JP2002034563A - Cathepsin k promoter and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for testing the therapy or prophylaxis for a variety of osteometabolic diseases including osteoporosis, osteoclasis in rheumatoid arthritis, bone metastasis and osteoclasis by cancer cells and the DNA that is used in the testing method. SOLUTION: In the nucleotide sequence represented in the sequence No.1 in the sequence table (please see the specification of this invention), the objective DNA comprises any nucleotide among the sequences from the nucleotide No.1 to the nucleotide No.1063 as the 5' terminus and the nucleotide of the nucleotide No.1675 as the 3' terminus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel DNA useful for testing an agent for treating or preventing osteoporosis.

[0002]

2. Description of the Related Art Bone is known as a dynamic organ that constantly repeats formation and resorption for remodeling in order to maintain its morphological change and blood calcium concentration. Since bone formation by osteoblasts and bone resorption by osteoclasts are in equilibrium, bone mass is usually kept constant.However, once this equilibrium is broken, bone metabolism abnormalities such as osteoporosis can occur. (Suda et al., Endocr. Rev. 13, 66-80 (199
2), Suda et al., In Principles of Bone Biology (Bi
lezikian et al. Eds), pp87-102, Academic Press, New
York (1996)).

[0003] Osteoporosis is roughly classified into primary osteoporosis having no underlying disease and secondary osteoporosis associated with other diseases such as various endocrine diseases and blood diseases. Primary osteoporosis is further classified into age-related degenerative osteoporosis and juvenile osteoporosis. On the other hand, secondary osteoporosis is associated with endocrine diseases such as hyperthyroidism and diabetes, with blood diseases such as multiple myeloma and malignant lymphoma, hereditary diseases such as osteogenesis imperfecta, and chronic joint disease. It is classified into those caused by other causes such as rheumatism and bone metastasis of malignant tumor (Experimental Medicine Series 16 for clinicians, “Regulatory system of bone and calcium metabolism and osteoporosis” pp 128-149 Yodosha).

[0004] Osteoclasts are multinucleated cells derived from hematopoietic stem cells, and their precursor cells are osteoclast differentiation factors (osteocla) expressed on the cell membrane of osteoblasts / stromal cells on the surface of bone.
st differentiation factor. (Hereinafter referred to as "ODF")
Has been shown to differentiate into osteoclasts (Yasuda et al., Proc. Natl. Acad. Sci. USA 95,
3597-3602 (1998), Lacey et al., Cell 93, 165-176
(1998)). As a result of experiments using recombinant soluble ODF lacking the intracellular domain and transmembrane domain of ODF,
Is a membrane-bound factor produced by osteoblasts / stromal cells, its expression is regulated by bone resorption factors, and ODF induces differentiation of osteoclast precursor cells into mononuclear osteoclasts (Yasuda et a
l., Proc. Natl. Acad. Sci. USA 95, 3597-3602 (199
8), Jimi et al., J. Bone Miner. Res. 23, S222 (Abst
ract) (1998)). Furthermore, since mice knocked out of ODF develop typical marble disease,
Is a physiological osteoclast differentiation inducer (Kong et al., Nature (London) 397, 315-323 (199
9)).

[0005] Bone resorption by osteoclasts is divided into a stage in which the bone adheres and becomes polar, a stage in which acids and enzymes are actively secreted from the upper edge of the wave, and a stage in which the cells undergo apoptosis and die. (Sakae Tanaka: Regulation of bone resorption function of osteoclasts. Bone signals and osteoporosis, pp36-4
1, Yodosha (1997)). Among them, lysosomal enzymes are involved in the destruction of bone matrix in resorption pits (Kenichi Tezuka, Masayoshi Kumegawa: Isolation method of osteoclasts and isolation and analysis of osteoclast-specific genes. Experimental medicine, 13 (4), 425-429 (199
Five)). In particular, cathepsin K is abundant in osteoclasts and has the activity of degrading bone matrix proteins such as type I collagen, osteopontin, and osteonectin (Bossard et al., J. Bol. Chem. 271, 12517-1
2524 (1996), Kafienah, et al., Biochem. J. 331, 72.
7-732 (1998)). In addition, concentrated dysostosis (pycnodysostosi
It has also been clarified that a cathepsin K gene abnormality exists in the hereditary human disease with reduced bone resorption called s) (Gelb et al., Science 273, 1236-1238 (1996), Jo).
hnson et al., Genome Res. 6, (1050-1055 (1996)).
In addition, it has been reported that bone resorption activity is impaired in mice knocked out of cathepsin K, causing marble disease (Saftig et al., Proc. Natl. Acad. Sci. USA 9
5, 13453-13458 (1998)), and the importance of cathepsin K in bone metabolism in vivo was proved.

[0006] Since the relationship between cathepsin K and bone metabolism has been elucidated, cathepsin K inhibitors have been developed for the purpose of treating osteoporosis. The search for low molecular weight compounds that directly inhibit enzymatic activity has been vigorously performed (for example, Yamashita and Dodds, Curr Pharm. Des. 6, 1-24
(2000)).

[0007] On the other hand, an approach of suppressing the expression of the cathepsin K gene itself is considered to be extremely effective, but only an expression inhibition experiment using an antisense oligonucleotide has been reported so far (Inuie
etal., J. Biol. Chem. 272, 8109-8112 (1997)). The transcriptional regulation mechanism of the cathepsin K gene is hardly elucidated, and humans (Rood et al., Genomics 41,
169-176 (1997), Gelb et al., Genomics 41, 258-262.
(1997)) and mice (Gelb et al., Biochem. Mol. Med. 5
9, 200-206 (1996), Li and Chen, J. Bone Miner. Re
s. 14, 487-499 (1999)) Although the DNA sequence of the cathepsin K chromosome gene has been reported, no specific DNA region (promoter / enhancer region) involved in the regulation of expression has been identified.

[0008]

An object of the present invention is to specify a promoter region involved in the transcriptional control of the mouse cathepsin K gene and to provide a method for screening a compound that regulates the activity of the promoter. More specifically, a method for testing a therapeutic or prophylactic agent for various bone metabolic diseases including osteoporosis, bone destruction in rheumatoid arthritis, bone metastasis of cancer cells and bone destruction, and DNA used in the method are described. Provided.

[0009]

