JP2006158339A - betaKlotho GENE, Cyp7al GENE, AND THEIR USE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain substances that promote the metabolism of cholesterol using a gene through elucidating the function of the gene involved in cholesterol metabolism. <P>SOLUTION: It has been found that βKlotho protein inhibits the expression of a gene encoding cholesterol 7α-hydroxylase and the cholesterol 7α-hydroxylase promotes synthesizing bile acid from cholesterol. The substances inhibiting the expression of a gene encoding the βKlotho protein and promoting the expression of the gene encoding the cholesterol 7α-hydroxylase, respectively, promote the metabolism of cholesterol respectively, being useful for preventing or treating arteriosclerosis, obesity and gallstone formation, etc. Antibodies to the βKlotho protein and the gene encoding the cholesterol 7α-hydroxylase are also usable per se for promoting cholesterol metabolism. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to βKlotho that regulates the synthesis of bile acids from cholesterol, cholesterol 7α hydroxylase (Cyp7a1), genes encoding these proteins, and a method for screening substances that promote cholesterol metabolism using these genes, The present invention relates to antibodies against these proteins and cholesterol metabolism promoters.

  Bile acids are synthesized from cholesterol in the liver. Bile acids play an important role in the absorption of fat from the small intestine and have the property of dissolving in fat. Furthermore, bile acid biosynthesis is essentially the only route to remove excess cholesterol.

  On the other hand, hydrophobic secondary bile acids produced by intestinal bacteria are toxic. Therefore, it is important for animals to have precise control of bile acids.

  The rate-limiting step of bile acid synthesis is catalyzed by cholesterol 7α hydroxylase (CYP7A1) and is strictly controlled by a number of factors. A representative example is negative feedback control using bile acids. Several nuclear receptor-mediated pathways have been shown to accompany this regulation (Endocr Rev 23, 443-463 (2002); J. Lipid Res 43, 533-543 (2002)). Bile acids are secreted from the liver, then reabsorbed from the small intestine and transported back to the liver. Bile membrane permeation is performed by several carrier proteins and ABC transporters.

  In order to properly control bile acid metabolism, such enterohepatic circulation must be completely performed.

  Here, the βKlotho gene is a gene expressed in liver cells. The mouse βKlotho gene encodes a protein similar in structure to the Klotho protein (Mech Dev 98, 115-119 (2000)). Both Klotho and βKlotho proteins are type I membrane proteins and have two glycosidase-like domains. In these proteins, glutamic acid residues are essential for activity expression, and it is considered that these two proteins constitute a subfamily in glycosidase family 1.

  βKlotho protein is expressed in liver, pancreas and adipose tissue (Mech Dev 98, 115-119 (2000)), but its molecular function is unknown.

    Thus, although the pathway involved in the synthesis of cholesterol into bile acids has not been elucidated, if a gene involved in cholesterol metabolism can be identified, it can be used to improve lifestyle-related diseases such as arteriosclerosis and obesity. It is useful for the development of drugs.

  An object of the present invention is to elucidate the function of a gene involved in cholesterol metabolism and to obtain a substance that promotes cholesterol metabolism using this gene.

  In order to achieve the above object, the present inventors have produced a knockout mouse of βKlotho gene, in which the serum cholesterol level is slightly reduced, and the amount of bile acid excreted per unit weight of stool is remarkably increased. It was found that the body weight decreased, the serum cholesterol level remained in the normal range even when excessive cholesterol was consumed, and that gallstones (cholesterol stones) were not generated in the gallbladder even when excessive cholesterol was consumed.

    We also found that the expression of Cyp7a1 gene was increased in knockout mice of βKlotho gene.

  The present invention has been completed based on the above findings, and provides the following genes and the like.

1. The following polynucleotide (1) or (2):
(1) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.
(2) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions and has promoter activity.

2. The following polynucleotide (3) or (4):
(3) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 2.
(4) A polynucleotide that encodes a polypeptide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and has an activity of inhibiting the expression of cholesterol 7α-hydroxylase.

    3. A vector comprising the polynucleotide according to item 1 and a structural gene linked so as to be expressed thereby.

    4). Item 4. The vector according to Item 3, wherein the structural gene is a reporter gene.

    5. Item 4. The vector according to Item 3, wherein the structural gene is the polynucleotide according to Item 2.

    6). Item 6. A test cell carrying the vector according to any one of items 3 to 5.

7). Contacting the test cell according to Item 6 with a test substance;
Comparing the expression level of the structural gene in the test cell with the expression level of the structural gene in the test cell not in contact with the test substance, and selecting a substance that inhibits the expression of the structural gene. Method.

8). The following polypeptide (5) or (6).
(5) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3.
(6) A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 3 and having an activity of inhibiting the expression of cholesterol 7α-hydroxylase.

    9. Item 9. An antibody that specifically recognizes the polypeptide of Item 8.

    10. Item 10. A cholesterol metabolism promoter comprising the antibody according to Item 9.

    11. An agent for preventing or treating arteriosclerosis, comprising the antibody according to Item 9.

    12 Item 10. A prophylactic or therapeutic agent for obesity comprising the antibody according to Item 9.

    13. An agent for preventing or treating gallstone formation, comprising the antibody according to Item 9.

14 The following polynucleotide (7) or (8):
(5) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 4.
(6) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 4 under stringent conditions and has promoter activity.

15. The following polynucleotide (9) or (10):
(9) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 5.
(10) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 under stringent conditions and encodes a polypeptide having cholesterol 7α-hydroxylase activity.

