in Aquasol II scintillation cocktail (Packard). The radioactivity of the filter was measured using LS6500 scintillation system (Beckman).

Cell culture
NHEK cells (Kurabo) were cultured using HuMedia-KB2 (Kurabo, basal medium) supplemented with growth supplements (Kurabo, growth medium).

Immunohistochemical staining Frozen sections of human skin (thickness 6 μm) were blocked with 10% BSA for 30 minutes at room temperature, and diluted with primary antibody diluted to 1.7 μg / ml with 1% BSA / PBS at 4 ° C. Incubate overnight. As the primary antibody, anti-human G2A antibody (Lifespan) or rabbit IgG (Santa-cruz) was used. Subsequently, it was incubated for 1 hour at room temperature using a secondary antibody (goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Molecular Probes)) diluted to 2 μg / ml with 1% BSA / PBS, and fluorescence microscope (Axioskop; Zeiss ) Was used for observation.

RT-PCR
Total RNA was extracted from NHEK cells treated with DNase (Qiagen) using RNeasy Mini kit (Qiagen). RT-PCR uses QIAquick one step RT-PCR kit (Qiagen), sense primer (5'-GGCTTTGCCATCCCTCTC-3 ': SEQ ID NO: 7) and antisense primer (5'-GACAGGCACAGAAACACC-3': SEQ ID NO: 8) went.

Intracellular calcium mobilization by 9 (S) -HODE in NHEK cells
Hepes-Tyrode's-BSA buffer containing 0.02% pluronic F127 (25 mM Hepes-NaOH, pH 7.4; 140 mM NaCl; 2.7 mM KCl; 1.0 mM CaCl ₂ ; 12 mM NaHCO ₃ ; 5.6 mM D-glucose; 0.37 mM NaH ₂ In PO ₄ ; 0.49 mM MgCl ₂ ; and 0.01% fatty acid-free BSA), 2.5 μMFura-2 / AM (Dojin) was added to NHEK cells and incubated at 37 ° C. for 1 hour. The cells were washed with Hepes-Tyrode's-BSA buffer, and the change in intracellular calcium concentration when stimulated with 9 (S) -HODE was measured using RF5300PC spectrofluorometer (Shimazu).

Measurement of cytokine concentration
After NHEK cells were stimulated with 9 (S) -HODE, the culture supernatant was collected and the cytokine concentration was measured using Bio-Plex ELISA system (Bio-Rad).

[Results] Intracellular calcium mobilization induced by 9 (S) -HODE in CHO cells expressing G2A As shown in Fig. 1, in CHO-G2A cells, 9 (S) -HODE is a concentration-dependent cell. Intrinsic calcium mobilization. The concentration of 9 (S) -HODE that gave half-maximal activity was about 2 μM. On the other hand, parental CHO-K1 cells did not react at all with 9 (S) -HODE.
G2A was thought to be a G protein coupled receptor. Therefore, evaluation was performed using cells in which the Gqi chimeric protein in which the C-terminal 9 peptide of the mouse Gq protein was replaced with the corresponding peptide of the mouse Gi protein was coexpressed with G2A. As shown in FIG. 1, a clone expressing both G2A and Gqi (CHO-G2A-Gqi) showed high reactivity to 9 (S) -HODE. On the other hand, CHO cells expressing only Gqi (CHO-Gqi) did not react with 9 (S) -HODE.

Ligand specificity of G2A 1 μM of various oxidized derivatives of linoleic acid and arachidonic acid were added, and the intracellular calcium concentration was measured (FIG. 2A).
As a result, among these, 9 (S) -HODE and 11-HETE showed the strongest intracellular calcium mobilization activity in CHO-G2A-Gqi cells. The structures of these two types of lipids are shown in parentheses in FIG. 2A. 9 (S) -Hydroperoxyoctadecadienoic acid (9 (S) -HPODE) showed activity similar to 9 (S) -HODE, but the activities of 13 (S) -HODE and 13 (S) -HPODE Was weak. These results suggest that the length of the carbon chain from the ω position and the position of the hydroxyl or hydroperoxy group are important for ligand recognition of G2A. On the other hand, 9 (S) -HODE cholesterol ester and 1-palmitoyl lysophosphatidylcholine showed little activity even at 10 μM. Also, it did not react with lysophosphatidylcholine, which was previously reported as a ligand.
Next, the optical specificity of the ligand was examined (FIG. 2B). As a result, 9 (R) -HODE (Cayman Chemical), which is an optical isomer of 9 (S) -HODE, also showed calcium mobilization activity, although not as much as 9 (S) -HODE. From this, it was found that 9 (R) -HODE is also a ligand.

Induction of GTPγS binding by 9 (S) -HODE
Whether 9 (S) -HODE promotes GDP-GTP exchange of Gα protein was examined using the membrane fraction of cells stably expressing G2A. As shown in FIG. 3A, the membrane fraction of G2A stably expressing cells showed 9 (S) -HODE-dependent [ ³⁵ S] GTPγS binding.
Furthermore, control vectors or pCXN2.1-G2A and pcDNA3.1 / Gi were transiently expressed in HEK293 cells and examined. 24 hours after transfection, membrane fractions were prepared and examined for binding of GTPγS binding. 9 (S) -HODE-dependent [ ³⁵ S] GTPγS binding was not observed in the membrane fraction of cells transfected with G2A alone (data not shown). As shown in FIG. 3B, 9 (S) -HODE-dependent GTPγS binding was observed when Gi was co-expressed with G2A. On the other hand, 9 (S) -HODE-dependent [ ³⁵ S] GTPγS binding was not observed when only Gi was transfected.
These results suggest that 9 (S) -HODE activates Gα protein via G2A.