Means for Solving the Problems The present invention provides (1) a DNA represented by any one of the following 1) to 4): 1) a nucleotide represented by SEQ ID NO: 1 in the sequence listing; A DNA having a nucleotide sequence having any one of Nos. 1 to 1063 at the 5 ′ end and nucleotide No. 1675 at the 3 ′ end; 2) a transformed E. coli strain E. coli; coli pUC18-mC
TSK-1.7 SANK 70500 (FERM B
P-7208), the DNA inserted into the pUC18 vector multiple cloning site in the recombinant plasmid vector; 3) hybridized with the DNA described in 1) or 2) above under stringent conditions, Osteocyte differentiation factor (os
teoclast differentiation factor: hereinafter referred to as "ODF")-dependent promoter activity; 4) an ODF-dependent promoter activity comprising a nucleotide sequence having 95% or more nucleotide sequence identity with the DNA described in 1) or 2) above. DNA having
(2) nucleotide number 106 of SEQ ID NO: 1 in the sequence listing
Comprising the nucleotide sequence shown from 3 to 1471;
DNA having DF-dependent promoter activity, (3)
Nucleotide numbers 1063 to 1 of SEQ ID NO: 1 in the sequence listing
DNA consisting of the nucleotide sequence represented by 675,
(4) a DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 1675 of SEQ ID NO: 1 in the sequence listing,
(5) The DN according to any one of (1) to (4).
(6) The vector according to (5), wherein a DNA encoding a protein is ligated downstream of the DNA according to any one of (1) to (4). , (7) Any one of (1) to (4)
The DNA linked downstream of the DNA described in (1) is a DNA encoding a protein selected from the group consisting of luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase and green fluorescent protein. (6) The DNA linked to the downstream of the DNA according to any one of (1) to (4) is a DNA encoding luciferase. ,
The vector according to (7), (9) the transformed E. coli strain E.
E. coli pUC18-mCTSK-1.7 SANK
70500 (FERM BP-7208), (10) a host cell transformed with the vector of any one of (5) to (9), (11) a transformed Escherichia coli Strain E. col
ipUC18-mCTSK-1.7SANK 705
00 (FERM BP-7208), (12) A method for testing a substance that regulates cathepsin K promoter activity, comprising the following steps i) and ii): i) any one of (6) to (8) Contacting a test substance with a host cell transformed with the vector of ii); (6) to (8) in the host cell of i) above.
Measuring the expression level of a protein encoded by a DNA linked downstream of the DNA according to any one of (1) to (4) in the vector according to any one of the above. (13) The method according to (12), wherein in step i) the host cell is contacted with a test substance and ODF, (14)
A method for testing a cDNA encoding a protein having an activity of regulating cathepsin K promoter activity, comprising: the vector according to any one of the following steps i) and ii): i) (6) to (8) C) in which the host cell transformed with
Ii) the vector according to any one of (6) to (8) in the host cell according to i) above, wherein the vector is transformed with the DNA; DN
Measuring the expression level of a protein encoded by DNA linked downstream of A, (15)
(14) The method according to (14), further comprising a step of contacting the host cell with ODF, (16) a method for screening a protein that regulates cathepsin K promoter activity, the method comprising: (17) a method comprising bringing a sample containing the protein into contact with the DNA according to any one of (4), and then separating a protein bound to the DNA; (17) cathepsin K
A method for testing a substance having an activity of regulating a promoter activity, comprising the steps i) and ii) below: i) a method in which the DNA according to any one of (1) to (4) is radiolabeled. , A sample to which a nuclear extract of mouse cells and a test substance are added, and a sample to which only a nuclear extract of mouse cells are added are prepared; ii) Each mixture sample of the above i) is subjected to electrophoresis, and mouse cells are subjected to electrophoresis. Comparing the mobility between a band found in a sample to which only a nuclear extract has been added and a band found in a sample to which a test substance has been added, (18) a nucleotide number 1 of SEQ ID NO: 1 in the sequence listing. Consisting of at least 10 contiguous nucleotides in a nucleotide sequence represented by
A, DNA having its antisense sequence or a derivative thereof.

[0010] Mouse monocyte-derived cell line RAW 264.7
Is known to strongly induce gene expression of various osteoclast differentiation markers including cathepsin K by stimulating with ODF (Hsu et al., Proc. Natl. Ac
ad. Sci. USA 96, 3540-3545 (1999)). Therefore, the present inventors constructed a reporter gene obtained by linking luciferase to the mouse cathepsin K gene 5'-terminal untranslated region of about 1.7 kilobase pairs (hereinafter, referred to as "1.7 kbp") in this cell. When introduced into a strain, it was found that ODF-dependent promoter activation was observed. The present inventors have also increased the size of the 5 'untranslated region linked to the luciferase gene from about 1.7 kbp to about 600 kbp.
It was also found that the ODF-dependent promoter activity was retained even when shortened from the bp and the 5 'end side, but the activity was lost when shortened to about 200 bp. The present inventors have further found that a compound that regulates the activity of the promoter can be screened by using the isolated promoter region, and completed the present invention.

The DNA of the present invention may contain any nucleotide sequence in the 5′-terminal upstream region of the mouse cathepsin K gene as long as it has promoter activity.
It is located at the 5 ′ end upstream of the mouse cathepsin K gene,
Preferable examples of DNA having ODF-dependent promoter activity include, for example, a nucleotide sequence corresponding to a position from -1670 to +5 at the 5 ′ end of the mouse cathepsin K gene (nucleotide No. 1 of SEQ ID No. 1 in the sequence listing). -1675) and also -60.
A DNA consisting of a nucleotide sequence corresponding to positions 8 to +5 (nucleotide numbers 1063 to 1675 of SEQ ID NO: 1 in the sequence listing) can be exemplified.

On the other hand, a nucleotide sequence corresponding to the -200 to +5 positions in the 5'-terminal upstream region of the mouse cathepsin K gene (nucleotide number 147 of SEQ ID NO: 1 in the sequence listing)
1-1167) has substantially no ODF-dependent promoter activity. Therefore, the nucleotide sequence corresponding to positions -608 to -200 in the upstream region of the 5 'end of the mouse cathepsin K gene (nucleotide numbers 1675-1471 of SEQ ID NO: 1 in the sequence listing) is:
Since the region essential for ODF-dependent promoter activity is considered to be included, the promoter DNA of the present invention
Preferably, a nucleotide sequence corresponding to positions -608 to -200 at the 5'-end upstream of the mouse cathepsin K gene (nucleotide number 1675-1 of SEQ ID NO: 1 in the sequence listing)
471).

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The upstream region of the 5 ′ end of the mouse cathepsin K gene is, for example, a nucleotide sequence of a chromosomal gene of mouse cathepsin K (Gelb et al., Bioche
m. Mol. Med. 59, 200-206 (1996), Li and Chen, J. B
one Miner. Res. 14, 487-499 (1999)) or a part thereof, or the nucleotide sequence of SEQ ID NO: 1 or a part thereof, by screening a mouse genomic library using the probe as a probe. Can be. Alternatively, the DNA of the present invention may be directly amplified without going through a library screening step by polymerase chain reaction (hereinafter referred to as “PCR”) using mouse genomic DNA as a template based on the information of these sequences. it can.

The transformed E. coli strain E. coli harboring the recombinant plasmid containing the DNA of the present invention. coli p
UC18-mCTSK-1.7 SANK 70500
Was internationally deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology on July 7, 2000 (Heisei 12).
RM BP-7208 is attached. Therefore, the DNA of the present invention can also be obtained from the strain.

Anyone having ordinary knowledge in the technical field to which the present invention pertains can use the natural promoter D
A DNA having a promoter activity equivalent to that of a natural promoter DNA can be prepared by modifying a part of the nucleotide sequence of NA with other nucleotides, or by deleting or adding nucleotides. Thus, the nucleotide in the natural nucleotide sequence has a nucleotide sequence substituted, deleted, or added,
DNA having a promoter activity equivalent to that of a natural promoter DNA is also included in the DNA of the present invention. Modification of the nucleotide sequence can be performed, for example, by restriction enzyme or DN.
Deletion introduction by A exonuclease (Toshimitsu Kishimoto et al., "Seismological Chemistry Laboratory Course 1: Gene Research Method II" 335-35
4), site-specific mutagenes
is / Mark, DF et al. (1984) Proc. Natl. Acad. Sc
i. USA 81, 5662-5666), PCR using mutation primers (Saiki, RK et al. (1988) Sci.
ence 239, 487-491),
It can be performed by a method such as direct introduction of a synthetic mutant DNA. . The DNA of the present invention includes not only mouse but also vertebrates including humans, particularly human cathepsin K promoter. Preferred among these are DNAs consisting of the nucleotide sequence represented by nucleotide numbers 1 to 1063 to nucleotide number 1675 of SEQ ID NO: 1 in the sequence listing, and more preferably from nucleotide number 1063 of SEQ ID NO: 1 in the sequence listing. DNA comprising the nucleotide sequence represented by 1675.