    16. Item 15. A vector comprising the polynucleotide according to Item 14 and a structural gene linked so as to be expressed thereby.

    17. Item 17. The vector according to Item 16, wherein the structural gene is a reporter gene.

    18. Item 17. The vector according to Item 16, wherein the structural gene is the polynucleotide according to Item 15.

    19. Item 19. A test cell carrying the vector according to any one of Items 16 to 18.

20. The step of bringing the test cell according to Item 19 into contact with the test substance;
Comparing the expression level of the structural gene in the test cell with the expression level of the structural gene in the test cell not contacted with the test substance, and selecting a substance that promotes the expression of the structural gene. Method.

    21. Item 16. A cholesterol metabolism promoter comprising the polynucleotide according to Item 15.

    22. Item 16. A preventive or therapeutic agent for arteriosclerosis comprising the polynucleotide according to Item 15.

    23. Item 18. A prophylactic or therapeutic agent for obesity comprising the polynucleotide according to Item 15.

    24. Item 16. A preventive or therapeutic agent for gallstone formation, comprising the polynucleotide according to Item 15.

25. The following polypeptide (11) or (12):
(11) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6.
(12) A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 6, and having cholesterol 7α-hydroxylase activity.

  According to the present invention, it has been found that inhibition of βKlotho gene expression enhances CYP7A1 gene expression and promotes bile acid synthesis from cholesterol.

    Therefore, substances that suppress the expression of βKlotho gene and antibodies against βKlotho protein are useful for promoting cholesterol metabolism.

    A substance that promotes the expression of the CYP7A1 gene is also useful for promoting cholesterol metabolism. Further, the Cyp7a1 gene can promote metabolism of cholesterol by using the Cyp7a1 gene itself as a gene therapy agent that is introduced into the body of a patient.

    Since these reduce serum cholesterol levels, they are useful as preventive or therapeutic agents for arteriosclerosis, preventive or therapeutic agents for obesity, and preventive or therapeutic agents for gallstone (gallbladder cholesterol stones) formation.

Hereinafter, the present invention will be described in detail.
βKlotho gene The first polynucleotide of the present invention is a regulatory region existing upstream of the βKlotho gene, and is shown in the following (1) or (2).
(1) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.
(2) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions and has promoter activity.

The second polynucleotide of the present invention is a βKlotho structural gene and is shown in the following (3) or (4).
(3) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 2.
(4) a polypeptide encoding a polypeptide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and has an activity of inhibiting the expression of cholesterol 7α-hydroxylase (Cyp7a1 protein) nucleotide.

  SEQ ID NO: 1 is the genomic sequence of the upstream adjacent regulatory region of the human βKlotho gene, and SEQ ID NO: 2 is the cDNA sequence of the human βKlotho gene.

    In the present invention, polynucleotides (including oligonucleotides) include polynucleotides having the base sequence and polynucleotides complementary thereto. Polynucleotides include both DNA and RNA unless otherwise specified. In addition to single-stranded DNA having the base sequence, DNA includes single-stranded DNA and double-stranded DNA complementary thereto. Unless otherwise stated, DNA includes cDNA, genomic DNA, and synthetic DNA. Unless otherwise specified, RNA includes total RNA, mRNA, rRNA and synthetic RNA.

  The polynucleotides (1) and (3) can be obtained, for example, from a human cDNA library library by hybridization screening using probes designed based on SEQ ID NOs: 1 and 2, respectively.

    In the present invention, stringent conditions include, for example, conditions of about 60 ° C. in 2 × SSC and 1 × Denhardt's solution.

  Regarding the method for obtaining the polynucleotide of (2) or (4) based on the polynucleotide of (1) or (3), modifications that do not lose biological function are, for example, the retention of the structure of the protein obtained after translation. From the viewpoint, the amino acid can be substituted with an amino acid having properties similar to the amino acid before substitution in terms of polarity, charge, solubility, hydrophilicity / hydrophobicity, polarity and the like. For example, glycine, alanine, valine, leucine, isoleucine, proline are classified as nonpolar amino acids; serine, threonine, cysteine, methionine, asparagine, glutamine are classified as polar amino acids; phenylalanine, tyrosine, tryptophan Lysine, arginine and histidine are classified as basic amino acids; aspartic acid and glutamic acid are classified as acidic amino acids. Accordingly, substitution can be made by selecting from the same group of amino acids.

  In addition, when one amino acid has several translation codons, it is of course possible to substitute a base within the translation codon. For example, alanine has four translation codons, GCA, GCC, GCG and GCT, and the third base of the codon can be substituted between ATGCs.

  The polynucleotides (2) and (4) include, for example, polynucleotides of other species corresponding to human polynucleotides. Such polynucleotides can be selected, for example, by NCBI blast search (www.ncbi.nlm.nih.gov/BLAST/). Specifically, for example, the base sequence of SEQ ID NO: 1 or 2 is subjected to NCBI blast search to search for sequences having higher homology than base sequence databases and EST databases of other species. By selecting a nucleotide sequence having a nucleotide sequence homology of 90% or more selected by the search, for example, a corresponding gene of another corresponding species can be selected. Further, it can be identified by screening a gene library of another species using the full length or a part of the polynucleotide of (1) or (3), 5'-RACE or the like.

  The βKlotho gene regulatory region can be used to screen for substances that inhibit gene expression by this regulatory region. The substance obtained by this screening inhibits the expression of βKlotho gene in humans, thereby promoting the expression of Cyp7a1 and promoting the synthesis of bile acids from cholesterol.