  Although data are not shown, 9 (S) -HODE-dependent calcium concentration increase reaction and cAMP increase suppression reaction in G2A expressing cells were inhibited by pertussis toxin (PTX), a Gi protein inhibitor, It was considered that the G2A receptor is coupled to the Gi protein. In addition, in CHO cells stably expressing G2A, it was found that JNK was activated in the MAP kinase system by 9-HODE stimulation.

Activation of G2A by ricinoleic acid Ricinoleic acid was added to CHO-G2A cells, and the intracellular calcium concentration was examined (FIG. 4). As a result, ricinoleic acid was found to cause intracellular calcium mobilization in cells expressing G2A to the same extent as 9 (S) -HODE. On the other hand, parental CHO-K1 cells did not react with 9 (S) -HODE (1 μM) or ricinoleic acid (10 μM). ATP is a positive control that causes intracellular calcium mobilization in many cells.

Analysis of the role of G2A in macrophage cells The mouse G2A gene was forcibly expressed or inhibited by RNA interference (RNAi) in J774 cells, which are mouse macrophage cells. Forced expression was performed using a lentiviral expression vector pLenti6 (Invitrogen). On the other hand, expression suppression was performed using the RNAi vector pBlock-iT (Invitrogen). When the expression level of G2AmRNA was confirmed by RT-PCR, it was increased about 4 times by G2A expression vector (pLenti6-mG2A) and decreased to about 1/5 or less by RNAi (pBlock-iT-mG2A). (Data omitted).

When LDL is added to J774 cells, cell foaming (fat droplet storage) occurs (FIG. 5a), and when oxidized LDL (LDL oxidized by copper ion treatment) is added, cell foaming further proceeds (FIG. 5d). . J774 cell foaming is an experimental system often used as a model of vascular wall fat accumulation in arteriosclerosis. J774 cells normally express the G2A receptor. When 9-HODE is allowed to act on this, foaming of LDL becomes remarkable (FIG. 5g). The action of oxidized LDL becomes more prominent by treatment with 9-HODE (FIG. 5j). In contrast, when G2A is forcibly expressed, the effects of LDL and oxidized LDL become significant (FIGS. 5b and 5e). Moreover, when G2A is forcibly expressed, the effect of 9-HODE on the foaming effect of LDL or oxidized LDL becomes more prominent (FIGS. 5h and k).
When expression of G2A was suppressed by RNAi, very intense foaming was observed under any condition (FIGS. 5c, f, i, l). In particular, the effect of oxidized LDL became remarkable (FIGS. 5f and 1).
Although the molecular mechanism of these phenomena is unclear, G2A and its ligand 9-HODE may be deeply involved in the pathogenesis of arteriosclerosis. Furthermore, G2A activity modulators have the potential to be useful as therapeutic agents for arteriosclerosis.
In addition, monitoring the expression of G2A and the production of oxidized fatty acids such as 9-HODE is considered useful for diagnosis of arteriosclerosis and determination of therapeutic effects.

Expression of G2A in human skin The expression of G2A in human skin was examined by immunohistochemical staining (FIG. 6A). G2A was abundant in the epithelium, especially squamous cells and granule layers, but less in the basal layer. In addition, the expression of G2A in cultured human keratinocyte NHEK cells was examined by RT-PCR (FIG. 6B). According to this, G2A expression was also observed in NHEK cells.

Intracellular calcium mobilization by 9 (S) -HODE in NHEK cells As shown in FIG. 7, 9 (S) -HODE also induced intracellular calcium mobilization in NHEK cells expressing endogenous G2A. The reaction with 3 μM 9 (S) -HODE was weak, but the reaction became significant by the addition of 10 μM 9 (S) -HODE. On the other hand, no reaction was induced even when 15 μM 9 (S) -HODE was added continuously. This was probably due to receptor desensitization, since cells still reacted with ATP.

Induction of cytokines by addition of 9 (S) -HODE in NHEK cells
The concentration of cytokine in the supernatant of NHEK cells treated with 9 (S) -HODE was examined using Bio-Plex ELISA system. As a result, 9 (S) -HODE induced IL-6 (FIG. 8A), IL-8 (FIG. 8B), and GM-CSF (FIG. 8C) in a concentration-dependent manner. The induction of IL-6 and IL-8 was significantly increased 4 hours after addition of 9 (S) -HODE, and the increase of GM-CSF was significantly increased 16 hours after addition of 9 (S) -HODE.

The G2A agonist of the present invention can be used as an active ingredient of a medicine or food having a therapeutic effect or a preventive effect on diseases such as cancer, immune diseases, inflammatory diseases, arteriosclerosis, and liver diseases. Moreover, according to the screening method of the present invention, a novel G2A activity modulator can be obtained.

Claims (3)

A method for evaluating whether a test compound has G2A binding ability, comprising 9-hydroxyoctadecadienoic acid, 9-hydroperoxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid And one or more compounds selected from ricinoleic acid as a control compound to evaluate the G2A binding ability of the test compound. Using at least one compound selected from 9-hydroxyoctadecadienoic acid, 9-hydroperoxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid and ricinoleic acid as a control compound, A screening method for a G2A activity modulator, characterized by evaluating the test compound's G2A activation ability or G2A activation inhibition ability. A method for evaluating whether a test compound has G2A activation ability or G2A activation inhibition ability, comprising 9-hydroxyoctadecadienoic acid, 9-hydroperoxyoctadecadienoic acid, 13-hydroxy It is characterized by evaluating the G2A activation ability or G2A activation inhibition ability of a test compound using at least one compound selected from octadecadienoic acid, hydroxyeicosatetraenoic acid and ricinoleic acid as a control compound. how to.