The DNA of the present invention includes a DNA capable of hybridizing under stringent conditions with a DNA having a nucleotide sequence represented by nucleotide numbers from 1 to 1063 to 1675 in SEQ ID NO: 1 in the sequence listing. Things. The DNA capable of hybridizing in this manner may be any DNA having ODF-dependent promoter activity. Such DNA
Are nucleotide numbers 1 to 10 of SEQ ID NO: 1 in the sequence listing.
It usually has a nucleotide sequence identity of at least 70%, preferably at least 80%, more preferably at least 95% with the nucleotide sequence from 63 to nucleotide number 1675. Such DNAs include mutant genes found in nature, artificially modified mutant genes, homologous genes derived from heterologous organisms, and the like.

In the present invention, hybridization under stringent conditions is carried out under ordinary stringent conditions (roast stringent conditions) by using 5 × SSC (0.75 M sodium chloride, 0.075 M Sodium citrate) or a hybridization solution with an equivalent salt concentration,
Perform for about 12 hours under the temperature condition of 37-42 ° C., 5 × SS
Preliminary washing may be performed with C or a solution having a salt concentration equivalent thereto, if necessary, followed by washing in 1 × SSC or a solution having a salt concentration equivalent thereto. Also,
In conditions having higher stringency (high stringency conditions), the washing was carried out at 0.1%.
× SSC or a solution having a salt concentration equivalent thereto.

Whether or not the thus obtained DNA of the present invention has an ODF-dependent promoter activity is determined by ligating a reporter gene such as luciferase downstream thereof as described in "5-2)" below. This can be confirmed by transforming a mammalian cell (having an ODF receptor) with the resulting product and examining whether or not the reporter gene expression in the transformed cell is induced by ODF stimulation.

Hereinafter, a method for using the DNA of the present invention will be described.

1) Use as Inducible Expression Vector The promoter DNA of the present invention is not limited to ODF stimulation,
When it is found that the gene is activated by a specific stimulus, a recombinant vector having the DNA of the present invention inserted upstream of a desired gene is prepared and introduced into somatic cells, and the stimulus is added. The desired gene can be inducibly expressed.

2) DN using antagonistic inhibition by DNA
TECHNICAL FIELD The present invention relates to a DNA containing at least a part of the sequence of the above promoter DNA (including a derivative thereof). Such DNA can antagonize the binding between the cathepsin K promoter and a protein (eg, a transcription factor) that can bind to it, regardless of whether it has promoter activity. Therefore, when the nucleotide sequence of the DNA corresponds to the binding site of a protein that inhibits cathepsin K promoter activity, the promoter activity can be promoted by this method, and conversely, the cathepsin K promoter activity can be promoted. In this case, the promoter activity can be inhibited in the case where it corresponds to the binding site of the protein to be treated. DNA used for competitive inhibition usually has a chain length of at least 6 nucleotides or more, preferably 10 nucleotides or more. Examples of DNA used for competitive inhibition include a sequence containing a transcription factor binding consensus site. Derivatives include those obtained by linking some of the above promoter DNAs in tandem, those linked to an adenovirus vector or the like to enable gene transfer into animal cells, Those in which biotin is linked to the '-or 5'-end can be used.

Since the cathepsin K protein has an activity of degrading bone matrix as described above, the promoter of the activity of the cathepsin K promoter DNA is useful for treating marble disease and the like. The activity inhibitor is effective for treating osteoporosis, rheumatoid arthritis, and the like.

3) Screening for Proteins that Regulate Promoter Activity To regulate the promoter activity of the DNA of the present invention, in addition to the competitive inhibition using the above-described partial sequences and the like, the D
A method utilizing a protein that regulates NA promoter activity can also be used. The DNA of the present invention can be used for screening a protein that regulates its promoter activity. Therefore, the present invention relates to a method for screening a protein that regulates cathepsin K promoter activity as described below.
Such proteins include proteins that directly bind to the DNA of the present invention to promote or inhibit its promoter activity, and also act on cell membrane receptors or intracellular proteins to indirectly promote the promoter activity of the DNA of the present invention. And proteins that promote or inhibit the activity.

3-1) Screening for Proteins That Bind to Promoter DNA This method includes a step of contacting a test protein sample with the DNA of the present invention to purify the protein that binds to the DNA of the present invention. Such a method includes, for example, affinity purification of a protein binding thereto using the promoter DNA of the present invention. As an example of a specific method, the promoter DNA of the present invention is biotinylated and bound to magnetic beads to which streptavidin has been bound to prepare DNA affinity beads. This is then incubated with the nuclear extract of the cells to purify the protein in the nuclear extract that specifically binds to the promoter DNA of the present invention, and its structure is determined. As a result, a protein that directly binds to the promoter DNA of the present invention and a protein that does not have DNA binding activity but forms a complex with the protein as a subunit and binds to the promoter DNA of the present invention can be purified (Gabrie
lsen OS et al., Nucleic acid Research 17, 6253-62
67 (1989), Savoysky E et al., Oncogene 9, 1839-184
6 (1994)).

3-2) Screening of Protein Using Promoter Activity as an Index In this method, a test DNA is introduced into cells having a reporter gene linked downstream of the DNA of the present invention to regulate the expression of the reporter gene. Selecting an expression product to be expressed. Such a method includes, for example, a one-hybrid method using yeast or animal cells. Specifically, a reporter gene into which the promoter DNA of the present invention has been inserted is stably introduced into cells, and then a gene library is introduced into the cells to select a clone that exhibits expression promotion or inhibition of expression of the reporter gene. Then, a protein that binds to the promoter DNA of the present invention is selected. According to this method, in addition to the protein directly binding to the promoter DNA of the present invention, a protein that acts on an intracellular protein and indirectly regulates the activity of the promoter DNA of the present invention can be obtained. The yeast one-hybrid method (Li
JJ and Herskowitz I., Science 262, 1870-1873 (1
993), Wang MMand Reed RR, Nature 364, 121-126
(1993)), and the "Matchmaker system (Clontech)" has already been released as a kit.

In still another embodiment, a test sample is contacted with cells containing a reporter gene linked downstream of the promoter DNA of the present invention, and a protein that indirectly regulates the expression of the reporter gene is selected. Process. As an example of a specific method, a cell into which a reporter gene into which a promoter DNA of the present invention has been stably introduced is incubated with a test sample (for example, a culture supernatant of a cell into which a gene library has been introduced). Then, a protein that promotes or inhibits expression of the reporter gene is selected. In this method, a protein that indirectly affects the activity of the promoter DNA of the present invention via a cell membrane receptor or the like is obtained.

4) Method for Screening DNA Encoding Protein That Regulates Promoter Activity Using the DNA of the present invention, it is also possible to directly isolate DNA encoding a protein that regulates promoter activity. The present invention also relates to a DNA encoding a protein that regulates the promoter activity of the DNA of the present invention.
A screening method. One embodiment of this screening method includes a step of bringing the expression product of a test DNA into contact with the DNA of the present invention and selecting a DNA encoding the expression product that binds to the DNA of the present invention. Such methods include, for example, the Southwestern method. Specifically, each protein is expressed in Escherichia coli into which a gene library has been introduced, and these are transferred to a filter membrane, and then directly blotted using the promoter DNA of the present invention as a probe to express a protein that binds to the DNA probe. A clone is selected and the gene encoding it is isolated. According to this method, a gene encoding a protein having an activity of binding to the DNA of the present invention can be obtained (see Experimental Medicine Separate Volume, Bio Manual Series, “Transcription Factor Research Method”, Yodosha Co., Ltd., pp. 177-188). .