The βKlotho structural gene can be used as a gene to be expressed by the regulatory region in the above screening, or can be inserted into an appropriate vector and used for mass production of βKlotho protein. Since the βKlotho protein antibody inhibits the activity of βKlotho protein, it is useful for promoting metabolism of cholesterol.
βKlotho protein The first polypeptide of the present invention is βKlotho protein, which is shown in the following (5) or (6).
(5) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3.
(6) It consists of an amino acid sequence in which one or more (preferably 3 or less) amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 3, and inhibits the expression of cholesterol 7α-hydroxylase. A polypeptide having activity.

  The polypeptide of (6) is a method of purifying from cells or tissues of human or other species by a known protein purification method, a method of isolating and purifying from the transformant (test cell) of the present invention described later, or It can be produced by a chemical synthesis method or the like.

 The method for obtaining the polypeptide (6) from the polypeptide (5) is as described for the polynucleotide.

Since the metabolism of cholesterol is promoted by inhibiting the activity of this polypeptide, this polypeptide can be used for screening for substances that inhibit the activity and for the production of antibodies.
Antibody The antibody of the present invention is an antibody that specifically recognizes the first polypeptide (βKlotho protein). This antibody may be a polyclonal antibody or a monoclonal antibody. The antibody of the present invention also includes an antibody having an antigen binding property to a polypeptide consisting of 5 amino acids, preferably 10 amino acids, in which the amino acid sequence constituting the βKlotho protein is continuous. Furthermore, derivatives of these antibodies (chimeric antibodies, etc.) and those labeled with an enzyme such as peroxidase are also included in the antibodies of the present invention.

    These antibodies can be produced according to well-known production methods (for example, Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.12 to 11.13).

  Specifically, when the antibody of the present invention is a polyclonal antibody, non-human animals such as rabbits can be immunized using βKlotho protein and obtained from the sera of the immunized animals according to a conventional method. In the case of a monoclonal antibody, for example, a non-human animal such as a mouse is immunized with a βKlotho protein or a polypeptide having a partial sequence thereof, and spleen cells and myeloma cells of the immunized animal are fused. It can be obtained from the resulting hybridoma (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).

Since this antibody inhibits the activity of βKlotho protein, it can be suitably used as a cholesterol metabolism promoter.
Cyp7a1 gene The third polynucleotide of the present invention is a regulatory region located upstream adjacent to the Cyp7a1 structural gene, as shown in (7) or (8) below.
(7) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 4.
(8) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 4 under stringent conditions and has promoter activity.

The fourth polynucleotide of the present invention is a Cyp7a1 structural gene, as shown in (9) or (10) below.
(9) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 5.
(10) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 under stringent conditions and encodes a polypeptide having cholesterol 7α-hydroxylase activity.

  SEQ ID NO: 4 is the genomic sequence of the upstream adjacent regulatory region of the human Cyp7a1 gene, and SEQ ID NO: 5 is the cDNA sequence of the human Cyp7a1 gene.

The Cyp7a regulatory region can be used to screen for substances that promote Cyp7a1 gene expression and promote cholesterol metabolism. The Cyp7a1 structural gene can be used as a structural gene expressed by the regulatory region in the above screening, as well as, for example, a gene for promoting cholesterol metabolism in patients whose Cyp7a1 expression is decreased or whose Cyp7a1 protein activity is decreased It can be used as a therapeutic agent.
Gene therapy agent for promoting cholesterol metabolism The gene therapy agent for promoting cholesterol metabolism of the present invention comprises the fourth polynucleotide of the present invention (Cyp7a1 structural gene and polynucleotide equivalent thereto). The fourth polynucleotide may be carried on a vector and introduced directly into the body, or cells may be collected from a human and introduced into the body and then returned to the body. As the vector, any of those known to be usable for gene therapy such as retrovirus, adenovirus, and vaccinia virus can be used without limitation, but a retrovirus is recommended.

Alternatively, the fourth polynucleotide of the present invention can be introduced into cells using microinjection or the like in which liposomes are encapsulated and delivered.
Cyp7a1 protein The 2nd polypeptide of this invention is Cyp7a1 protein, and is the following polypeptide of (11) or (12).
(11) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6.
(12) A polypeptide comprising an amino acid sequence in which one or more (preferably 3 or less) amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 6 and having cholesterol 7α-hydroxylase activity .

Substances that promote the activity of the polypeptide promote the synthesis of bile acids from cholesterol. Therefore, this polypeptide can be used for screening for substances that promote cholesterol metabolism.
Recombinant Vector / Transformant The vector of the present invention comprises a second polynucleotide (βKlotho structural gene or a polynucleotide equivalent thereto, DNA here) or a fourth polynucleotide (Cyp7a1 structural gene or a polynucleotide equivalent thereto) of the present invention. Nucleotide, here a vector that has been integrated so that DNA) can be expressed.

    As a vector into which the polynucleotide of the present invention is introduced, a known vector can be selected from a wide range depending on the host. For example, pBR322, pUC12, pUC119, pBluescript and the like are used when Escherichia coli is used as a host, and pUB110 and pC194 are used when a Bacillus bacterium is used as a host. When using yeast as a host, Yip5, Yep24 and the like can be mentioned. AcNPV etc. are mentioned when using an insect cell as a host. When animal cells are used as hosts, examples include pUC18 and pUC19.

  Any known host can be used without limitation. For example, bacteria such as E. coli (eg K12), bacteria such as Bacillus (eg MI114), yeast (eg AH22), insect cells (eg Sf cells), animal cells (eg Vero cells, Hela cells, CV1 cells, COS1 cells, CHO Cell etc.).