In another embodiment, a test D cell is added to a cell carrying a reporter gene linked downstream of the DNA of the present invention.
A step of introducing NA and selecting a DNA encoding an expression product that regulates the expression of the reporter gene. Such methods include, for example, the above-described one-hybrid method using yeast or animal cells. That is, a reporter gene into which the promoter DNA of the present invention has been inserted is stably introduced into cells, and then a gene library is introduced into the cells to select a clone that exhibits the promotion or inhibition of expression of the reporter gene. And a gene encoding a protein that binds to the promoter DNA. By this method, in addition to a gene encoding a protein that directly binds to the promoter DNA of the present invention, a gene encoding a protein that acts on an endogenous protein in a cell to indirectly regulate the activity of the promoter DNA of the present invention Can be obtained.

In still another embodiment, the DNA of the present invention
Contacting the expression product of the test DNA with a cell carrying a reporter gene linked downstream of the method, and selecting a DNA encoding the expression product that regulates the expression of the reporter gene. As an example of a specific method, a cell into which a reporter gene into which a promoter DNA of the present invention is inserted is stably introduced is incubated with a test protein (for example, a culture supernatant of a cell into which a gene library has been introduced). Thus, a protein or a gene encoding the protein that promotes or inhibits expression of the reporter gene is isolated. According to this method, a gene encoding a protein that acts on the activity of the promoter DNA of the present invention indirectly via a cell membrane receptor or the like can be obtained.

5) Screening for a compound that regulates promoter activity Using the DNA of the present invention, it is possible to screen for a synthetic compound that regulates the promoter activity. That is, the present invention relates to a method for screening a compound that regulates cathepsin K promoter activity as described below.

5-1) Screening Using Binding between Promoter and Protein as an Index In this method, the DNA of the present invention is brought into contact with the test sample in the presence of the test compound, Selecting a compound that promotes or inhibits binding to the protein therein. Specifically, for example, a cell nuclear extract is
After binding to the promoter DNA probe of the present invention labeled with an isotope or the like, a band of a complex between the protein in the nuclear extract and the promoter DNA of the present invention is detected by gel shift method by polyacrylamide gel electrophoresis (Experimental Medicine Supplement. Bio-manual series “Transcription factor research method”, published by Yodosha Co., Ltd., pp. 107-112). When the DNA probe is added, the test compound is also added, and the protein in the nuclear extract and the promoter D of the present invention are added.
A compound that promotes or inhibits the formation of a complex band with NA is selected. In this method, a compound that directly acts on the promoter DNA of the present invention and a compound that acts on a protein that binds to the promoter DNA of the present invention can be obtained. For example, when a protein that binds to the promoter DNA of the present invention inhibits the activity of the promoter DNA of the present invention in vivo, a compound that inhibits the binding of this protein to the promoter DNA of the present invention may be a compound of the present invention. It is considered that the activity of the promoter DNA can be promoted. If a protein that binds to the promoter DNA of the present invention has already been isolated, a recombinant protein of the protein can be used instead of the cell nuclear extract.

5-2) Screening using promoter activity as an index In this method, a test compound is brought into contact with a cell holding a reporter gene linked downstream of the promoter DNA of the present invention to regulate the expression of the reporter gene. Selecting a compound. For example, the promoter D of the present invention
A luciferase gene vector into which NA has been inserted upstream is introduced into cells to prepare cells that stably express luciferase activity dependent on the promoter DNA of the present invention, and the cells and the test compound are incubated in a culture solution. Promoter DNA of the present invention using luciferase activity as an indicator
Is measured, and a compound that promotes or inhibits the promoter activity is selected. In this method, a compound that directly or indirectly regulates the activity of the promoter DNA of the present invention can be obtained.

When a protein that regulates the activity of the promoter DNA of the present invention has already been obtained, the protein (or a derivative thereof) can be obtained in the presence of a test compound.
Is brought into contact with the promoter DNA of the present invention, and the protein (or derivative thereof) is brought into contact with the promoter DN of the present invention.
By selecting a compound that promotes or inhibits the binding to A, a compound that regulates the activity of the promoter DNA of the present invention can be screened. Specifically, for example, a protein that binds to the promoter DNA of the present invention fused with glutathione S-transferase (DN
A binding domain may be purified), bound to a microplate covered with an anti-glutathione S-transferase antibody, and then contacted with the biotinylated promoter DNA of the present invention. Binding to DNA is detected with streptavidinated alkaline phosphatase. When adding the promoter DNA of the present invention, a test compound is also added, and a compound that promotes or inhibits the binding between the protein and the promoter DNA of the present invention is selected. in this way,
Compounds that act directly on the promoter DNA of the present invention and compounds that act on proteins that bind to the promoter of the present invention are obtained. For example, the promoter D of the present invention
When a protein that binds to NA inhibits the activity of the promoter DNA of the present invention in vivo, it is thought that a compound that inhibits the binding of the protein to the promoter of the present invention can promote the activity of the promoter DNA of the present invention. Can be

[0034]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

Embodiment 1 Mouse cathepsin K gene D
Isolation of NA and construction of luciferase expression plasmid vector a) Construction of plasmid pGL3-mCTSK-1.7 The mouse chromosomal DNA contained approximately 2 × 10 ⁷ RAW26
Using a 4.7 cell (purchased from Dainippon Pharmaceutical Co., Ltd.) as a starting material, a DNA extraction and purification kit (Blood & Cell Culture)
DNA Midi Kit (Qiagen)) was used according to the attached protocol.

On the other hand, the following nucleotide sequence was used as a PCR primer: 5′-atggtacctc gactcgatct gggctctctc tactca-3 ′
(Primer 1: SEQ ID NO: 2 in the sequence listing) and 5'-taagatctgt gagcggaaga ctaagggtgc tagat -3 '
An oligonucleotide having (Primer 2: SEQ ID NO: 3 in Sequence Listing) was chemically synthesized.

The above primers (final concentration: 0.2 μM each)
And 25 μl of PCR reagent mixture (One-shot LA
PCR mix (manufactured by Takara Shuzo Co., Ltd.)), 1 μg of the above mouse chromosomal DNA and sterilized water, and the total volume was 50 μl.
PCR was performed using a thermal cycler (Gene Amp PCR System 9600, manufactured by Perkin Elmer Japan Applied Biosystems Division) (temperature conditions: heated at 94 ° C. for 2 minutes, then 9
A temperature cycle of 20 seconds at 8 ° C and 2 minutes 30 seconds at 68 ° C
This was repeated 0 times, further heated at 72 ° C. for 10 minutes, and then cooled and stored at 4 ° C.).

The reaction product was electrophoresed on a 0.8% agarose gel (manufactured by Gibco BRL). After the gel after electrophoresis was stained with ethidium bromide, a band portion corresponding to about 1.7 kbp was separated using a razor blade under ultraviolet irradiation. The DNA contained in this gel was purified using an extraction purification kit (QIAquick Gel Extraction Kit (Qiagen)) according to the attached protocol.