  The transformant can be used as a test cell for screening described later, and can also be used for mass production of βKlotho protein or Cyp7a1 protein. When used for screening, it is preferable to use animal cells, particularly human-derived cells.

The host transformation method using the recombinant vector of the present invention may be selected from known methods according to the host. Known introduction methods include calcium phosphate method, electroporation method, lipofection method, DEAE dextran method and the like. What is necessary is just to select a transformant from the transformant using the drug resistance marker etc. which a vector has as a parameter | index.
Production Method of Polypeptide of the Present Invention The polypeptide of the present invention is obtained by culturing the above transformant and recovering from the cultured transformant.

  What is necessary is just to select suitably the culture | cultivation conditions of a transformant according to the kind of transformant. When the transformant is a microorganism, it may be cultured using a liquid medium or a plate medium usually used for culturing the microorganism. The culture temperature should just be in the temperature which can grow microorganisms, for example, about 15-40 degreeC is mentioned. The pH of the medium may be within the range in which microorganisms can grow, and examples thereof include about 6 to 8. The culture time varies depending on other culture conditions, but is usually about 1 to 5 days, particularly about 1 to 2 days. When an induction type expression vector such as a temperature shift type or an IPTG induction type is used, the induction time may be within one day, particularly about several hours.

Even when the transformant is a mammalian cell, it may be cultured under conditions suitable for the cell. For example, DMEM medium (Nissui, etc.) supplemented with FBS can be used and cultured at a temperature of about 36 to 38 ° C. in the presence of 5% CO ₂ while being replaced with a new medium every few days. Once the cells have grown to confluence, for example, trypsin PBS solution may be added to disperse the cells, and the resulting cell suspension may be diluted several times and seeded in a new petri dish to continue passage. The culture days are usually about 2 to 5 days, particularly about 2 to 3 days.

Even when the transformant is an insect cell, the culture conditions may be suitable depending on the cell type. For example, it can culture | cultivate at about 25-35 degreeC using culture media for insect cells, such as Grace's medium containing FBS and Yeastlate. The culture time is preferably about 1 to 5 days, particularly about 2 to 3 days. Further, in the case of a transformant containing a virus such as Baculovirus as a vector, the culture time should be until the cytoplasmic effect appears and the cells die (for example, about 3 to 7 days, particularly about 4 to 6 days). preferable.
After completion of the culture, the transformant cells are collected by centrifugation or the like, and if necessary, the cells are suspended in an appropriate buffer and then disrupted with polytron, ultrasonic treatment, homogenizer or the like. The polypeptide of the present invention can be purified from the obtained disrupted solution by subjecting it to known protein purification methods such as ion chromatography, hydrophobicity, gel filtration, affinity chromatography and the like.
Cholesterol metabolism promoter screening method
First method (using βKlotho regulatory region)
In the first method, a cell holding a vector containing the first polynucleotide of the present invention (βKlotho regulatory region or a polynucleotide equivalent thereto) and a structural gene linked so as to be expressed thereby is used as a test cell. use.

That is, the first method for screening a substance for promoting cholesterol metabolism according to the present invention comprises a step of contacting a test cell with a test substance,
Comparing the expression level of the structural gene in the test cell with the expression level of the structural gene in the test cell not contacted with the test substance, and selecting a substance that inhibits the expression of the structural gene.

As a structural gene, an appropriate structural gene such as a βKlotho structural gene or a polynucleotide equivalent thereto (second polynucleotide of the present invention) may be used, but its expression level can be easily measured by using a reporter gene. it can.
Second method (using Cyp7a1 regulatory region)
In the second method, a cell holding a vector containing the second polynucleotide of the present invention (Cyp7a1 regulatory region or a polynucleotide equivalent thereto) and a structural gene linked so as to be expressed thereby is used as a test cell. use.

That is, the second method for screening a substance for promoting cholesterol metabolism according to the present invention comprises a step of bringing a test cell into contact with a test substance,
Comparing the expression level of the structural gene in the test cell with the expression level of the structural gene in the test cell not contacted with the test substance, and selecting a substance that promotes the expression of the structural gene.

As a structural gene, an appropriate structural gene such as Cyp7a1 structural gene or a polynucleotide equivalent thereto (the fourth polynucleotide of the present invention) may be used, but its expression level can be easily measured by using a reporter gene. it can.
<Reporter gene>
In the method of the present invention, the reporter gene refers to a gene that can qualitatively or quantitatively confirm the expression of the gene.

  As the reporter gene, for example, known reporter genes can be widely used. Such known reporter genes include luciferase gene, chloramphenicol acetyltransferase (CAT) gene, secreted alkaline phosphatase (SEAP), human growth hormone (hGH), β-glucuronidase (GUS), green fluorescent protein ( GFP), an enzyme capable of quantifying a product by the enzyme, a photoprotein, a fluorescent protein, an extracellular secreted protein, a colored protein, a protein that causes cell death, and the like. Of these, the luciferase gene is preferable because of its high sensitivity and easy activity measurement.

  These are commercially available as reporter plasmids lacking the endogenous promoter region or reporter plasmids lacking the endogenous enhancer. As luciferase plasmids, pT811uc, pSluc2, pXP1 / pXP2 (all of which are Nordeen, 1988) are commercially available.

  In measuring the expression level of the reporter gene, among the above reporter proteins, SEAP and hGH are secreted proteins, so the amount of protein expression in the cell culture supernatant can be quantified. What is necessary is just to measure the protein expression level in the cell lysate melt | dissolved using the agent etc. by the method according to these proteins.

  The expression level of the luciferase gene can be quantified in the cell lysate by detecting the fluorescence due to the degradation of luciferin catalyzed by luciferase using a luminometer. Moreover, the fluorescence emitted by the cells can also be detected using a luminometer.

  As will be described later, the expression level of the reporter gene is not necessarily quantified accurately, but can be roughly quantified (confirmed qualitatively) by visual observation or the like.

Although these reporter assays can be performed manually, they can be performed quickly and easily by using so-called high-throughput screening (tissue culture engineering, Vol23, No.13, 521-524, US Pat. It can be carried out.
<Other structural genes>
In the case of using a structural gene other than the reporter gene, for example, βKlotho gene, its expression level can be measured by measuring the corresponding mRNA level. The amount of mRNA can be measured by a known method such as Northern blot using a probe. Specifically, when using Northern blotting, RNA is prepared from test cells according to a conventional method, transferred to a nylon membrane, etc., and hybridized with a probe labeled with a radioisotope or fluorescent substance. What is necessary is just to detect the double chain | strand of a probe and RNA by the method according to a label | marker.

The expression level of the structural gene can also be measured by directly quantifying the protein. The amount of this protein can be measured by a known method such as Western blotting using an antibody.
<Test cell>
Test cells include, but are not limited to, mammalian cells or insect cells.

Examples of mammalian cells that can be used include known cells such as Vero cells, HeLa cells, CV1 cells, Namalva cells, COS1 cells, and CHO cells. As insect cells, known cells such as Sf cells, MG1 cells, and High Five ^™ cells can be used. In order to more accurately reflect the regulation of βKlotho expression in humans, it is preferable to use human cells.

In such a cell, a first polynucleotide (expression control region of βKlotho gene) or a second polynucleotide (expression control region of Cyp7a1 gene) and a structural gene linked so as to be expressed thereby What is necessary is just to use what introduce | transduced the appropriate vector containing this as a test cell.
<Test substance>
In the screening method of the present invention, the type of test substance is not particularly limited. For example, proteins, peptides, DNA encoding these, non-peptide substances (saccharides, lipids, etc.), organic low molecular weight compounds, inorganic low molecular weight compounds, fermentation products, cell extracts, cell nuclear extracts, plant extracts Animal tissue extracts, microbial culture supernatants, and the like can be used. These may be isolated from nature or may be artificially synthesized. The kind and molecular weight of these substances are not particularly limited, but a low molecular weight compound is preferable in consideration of transferability into cells.
<Contact / Judgment>
The contact between the test cell and the test substance can be performed, for example, as follows. That is, the cells carrying the vector are subcultured using a medium suitable for the cells and cultured so as to be in a logarithmic growth phase. When contacting the test substance, the cells are seeded so as to be about 20-30% confluent and cultured for about 24 hours. Next, the test substance is added to the cell culture so as to have a concentration of about 1 nM to 100 μM, for example, depending on the type. Next, it is incubated at about 37 ° C. for about 12 to 48 hours.

  Thereafter, the expression level of the structural gene in the test cell contacted with the test substance may be compared with the expression level of the structural gene in the test cell not contacted with the test substance. The comparison may be performed after measuring the expression level in each case, but may be performed directly by visual observation or the like.

  If the expression level of the structural gene is, for example, 70% or less, preferably 50% or less of the control in which the test substance is not contacted by using the test substance, it may be determined that the expression of the structural gene is suppressed. Further, when the expression level of the structural gene is, for example, 150% or more, preferably 200% or more of the control, it may be determined that the expression is promoted.

  According to this method, in addition to substances that indirectly affect the activity of the regulatory region of βKlotho gene or Cyp7a1 gene via cell membrane receptors etc., it is taken into the cell and directly binds to the regulatory region to affect its activity A low molecular weight compound that can be taken into the cell and act on other intracellular proteins to act indirectly on the regulatory region.

A substance that inhibits the expression of the structural gene obtained by the first method using the βKlotho regulatory region and a substance that promotes the expression of the structural gene obtained by the second method using the regulatory region of the Cyp7a1 gene are cholesterol Therefore, it can be suitably used as a prophylactic or therapeutic agent for arteriosclerosis, a prophylactic or therapeutic agent for obesity, a prophylactic or therapeutic agent for gallstone (gallbladder cholesterol stone) formation, and the like.
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples.
<Animals and food>
Mice were bred under special aseptic conditions and in the following examples all experiments were performed according to Institute's animal care guidelines. The standard diet is an NMF diet (Oriental Yesast). Other diets, Basal diet (code No.5795), 1% CA diet (catalog No.58794), 1% CDCA diet (code No.5T11), and edema-producing diet (code No.5806C-Q) Purchased from PMI Feed Inc. In metabolic experiments, mice were housed separately in metabolic cages and feces and urine collected every 24 hours for 3 days.
<Antibody / protein analysis>
A part of βKlotho cDNA (corresponding to amino acids 814-967) was cloned into the EcoRI site of pGEX4T-1 and introduced into E. coli BL21. After further separation by SDS-PAGE, a band corresponding to βKlotho-GST was extracted from the gel and injected into rats. Immunized rat thymocytes were used to generate monoclonal antibodies after selection of hybridomas.
<Northern blotting>
Total RNA was extracted using Total RNA separation system (Promega), and 30 μg was used for Northern blotting. The blot was exposed to an imaging plate (Fuji Film) and analyzed with a STORM860 scanner (Molecular Dynamics). A cDNA fragment amplified by PT-PCR was used as a detection probe. Each fragment was cloned into pT7Blue T-vector (Novagen) and sequenced.