Next, a part of the purified DNA fragment was digested with restriction enzymes KpnI and BglII, and kept at 70 ° C. for 15 minutes to inactivate the enzyme. On the other hand, the luciferase expression vector pGL3-Basic (manufactured by Promega) is similarly digested with KpnI and BglII, and the end is dephosphorylated using bovine intestinal alkaline phosphatase (manufactured by Toyobo Co., Ltd.). Ligation was performed using a DNA ligation kit (version 2, manufactured by Takara Shuzo Co., Ltd.). Escherichia coli DH5α competent cells (manufactured by Toyobo Co., Ltd.) were transformed with this DNA to obtain ampicillin-resistant colonies.

Plasmids were extracted from cultured cells of several colonies and double-cut with the restriction enzymes KpnI and BglII, and mouse cathepsin was used as an indicator that fragments of about 1.7 kbp and about 4.8 kbp were generated. Plasmid DNA into which DNA upstream of the 5 'end of the K gene has been inserted
I got The nucleotide sequence inserted into the obtained plasmid DNA was analyzed using a DNA sequencer (Model 3700, manufactured by PerkinElmer Japan Applied Biosystems Division).
It turned out that it is the sequence shown by sequence number 1 of a sequence table. This sequence was obtained from the purified approximately 1.7 kbp PCR
Although the fragment was identical to the nucleotide sequence decoded by direct sequencing, Li, YI and Chen,
W. reports (J. Bone. Miner. Res. 14, 487-499 (199
9)), and 9 differences from the sequence registered as a mouse cathepsin K gene in the GenBank database (accession number: AJ006033). This plasmid is called pGL3-m
It was named CTSK-1.7. The transformed Escherichia coli carrying this plasmid was cultured overnight at 37 ° C. in 100 ml of a liquid LB medium containing 50 μg / ml of ampicillin, and a plasmid extraction and purification kit (Endfree Plasmid Maxi Kit) (Manufactured by Qiagen)) to obtain pGL3-mCTSK-1.7 DNA
Was collected and purified.

This plasmid was replaced with the restriction enzyme Kp.
Insert D digested with nI and BglII and cut out
NA was pUC1 similarly treated with KpnI and BamHI.
E. coli E. coli harboring a plasmid ligated to the E.8 vector. E. coli pUC18-mCTSK-1.7
SANK 70500 was internationally deposited on July 7, 2000 (Heisei 12) at the Institute of Biotechnology and Industrial Technology, and given the accession number FERM BP-7208.

B) Plasmid pGL3-mCTSK-
Construction of 0.6 A part of the pGL3-mCTSK-1.7 plasmid obtained in the above a) was digested with the restriction enzymes PmaCI and BglII, subjected to 1.0% agarose gel electrophoresis, and described in a). In this manner, a DNA fragment containing about 600 bp of the upstream region of the 5 'end of mouse cathepsin K was isolated and purified.

On the other hand, the luciferase expression vector pGL
3-Basic (manufactured by Promega) was also converted to SmaI and B
After digestion with glII and dephosphorylating the terminal using bovine intestinal alkaline phosphatase (manufactured by Toyobo Co., Ltd.),
The above DNA fragment was ligated with a DNA ligation kit (version 2, Takara Shuzo Co., Ltd.). This D
E. coli DH5α competent cells (manufactured by Toyobo Co., Ltd.) were transformed with NA to obtain ampicillin-resistant colonies. Plasmids were extracted from cultured cells of several colonies and double-cut with the restriction enzymes KpnI and BglII to generate fragments of about 0.6 kbp and about 4.8 kbp. '
A plasmid DNA into which about 600 bp DNA in the terminal upstream region was inserted was obtained to obtain pGL3-mCTSK-0.6. 50 transformed E. coli harboring this plasmid
100 ml of liquid LB containing μg / ml of ampicillin
The cells were cultured overnight at 37 ° C. in a medium, and pGL3- was purified from the culture using a plasmid extraction and purification kit (Endfree Plasmid Maxi Kit (Qiagen)).
mCTSK-0.6 DNA was recovered and purified.

C) Plasmid pGL3-mCTSK-
Construction of 0.2 As a forward PCR primer, an oligonucleotide having the following nucleotide sequence: 5′-atggtaccga aactaaacac ttagcagcta-3 ′ (primer 3: SEQ ID NO: 4 in the sequence listing) was synthesized (Amersham
Pharmacia).

The above forward PCR primer (final concentration: 0.
2 μM), reverse PCR primer (primer 2) (final concentration 0.2 μM) used in a) of Example 1, 25 μl
PCR reagent mixture (one-shot LA PCR mix (Takara Shuzo Co., Ltd.)), pGL obtained in a) above
100 ng of 3-mCTSK-1.7 plasmid DNA
And sterilized water to make a total volume of 50 μl. Thermal cycler (GeneAmp PCR System 9600, Inc.)
PCR was performed using PerkinElmer Japan Applied Biosystems Division (temperature condition: 9).
Heat at 4 ° C for 2 minutes, then at 94 ° C for 30 seconds, 6
A temperature cycle at 0 ° C. for 30 seconds and 72 ° C. for 2 minutes was repeated 28 times, and further heated at 72 ° C. for 10 minutes, and then cooled and stored at 4 ° C.). This reaction product was electrophoresed on a 1.5% agarose gel (manufactured by Gibco BRL). After the gel after electrophoresis was stained with ethidium bromide, a band portion corresponding to about 200 bp was separated using a razor blade under ultraviolet irradiation. The DNA fragment contained in this gel piece was
NA extraction and purification kit (QIAquick Gel Extraction Kit
(Manufactured by Qiagen)) according to the attached protocol.

About one-eighth of the purified DNA fragment was digested with restriction enzymes KpnI and BglII,
The restriction enzyme was inactivated by heating for 5 minutes. On the other hand, the luciferase expression vector pGL3-Basic
(Promega) is similarly digested with KpnI and BglII, the ends are dephosphorylated using bovine intestinal alkaline phosphatase (manufactured by Toyobo Co., Ltd.), and then the above DNA fragment and DNA ligation kit (version 2, Takara Shuzo) (Manufactured by K.K.). Escherichia coli DH5α competent cells (manufactured by Toyobo Co., Ltd.) were transformed with this DNA to obtain ampicillin-resistant colonies. Plasmids were extracted from cultured cells of several colonies and double-cut with restriction enzymes KpnI and BglII.
Using the occurrence of fragments of 2 kbp and about 4.8 kbp as an index, the mouse cathepsin K gene 5′-terminal upstream region of about 2 kbp was used.
The plasmid DNA into which 00 bp DNA was inserted was obtained to obtain pGL3-mCTSK-0.2. The nucleotide sequence inserted into the obtained plasmid DNA was determined by a DNA sequencer (Model 3700, Inc.).
(PerkinElmer Japan Applied Biosystems Division), and it was confirmed that the nucleotide sequence coincided with the nucleotide sequence shown in nucleotide numbers 1471-1675 of SEQ ID NO: 1 in the sequence listing. The transformed Escherichia coli carrying this plasmid was cultured overnight at 37 ° C. in 100 ml of a liquid LB medium containing 50 μg / ml of ampicillin, and a plasmid extraction and purification kit (Endfree Plasmid Maxi Kit) (Manufactured by Qiagen)) to obtain pGL3-mCTSK-0.2
DNA was recovered and purified.