Generation of a mouse completely lacking βKlotho gene The mouse βKlotho site consists of at least 8 exons. In ES cells, a gene targeting vector in which most of the putative exon 1 was deleted was constructed by homologous recombination (FIG. 1A).

  Genomic DNA containing the βKlotho site was isolated from the mouse 129SVJ genomic library. The 5′-arm of the target vector covered a 6.4 kb EcoRI fragment containing the exon part containing the putative first ATG. An EGFP gene without a promoter and a PGKneo cassette were inserted between these two arms together with the loxP5171 (5 ′ side) and the EGFP gene whose front is adjacent to the loxP sequence. The PGKneo cassette is present in the adjacent region on the opposite side of the loxP sequence. Vector DNA was linearized and introduced into TTS ES cells by electroporation. G418 resistant clones were selected and selected by Southern blotting. Three clones were selected from 439 clones and injected into stage ICR embryo cells. Embryo transmission occurred in two chimeras derived from one clone. Male chimeras were mated with female C57B1 / 6 mice, and heterozygous offspring were cross-fertilized to yield βKlotho complete deletion mice.

  Offspring born from these heterozygous parents were used in this study. However, the same phenotype was also observed in offspring from heterozygous parents by 5 backcrosses to C57B1 / 6.

    In order to confirm that the βKlotho site was destroyed in the genomic DNA, Southern analysis was performed on heterozygous mice and homozygous mice using a probe in the upstream region adjacent to the targeting vector. The results are shown in FIGS. 1A and 1B.

    Further, the removal of βKlotho mRNA expression was confirmed by Northern blotting (FIG. 1C). Furthermore, it was also confirmed by RT-PCR using several primer sets covering different regions of the remaining exons (data not shown).

    Deletion of βKlotho protein was also confirmed by Western blotting using a monoclonal antibody that recognizes the C-terminal part of the second internal repeat (βKL2) in βKlotho protein (FIG. 1D). A deletion of EGFP transcription showing the same tissue distribution as βKlotho was detected, but no deletion of EGFP protein was detected by Western blotting (data not shown).

βKlotho ^{− / −} mice were viable and fertile under normal conditions and were generally normal. The number of homozygous mice (+ / +: +/-:-/-= 297: 624: 193) was less than Mendelian. Body weight was significantly less than wild-type and heterozygous littered mice (FIG. 1E). A statistically significant decrease was not observed before weaning in both sexes. In males, it was not observed thereafter. Weight loss was not due to differences in liver and gonadal fat sizes. There was no reproducibility of the slight decrease in body weight in the group that was backcrossed 5 times to C57B1 / 6 (no data).

Various tissues including βKlotho expressing tissues stained with hematoxylin and eosin were observed well. However, there was no difference in tissue between βKlotho ^{− / −} mice and wild type mice. In both cases, serum triglycerides, fatty acids, cholesterol, phospholipids, and serum markers worthy of βKlotho-expressing tissues such as enzyme activity derived from the liver and pancreas were not significantly different between the two phenotypes.

Evaluation of bile acid synthesis and excretion βKlotho expressing tissues are involved in lipid metabolism. Therefore, it was predicted that this molecule is involved in lipid metabolism in some way. However, serum markers of lipid metabolism such as triglycerides, fatty acids and phospholipids did not change significantly in βKlotho ^{− / −} mice (data not shown).

Serum cholesterol levels were slightly decreased, although no statistically significant difference was observed (FIG. 2A). This led to a focus on cholesterol metabolism in βKlotho ^{− / −} mice. Since βKlotho is strongly expressed in the liver where cholesterol is converted into bile acids, we decided to investigate the metabolism of cholesterol in βKlotho ^{− / −} mice.

In βKlotho ^{− / −} mice, the amount of bile acid excreted in feces was significantly higher than that in wild-type mice (FIG. 2B). Daily food intake and total intake were not different in both groups (Figure 2C). However, bile acid accumulation was slightly higher in βKlotho ^{− / −} mice (FIG. 2D).

CY7A1 is the most important enzyme in the production of bile acids because it catalyzes the first step of the major bile acid synthesis pathway and the rate-limiting step (nnu Rev Biochem 72,137-174 (2003)). Cyp7a1 was markedly enhanced in βKlotho ^{− / −} mice compared to wild-type litter mice (FIG. 2E). This was reproducible 5 times. In addition, the same results were obtained for 20 or more mice with no stomach (data not shown).

Other enzymes, such as sterol 12α-hydroxylase (CYP8B1) are essential and important for the production of coric acid (CA), the most abundant bile acid. Therefore, when the expression of this gene was examined, Cyp8b1 was also significantly enhanced in βKlotho ^{− / −} mice (FIG. 2E).

It has been reported that bile acid synthesis can be evaluated by inhibition of enterohepatic circulation (J. Biol. Chem. 278, 33920-33927 (2003)). Therefore, the state of circulation was examined. Expression of I-babp (intestinal bile acid binding protein) is known to be induced in response to bile acid absorption (FEBS Lett 384, 131-134 (1996)). I-babp expression was markedly enhanced in βKlotho ^{− / −} mice (FIG. 2F). This is thought to reflect the increased secretion of bile acids into the duodenum, and therefore bile acid absorption is considered to be complete in βKlotho ^{− / −} mice.

No genotype differences were detected in the expression of bile acid transporters in liver cells and intestinal cells (data not shown). Furthermore, bile acid accumulation was decreased in mice lacking intestinal circulation, but slightly increased in βKlotho ^{− / −} mice.