Embodiment 2 FIG. Glutathione-S-transferase (GST) and mouse osteoclast differentiation factor (ODF)
Preparation of extracellular region fusion protein (GST-mODF) a) Construction of plasmid GST-mODF Mouse O registered in GenBank database
In order to obtain a cDNA corresponding to the extracellular region of the DF gene sequence (registration number: 019048) and to add a sequence recognized by the restriction enzyme SmaI, the following nucleotide sequence: 5′-gtgcccgggc agcgcttctc agg -3 '
(S1: SEQ ID NO: 5 in the sequence listing); and 5′-catcccgggt
Two types of oligonucleotide primers having cagtctatgt cctgaact -3 ′ (AS1: SEQ ID NO: 6 in the sequence listing) were synthesized using a DNA synthesizer (Oligo 1000 (manufactured by Beckman)).

The total RN was obtained by using an RNA extraction reagent (ISOGEN (manufactured by Nippon Gene Co., Ltd.)) from primary mouse bone marrow cells according to the attached protocol.
A was obtained, and RT-PCR was performed using a kit (RNA PCR kit AMV ver2.1 (manufactured by Takara Shuzo Co., Ltd.)). That is, first, a reverse transcription reaction was performed on this total RNA at 42 ° C. for 30 minutes to synthesize single-stranded DNA (reaction volume: 20 μl). As a primer for the reverse transcription reaction, a random primer attached to the kit was used.

Next, using this single-stranded DNA as a template,
PCR was carried out using the above oligonucleotides S1 and AS1 as primers (reaction volume 100 μl).
l. Temperature conditions: 94 ° C for 1 minute, 55 ° C for 30 seconds, 72 ° C
And the temperature cycle of 30 seconds was repeated 35 times). This P
A part of the reaction solution after CR is taken, and the amplified DNA fragment is converted into a plasmid (pCR) using a TA cloning kit (manufactured by Invitrogen) according to the attached protocol.
By cloning into the plasmid pC
RII-mODF was obtained. The nucleotide sequence of the inserted DNA fragment was confirmed by the dideoxynucleotide chain termination method.

The obtained plasmid pCRII-mODF
Was digested with a restriction enzyme SmaI, separated by 1.0% agarose gel electrophoresis, and then the mODF DNA fragment was purified. On the other hand, a plasmid pGEX- for expressing glutathione-S-transferase (hereinafter referred to as “GST”)
3X (Amersham Pharmacia) also has the restriction enzyme Sm
Similarly digested with aI and purified by 1.0% agarose gel electrophoresis. Then, these fragments were converted into a DNA ligation kit (version 2, Takara Shuzo Co., Ltd.).
The GST-mODF expression plasmid pG
EX-3X-mODF was obtained.

B) Expression and purification of GST-mODF protein in Escherichia coli The plasmid of pGEX-3X-mODF obtained above was p
GST under the control of the lac promoter from GEX-3X
Since the mODF gene is linked so that the reading frame matches the gene, the GST-mODF fusion protein can be expressed by introducing this plasmid into a host. In addition, pGEX-
The amino acid sequence of the fusion protein expressed by 3X-mODF is shown.

The above plasmid pGEX-3X-mOD
F. was used to transform E. coli TOP10 strain by an ordinary method.
TOP10 strain carrying pGEX-3X-mODF was
In an L-broth medium (100 g of tryptone (manufactured by Difco), 5 g of yeast extract (manufactured by Difco), and 5 g of sodium chloride in a 1-liter aqueous solution each containing 100 μg / ml of ampicillin)
After shaking culture at 7 ° C for 4 hours, ampicillin 1 was added.
Three liters of L-broth medium containing 00 μg / ml was inoculated with 5.0% (v / v), and cultured with shaking at 37 ° C. for 3 hours. Thereafter, isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.1 mM, and shaking culture was performed at 25 ° C. for 20 hours. After completion of the culture, the culture solution was centrifuged (8000 × g, 10 minutes) to collect the precipitated cells, which were suspended in 150 ml of Dulbecco's phosphate buffer (hereinafter referred to as “PBS”). Then, the cells were disrupted by ultrasonication, and Triton X-10 was used.
0 to a final concentration of 1% (v / v), and
Shake gently for 0 minutes. High speed centrifugation (11000 ×
g, 15 minutes), and 1.5 ml of glutathione Sepharose 4B gel was added to the supernatant, followed by gentle shaking for 30 minutes. By the batch method, 10
mM reduced glutathione solution (dissolved in PBS)
Elution was performed using 7.5 ml. The eluate was added to a DEAE Toyopearl 650M column equilibrated with 10 mM Tris-hydrochloric acid (pH 7.0), and anion exchange chromatography was performed using a linear gradient of sodium chloride (0-1).
M). A part of the eluate was taken and subjected to SDS polyacrylamide gel (gel concentration 12.5
%) An electrophoresis was performed, and a fraction having a band at a position corresponding to the expected molecular weight (46000) of the fusion protein was collected. The recovered crude product is further
Purification was performed by gel filtration using Sephacryl S-100 equilibrated with PBS. Take a part of the eluate and use SD
An S-polyacrylamide gel (gel concentration: 12.5%) was subjected to electrophoresis (prestained marker SP-0120 manufactured by Apro) was used as a molecular weight marker. Were collected. The fusion protein thus obtained was used in the following examples.

Embodiment 3 FIG. Measurement of Cathepsin K Gene Promoter Activity The mouse cathepsin K gene 5′-terminal upstream region isolated in Example 1 (about 1.7 kbp, about 600 bp and about 20 kbp)
In order to evaluate the transcription activity possessed by 0 bp), an assay was performed by the reporter gene method. That is, the firefly luciferase expression plasmids (pGL3-mCTSK-1.7, pGL3-mCTS
K-0.6 or pGL3-mCTSK-0.2)
Mouse RAW 264.7 cells (purchased from Dainippon Pharmaceutical Co., Ltd.), which are known to express the cathepsin K gene, were transfected to examine changes in firefly luciferase activity. In addition, Renilla luciferase expression plasmid pRL-SV40 (promega) was used as an internal standard for correcting experimental lot differences between plasmids in transfection efficiency into cells.

First, mouse RAW 264.7 cells were
0% fetal bovine serum (manufactured by Gibco BRL) and
Medium containing penicillin / streptomycin
(Manufactured by Gibco BRL) at 37 ° C with 5% charcoal
The cells were cultured in an acid gas incubator. Semicon full
Medium-angle flask grown to an ent (Sumitomo Bake
Light (manufactured by Light Inc.), PBS (-) (Dulbecco PBS)
(-) "Nissui", manufactured by Nissui Pharmaceutical Co., Ltd.)
Use cell scraper (Sumitomo Bakelite Co., Ltd.)
The cells were scraped off and collected. Transfer the cells to 1x TBS
(++) (25 mM Tris-HCl (pH 7.5), 13
7 mM sodium chloride, 5 mM potassium chloride, 0.1 mM
5 mM disodium phosphate, 0.49 mM Mag chloride
Nesium, 0.68 mM calcium chloride)
Count the number of cells, 2.5 × 10 ⁶15 ml each
The cells were transferred to a centrifuge tube and centrifuged to collect the cells. on the other hand
The above firefly luciferase expression plasmid
g, the above Renilla expression plasmid 0.4 μg,
0.5 mg / ml DEAE-dextran (Promega
0.4 ml of 1 × TBS (++) solution containing
did. This DNA / DEAE-dextran solution was
2.5 × 10⁶Resuspend cells for 10 minutes and lighten every 10 minutes
Incubate for 30 minutes at room temperature with good agitation.
Was. Then, the cells were added to 10 ml of 1 × TBS (++).
, And collected by centrifugation.
Suspended in DMEM medium containing 10% fetal bovine serum, 9
0.1 ml / well in a 6-well plate (Corning)
Sowed with le. After culturing overnight at 37 ° C,
Or GST-mODF diluted with medium (final concentration 100
ng / ml) at 37 ° C. for another 2 μl.
After culturing for 4 hours, the cells were washed twice with 100 μl of PBS (−).
After washing, 50 μl of cell lysing agent (Passive Lysis Buffer)
(Promega)) to prepare a cell extract.
Was. Then, using 10 μl of the mixture,
Luciferase reporter assay system (pro
Romega) according to the attached protocol.
And firefly and Renilla luciferase activity
Was measured (Luminoscan (Lab Systems))
use). The protein concentration in the cell extract is
Reagents for measuring protein concentration (Bio-Rad, Protein,
Assay (manufactured by Bio-Rad). Real
For the test results, see the internal standard Renilla lucifera
Firefly luciferase activity corrected for
did.