These results show that in βKlotho ^{− / −} mice, the intestinal circulation of bile acids has not received much loss.

Cholesterol stone formation
C57B1 / 6α mice have been reported to produce cholesterol stones when fed a special diet containing cholesterol and bile acids. This is more pronounced in males than in females. It has been reported that stone formation is suppressed by overexpression of rat CYP7A1 in mice (Thromb Vasc Biol 22,121-126 (2002)).

Here, the response to a special diet containing cholesterol and bile acids was examined in βKlotho ^{− / −} mice. Similar to the above document, calculus formation was promoted in wild-type mice. The number varied but was effective in males (FIG. 3A). In contrast, in βKlotho ^{− / −} mice, stone formation was not observed even in males (FIG. 3A). Over 14 mice were examined for each genotype group with no exceptions. In both sexes, the gallbladder size was smaller in βKlotho ^{− / −} mice than in wild type mice (FIG. 3A arrow).

    During bile supersaturation, cholesterol crystals form stones. Many factors are known to accompany the initiation of stone formation (Gastroenterol Clin North Am 28,99-115 (1999)). However, the most important factor is the presence of excess cholesterol relative to bile acids and phospholipids (Z Ernahrungswiss 10, 239-252 (1971)). Mutant mice with increased bile acid synthesis appear to be resistant to stone formation or increased bile acid synthesis appears to be sufficient for prevention. This is supported by the fact that long-term administration of ursodeoxycoric acid (UDCA) dissolves stones in bile.

To elucidate the mechanism of calculus resistance, we investigated the behavior of cholesterol in βKlotho ^{− / −} mice. The amount of liver and serum and fecal excretion were examined. Under normal dietary conditions, hepatic cholesterol was slightly elevated in both sexes in βKlotho ^{− / −} mice. However, no statistically significant difference was observed (Figure 3B). Fecal cholesterol excretion was not significantly different (Figure 3C). Changing to the above special diet significantly increased cholesterol in the liver and feces. However, there was no gender difference in any genotype group (Fig. 3B, Fig. 3C). Serum cholesterol was significantly increased by the special diet. However, genotype-dependent significant differences were observed only in females (FIG. 3D).

When the expression of Abcg5 and Abcg8, which are free cholesterol transporters in the liver, was examined, no difference was found in the expression level of mRNA between the two genotypes. Therefore, it is considered that cholesterol excretion into bile is not enhanced in βKlotho ^{− / −} mice.

Gene expression profiles under different dietary conditions Gene expression profiles in the liver were compared for both genotypes. Northern blotting of the same sex of each sex difference between more than 30 genes encoding lipids, steroids and xenobiotic nuclear receptors, transporters, P-450s, other enzymes, lipoproteins, and apolipoproteins I examined the difference of the pair.

In addition, several genes whose expression changes depending on the genotype were identified. Hmg-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgr)), an enzyme that catalyzes the rate-limiting step of cholesterol biosynthesis, is strongly enhanced in both females and males in βKlotho ^{− / −} mice. (FIG. 4A). The expression of Fxr (farnesoid X receptor, NR1H4) and Ldlr (low density lipoprotein receptor) was also slightly increased (data not shown). Shp (small heterodimer partner, NRB2) expression was almost always reduced in βKlotho ^{− / −} mice (in one case, the same for both genotypes) (FIG. 4B).

In βKlotho ^{− / −} mice, metabolism of cholesterol and bile acids was altered, so the effect of dietary conditions on these parameters was tested. Mice were given a basal diet from the weaning period and then shifted to a bile acid-containing diet (top of FIG. 4C). Cyp7a1 expression was not detected in wild-type mice under these conditions. However, there was no substantial decrease in βKlotho ^{− / −} mice. On the other hand, Cyp8b1 expression was completely reduced by dietary bile acids regardless of genotype (FIG. 4C). The expression of Shp and Bsep, which are direct targets of FXR, was also examined. Bsep expression was significantly enhanced by dietary CDCA regardless of genotype. On the other hand, CA was not so clear (Figure 4C).

Shp expression was slightly higher in mutant mice compared to wild-type mice fed the basic diet. In both genotypes, expression levels were increased by adding dietary bile acids. In the mutant form, this induction was mild. There was an inverse relationship between the expression level of Shp and the expression level of Cyp7a1 (FIG. 4C). βKlotho ^{− / −} expression was not significantly affected by dietary switching (data not shown).

  From these results, it is considered that the influence of bile acids on the inhibition of Cyp7a1 is divided into two signal pathways. One is an SHP-independent pathway, the other is an SHP-dependent pathway, and βKlotho is involved in the former pathway. ΒKlotho is specifically involved in the transcriptional regulation of Cyp7a1 and is not considered to be involved in the transcriptional regulation of Cyp8b1.

A is a diagram showing a strategy for destruction of mouse βKlotho. The upper row shows the target vector, the middle row shows the wild type allele, and the lower row shows the destroyed allele. The numbers indicate the estimated distance from the translation start position. B is a diagram showing the results of Southern blot analysis of Eco-RV digested genomic DNA. FIG. C is a diagram showing the results of Northern blot analysis of βKlotho gene expression. D is a figure which shows the result of the Western blot analysis of the expression of (beta) Klotho protein. WAT indicates adipose tissue. WAT indicates white adipose tissue. E is a graph comparing growth curves among βKlotho (− / −) (− / +) (+ / +) mice. 5-12 mice in each group were traced over 2-6 weeks. Error bars indicate standard deviation. FIG. 6 compares several parameters between wild type and knockout mice. A shows a comparison of serum cholesterol levels between 4-6 animals in each group. B shows stool collected from 4-6 mice in each group for 3 days, and the amount of total bile acid excretion was measured and averaged for 12-18 samples in each group. C shows the daily food intake and fecal excretion of each of the four phenotypes for 3 days. D shows a comparison of bile acid retention in 4-10 mice in each group. E shows a comparison of the expression of Cyp7a1 and Cyp8b1 between βKlotho ^{− / −} mice and wild type mice. 30 μg of total RNA from each phenotype liver was used. Representative values from two independent experiments were used. The Northern blot is shown on the left and the relative expression level that complements the Gapdh level is shown on the right. F shows expression of intestinal bile acid binding protein (I-babp). 30 μg of total RNA obtained from the whole small intestine was used. Samples were taken from animals of the same abdominal age and sex. The animals used were 7 weeks old on a standard diet. M represents a male and F represents a female. Error bars indicate standard deviation. * P <0.05. A shows that cholesterol stones were formed in wild-type mice (left) and not formed in βKlotho ^{− / −} mice (right) by an atherogenic diet. The dietary administration schedule is shown above. Mice were killed after each schedule. The gallbladder is indicated by an arrow. Inserted in the left view is an enlarged photo of a wild-type gallbladder with several stones. Examples of gallbladder from other individuals are shown below. The left panel of B shows the average liver cholesterol level of 7-week-old mice fed a standard diet (NMF). The average liver cholesterol level of 14-week-old mice fed an atherogenic diet (Athero) is shown on the right. 4-7 mice were used for each group. C is a figure which shows the average cholesterol density | concentration in feces. The left view is the result of collecting stool for 3 days for a 7-week-old mouse fed the standard diet, and the right view is the result of collecting stool for 3 days for a 14-week-old mouse fed the atherogenic diet. is there. 3-4 mice were used for each group. Feces from the same individual were used for analysis. D is a figure which shows the serum cholesterol level of the 14-week-old mouse | mouth ingesting the atherogenic diet. 4-7 mice were used for each group. Error bars indicate standard deviation. * P <0.05, ** P <0.005. A is a graph showing increased Hmgr mRNA expression in βKlotho ^{− / −} mice. FIG. Representative data from two independent experiments are shown. The left column is a Northern blot, and the right column shows the relative expression level supplementing Gapdh. B is a figure which shows Shp expression level. The experiment was performed in the same manner as A. # Indicates a case where expression levels are similar between phenotypes. The top row of C shows the diet schedule used to test the effect of bile acids in the diet. Mice were killed at the end of each schedule. The effects of CA and CDCA in the diet on the expression of Cyp7a1, Cyp8b1, SHP target genes and Shp, Bsep, FXR target genes are shown. 30 μg of total RNA obtained from the liver was used for Northern blot. The results obtained using two independent individuals for each condition are shown. The expression level of Gapdh was used as an internal control.

Claims (25)

The following polynucleotide (1) or (2):
(1) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.
(2) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions and has promoter activity. The following polynucleotide (3) or (4):
(3) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 2.
(4) A polynucleotide that encodes a polypeptide that hybridizes with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and has an activity of inhibiting the expression of cholesterol 7α-hydroxylase. A vector comprising the polynucleotide according to claim 1 and a structural gene linked so as to be expressed thereby. The vector according to claim 3, wherein the structural gene is a reporter gene. The vector according to claim 3, wherein the structural gene is the polynucleotide according to claim 2. A test cell carrying the vector according to claim 3. Contacting the test cell according to claim 6 with a test substance;
Comparing the expression level of the structural gene in the test cell with the expression level of the structural gene in the test cell not in contact with the test substance, and selecting a substance that inhibits the expression of the structural gene. Method. The following polypeptide (5) or (6).
(5) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3.
(6) A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 3 and having an activity of inhibiting the expression of cholesterol 7α-hydroxylase. An antibody that specifically recognizes the polypeptide according to claim 8. A cholesterol metabolism promoter comprising the antibody according to claim 9. A prophylactic or therapeutic agent for arteriosclerosis comprising the antibody according to claim 9. A preventive or therapeutic agent for obesity comprising the antibody according to claim 9. A prophylactic or therapeutic agent for gallstone formation, comprising the antibody according to claim 9. The following polynucleotide (7) or (8):
(5) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 4.
(6) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 4 under stringent conditions and has promoter activity. The following polynucleotide (9) or (10):
(9) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 5.
(10) A polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 5 under stringent conditions and encodes a polypeptide having cholesterol 7α-hydroxylase activity. A vector comprising the polynucleotide according to claim 14 and a structural gene ligated so as to be expressed thereby. The vector according to claim 16, wherein the structural gene is a reporter gene. The vector according to claim 16, wherein the structural gene is the polynucleotide according to claim 15. A test cell carrying the vector according to any one of claims 16 to 18. Contacting the test cell of claim 19 with a test substance;
Comparing the expression level of the structural gene in the test cell with the expression level of the structural gene in the test cell not contacted with the test substance, and selecting a substance that promotes the expression of the structural gene. Method. A cholesterol metabolism promoter comprising the polynucleotide according to claim 15. An agent for preventing or treating arteriosclerosis comprising the polynucleotide according to claim 15. A preventive or therapeutic agent for obesity comprising the polynucleotide according to claim 15. A preventive or therapeutic agent for gallstone formation comprising the polynucleotide according to claim 15. The following polypeptide (11) or (12):
(11) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6.
(12) A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 6, and having cholesterol 7α-hydroxylase activity.

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