The results are shown in FIG. 1 (in FIG. 1, "-1650 to +
5 "," -608 to +5 "and" -200 to +5 "are pG
L3-mCTSK-1.7, pGL3-mCTSK-
0.6 and pGL3-mCTSK-0.2 were introduced). The D-encoding luciferase is located in the cathepsin K 5 'terminal upstream region about 1.7 kbp constructed this time.
When ligated NA was introduced into RAW 264.7 cells, luciferase activity was observed even without stimulation. As a result, it was revealed that the promoter activity was present in this region. Furthermore, pGL3-mC
When cells transformed with TSK-1.7 or pGL3-mCTSK-0.6 are stimulated with GST-mODF, which is known to differentiate into osteoclasts, the expression of cathepsin K mRNA is stimulated in parallel. In addition, remarkable induction of luciferase activity was observed. The 5 'terminal upstream region is 1.
7 kbp (pGL3-mCTSK-1.7) to 0.6
When shortened to kbp (pGL3-mCTSK-0.6), the activity itself when unstimulated was reduced to about one-fourth to one-fifth, but the ability to induce activity by GST-mODF stimulation (fold value ) Did not make a big difference. However, the 5 'terminal upstream region was 0.2 kbp (pGL3-mC
Further shortening to TSK-0.2) significantly reduced both the unstimulated activity and the induction of activity by GST-mODF stimulation.

As a result, the cathepsin K gene 5'-terminal upstream region was about 0.6 kbp to 0.2 kbp, and O
It was revealed that DF stimulation-responsive promoter activity was present.

In the method of this embodiment, for example, pGL3
-MCTSK-1.7 or pGL3-mCTSK-
RAW264.7 cells transformed with 0.6 were transformed into GST-
The test substance is allowed to coexist when stimulated with mODF, or the cDD integrated into the expression vector is added to the transformed cell.
After the forced expression by introducing NA, a substance that inhibits the cathepsin K promoter activity can be tested by measuring the luciferase activity.

[0058]

As described above, according to the present invention, the ODF
Novel D with dependent cathepsin K promoter activity
NA was provided. By using the DNA of the present invention, it is possible to search for agents for treating or preventing various bone metabolic diseases including osteoporosis, bone destruction in rheumatoid arthritis, bone metastasis of cancer cells and bone destruction.

[Brief description of the drawings]

FIG. 1 is a view showing the results of detecting mouse cathepsin K gene promoter activity in RAW 264.7 cells by a reporter gene method.

[Sequence List Free Text]

SEQ ID NO: 2: PCR primer for amplifying mouse cathepsin K promoter DNA fragment SEQ ID NO: 3: PCR primer for amplifying mouse cathepsin K promoter DNA fragment SEQ ID NO: 5 5 ′ untranslated region of mouse cathepsin K gene Primer for amplifying a DNA fragment in the DNA SEQ ID NO: 5: cD encoding mouse osteoclast differentiation factor
PCR primers for amplifying NA SEQ ID NO: 6: cD encoding mouse osteoclast differentiation factor
PCR primer for amplifying NA SEQ ID NO: 7: Fusion protein consisting of glutathione-S-transferase and mouse osteoclast differentiation factor

[Sequence List] SEQUENCE LISTING <110> Sankyo Company, Limited <120> Cathepsin K Promoter and Uses Thereof <130> 2000 122SS <140> <141> <160> 7 <170> PatentIn Ver. 2.0 <210> 1 <211> 1675 <212> DNA <213> Mus musculus <400> 1 tcgactcgat ctgggctctc tctactcagg tggtacattg agatcctggg gtctagaaat 60 cctggggtct gtgccaggaa tccagctagg aactgaatct atatttgtgg taatatcctt 120 tgtgcttcac agtccctcat ttcctaattc ctcacatgtg tttatggaag taaatggagt 180 ttagggtgtg gttggccaag tcagactctt atgatctgtc acatgcctgg attttgggga 240 agcgcttgga gactcttgta actggaccta gtttgggatt ttaatgattg agatgcagta 300 ctcatcagac attctaccac ccgagagtct agcagtagag tcccacgtgt atccattaat 360 gtagattcaa gtgttgtaaa taattaggtg ctacattggg tcagttgtct ttccttactg 420 tcatgttggg tgttgatcct tgggcctcat aatgttagga aagcccacca ccgctgagtc 480 atgttcctca accctctttt agctttttgt aaattacttt tatcctccat ttaagcaact 540 agaatctttt tgtaaagttt atgtatatga gtacactatc tctgtcttca gacacaccag 600 aagagggtat cagatcccat tacagatggt tttgagccac catgtggttg ctgggaattg 660 aacttagg ac ctctggaaga ataggcagtg cttttaactg ctgagccatc tccctggccc 720 tgtagggcat ttcttagtgg ttgatgggag agagcccagc tcactgtgga tggtgccatt 780 cctgggctgg tgccccagcc gttgttattt ttagacagga tctcactaaa ttgcctagag 840 taggttgacc ttgaaagtaa gttcttactt agagacgtct cactgtatgg ctctggctga 900 ctttggactc actctgtaga ctgttgtggc attgacctca cagagaacca tccacccctg 960 ccatccagtg tgtatgttga ggggacagag gtgtgcccat agccagtgat ggctttgctt 1020 tagcttcctg aatatctggg attactgcac tgtctcacac acgtgacttt ctggccactt 1080 ttgaaccatc tcacctggac tttgtgggca gctcctccct tgatgtacag ccataatggg 1140 ttagaaaaac tactccatgc ccagaagcag ttaatagcaa acagtcacaa aacggtttta 1200 aatgtttaaa ttagagcctt acatttctag cctttcctcc cctctccctc actccacccc 1260 catcatctca gaagaggatc tgacacaacg ttggaaatgg tgcagagtaa ggaggtgggt 1320 cagaactaga tgagttgaaa agatggaaat tgccagtcag acattttgag gaagaatgga 1380 gacaactagg gctgtgttct taaatctccc accaaagtct caatttgaat attgtgctat 1440 cttatttttg ccccccaaag tcagtcagat gaaactaaac acttagcagc tatccacact 1500 ggcatatgat actgtat aca cacattgtgc aaatgtgtgt ccttcctccc taacccctcc 1560 tccttttcat cctatccctg actccctccc ttatccagat tttccagcca ctgctggagt 1620 tgacttccgc aatccttacc gaataaatct agcaccctta gtct <br> <br> <br> <br> to amplify a mouse cathepsin K promoter DNA fragment <400> 2 atggtacctc gactcgatct gggctctctc tactca 36 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a mouse cathepsin K promoter DNA fragment <400> 3 taagatctgt gagcggaaga ctaagggtgc tagat 35 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment in the 5 'non-coding region of mouse cathepsin K gene <400> 4 atggtaccga aactaaacac ttagcagcta 30 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a cDNA encoding mous e osteoclast differentiation factor <400> 5 gtgcccgggc agcgcttctc agg 23 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a cDNA encoding mouse osteoclast differentiation factor <400> 6 catcccgggt cagtctatgt cctgaact 28 <210> 7 <211> 409 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: a fusion protein consisting of a glutathione-S-transferase and a mouse osteoclast differentiation factor <400> 7 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg I le Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Ile Glu Gly 210 215 220 Arg Gly Ile Pro Gly Gln Arg Phe Ser Gly Ala Pro Ala Met Met Glu 225 230 235 240 Gly Ser Trp Leu Asp Val Ala Gln Arg Gly Lys Pro Glu Ala Gln Pro 245 250 255 Phe Ala His Leu Thr Ile Asn Ala Ala Ser Ile Pro Ser Gly Ser His 260 265 270 Lys Val Thr Leu Ser Ser Trp Tyr His Asp Arg Gly Trp Ala Lys Ile 275 280 285 Ser Asn Met Thr Leu Ser Asn Gly Lys Leu Arg Val Asn Gln Asp Gly 290 295 300 Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His His G lu Thr Ser 305 310 315 320 Gly Ser Val Pro Thr Asp Tyr Leu Gln Leu Met Val Tyr Val Val Lys 325 330 335 Thr Ser Ile Lys Ile Pro Ser Ser His Asn Leu Met Lys Gly Gly Ser 340 345 350 Thrys Lys Asn Trp Ser Gly Asn Ser Glu Phe His Phe Tyr Ser Ile Asn 355 360 365 Val Gly Gly Phe Phe Lys Leu Arg Ala Gly Glu Glu Ile Ser Ile Gln 370 375 380 Val Ser Asn Pro Ser Leu Leu Asp Pro Asp Gln Asp Ala Thr Tyr Phe 385 390 395 400 Gly Ala Phe Lys Val Gln Asp Ile Asp 405

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/66 C12Q 1/68 A 1/68 (C12N 1/21 // (C12N 1/21 C12R 1: 69) C12R 1:69) (C12Q 1/28 (C12Q 1/28 C12R 1:19) C12R 1:19) (C12Q 1/42 (C12Q 1/42 C12R 1:19) C12R 1:19) (C12Q 1 / 54 (C12Q 1/54 C12R 1:19) C12R 1:19) (C12Q 1/66 (C12Q 1/66 C12R 1:19) C12R 1:19) (C12Q 1/68 A (C12Q 1/68 C12R 1 : 19) C12R 1:19) C12N 15/00 ZNAA F term (reference) 4B024 AA01 AA11 BA07 CA04 DA06 EA04 GA11 HA11 HA14 HA15 4B063 QA01 QA20 QQ22 QQ33 QQ35 QQ42 QR08 QR32 QR42 QR66 QS25 QS33 QS34 Q0202B04A CA27 CA46

Claims (18)

[Claims] 1. A DNA represented by any one of the following 1) to 4): 1) In the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing, any one of nucleotides 1 to 1063 is replaced by 5 A DNA consisting of a nucleotide sequence having a 3'-terminal at nucleotide number 1675 at the 2'-terminal; 2) a transformed E. coli strain coli pUC18-mC
TSK-1.7 SANK 70500 (FERM B
P-7208), the DNA inserted into the pUC18 vector multiple cloning site in the recombinant plasmid vector; 3) hybridized with the DNA described in 1) or 2) above under stringent conditions, Osteocyte differentiation factor (os
teoclast differentiation factor: hereinafter referred to as "ODF")-dependent promoter activity; 4) an ODF-dependent promoter activity comprising a nucleotide sequence having 95% or more nucleotide sequence identity with the DNA described in 1) or 2) above. DNA having the formula: 2. A DNA comprising a nucleotide sequence represented by nucleotide numbers 1063 to 1471 of SEQ ID NO: 1 in the sequence listing and having ODF-dependent promoter activity. 3. A DNA comprising a nucleotide sequence represented by nucleotide numbers 1063 to 1675 of SEQ ID NO: 1 in the sequence listing. 4. A DNA comprising a nucleotide sequence represented by nucleotide numbers 1 to 1675 of SEQ ID NO: 1 in the sequence listing.
NA. A vector comprising the DNA according to any one of claims 1 to 4. 6. The vector according to claim 5, wherein a DNA encoding a protein is ligated downstream of the DNA according to any one of claims 1 to 4. 7. The DNA linked downstream of any one of claims 1 to 4 is selected from the group consisting of luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase and green fluorescent protein. D that encodes a protein
The vector according to claim 6, wherein the vector is NA. 8. The vector according to claim 7, wherein the DNA linked downstream of the DNA according to any one of claims 1 to 4 is a DNA encoding luciferase. 9. The transformed E. coli strain E. coli. coli pUC
18-mCTSK-1.7 SANK 70500 (F
ERM BP-7208). 10. A host cell transformed with the vector according to any one of claims 5 to 9. 11. The transformed E. coli strain E. coli. coli pU
C18-mCTSK-1.7 SANK 70500
(FERM BP-7208). 12. A method for testing a substance that regulates cathepsin K promoter activity, comprising the steps i) and ii): i) transforming with the vector according to any one of claims 6 to 8. Ii) in the vector according to any one of claims 6 to 8 in a host cell according to i) above, in a vector according to any one of claims 6 to 8. D linked downstream of DNA
Measuring the expression level of the protein encoded by NA. 13. The method according to claim 12, wherein the host cell is contacted with a test substance and ODF in step i). 14. A method for testing a cDNA encoding a protein having an activity of regulating cathepsin K promoter activity, comprising the following steps i) and ii): i) any one of claims 6 to 8 A host cell transformed with the vector according to the above, is transformed with the cDNA incorporated in the expression vector for mammals; ii) The vector according to any one of claims 6 to 8 in the host cell of the above i). Wherein D is linked downstream of the DNA according to any one of claims 1 to 4.
Measuring the expression level of the protein encoded by NA. 15. The method of claim 14, comprising the step of contacting the host cell with ODF. 16. A method for screening a protein that regulates cathepsin K promoter activity, comprising contacting a sample containing the protein with the DNA according to any one of claims 1 to 4, and then contacting the DNA with the DNA. A method comprising separating bound proteins. 17. A method for testing a substance having an activity of regulating cathepsin K promoter activity, comprising the steps i) and ii): i) using the DNA according to any one of claims 1 to 4 A radiolabeled product obtained by adding a nuclear extract of mouse cells and a test substance, and a product obtained by adding only a nuclear extract of mouse cells are prepared; ii) Electrophoresis of each mixture sample of the above i) And comparing the mobility between a band found in a sample to which only a nuclear extract of mouse cells has been added and a band found in a sample to which a test substance has been added. 18. A DNA comprising at least 10 contiguous nucleotides, a DNA having an antisense sequence thereof or a derivative thereof in the nucleotide sequence represented by nucleotide numbers 1 to 1675 of SEQ ID NO: 1 in the sequence listing.