WO2015147506A1 - Novel biomarker piro for osteoclast fusion - Google Patents

Novel biomarker piro for osteoclast fusion Download PDF

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WO2015147506A1
WO2015147506A1 PCT/KR2015/002823 KR2015002823W WO2015147506A1 WO 2015147506 A1 WO2015147506 A1 WO 2015147506A1 KR 2015002823 W KR2015002823 W KR 2015002823W WO 2015147506 A1 WO2015147506 A1 WO 2015147506A1
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protein
seq
amino acid
osteoporosis
acid sequence
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윤병수
오재민
김주영
이명수
윤권하
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(주)오스티오뉴로젠
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to a new biomarker of osteoclast fusion, PIRO (Progranulin induced receptor like gene during Osteoclastogenesis), and specifically, an inhibitor thereof is effective by newly discovering PIRO as a factor involved in the fusion of osteoclasts causing osteoporosis.
  • PIRO Progranulin induced receptor like gene during Osteoclastogenesis
  • the present invention relates to a composition for preventing or treating osteoporosis as a component and inducing fusion of osteoclasts using this PIRO as an active ingredient.
  • osteoclast progenitors cells belong to the myeloid cell lineage, and when treated with a macrophage-colony stimulating factor (M-CSF) on the bone marrow cells, they differentiate into preosteoclasts and macrophages.
  • M-CSF macrophage-colony stimulating factor
  • RANK-ligand the master regulator of osteoclast differentiation, binds to RANK, a receptor, and produces a variety of signaling pathways, resulting in Ca 2+ -dependent kinases caused by changes in calcium oscillators in cells ( Activation of the transcription factor NFAT-c1 through activation of Ca 2+ -dependent kinase leads to the completion of expression of several proteins required for osteoclast differentiation.
  • cell-communication (coupling) factors that express osteoblasts or osteoclasts such as TGF- ⁇ , IGF, IFN- ⁇ , TNF- ⁇ and SemaD to regulate the amount and rate of osteoclast differentiation act like RANKL. Regulates osteoclast differentiation and death in homeostasis and pathology.
  • Progranulin is a 88kD glycoprotein consisting of seven half-granulin (Grn) domains consisting of twelve "cysteine-rich motifs".
  • the human proteome atlas is present as 80 kD glycosylated protein in serum or plasma. Although it has been found and called proepithelin and PC cell-derived growth factor in some groups, the official name of HUGO is "GRN".
  • Granulin peptides were first discovered as peptides secreted from white blood cells (Bateman et.al., BBRC, 1990). So far, it has been reported that it acts as a wound-healing factor on the function of granulin (Bateman et. Al., Nature Medicine, 2003), and also acts as a neuronal growth factor. Reported (Bateman et. Al., BMC Neuroscience, 2009; and Van Damme et, al., JCB, 2008). In addition, granulin is the causative gene of tau-negative familial FTD (Baker & Cruts et. Al., Nature, 2006) and metabolic hormone (Youn et. Al. , Diabetes, 2009), appetite suppressing hormone (Kim et al., Endocrinology, 2011), and insulin resistance factor (Matsubara et. Al., Cell Metabolism, 2012).
  • osteoporosis and Progenullin is an osteoclast differentiation factor and is a osteoporosis biomarker or a serum biomarker of prostate cancer.
  • PIRO progranulin-induced receptor-like molecules during Osteoclastogenesis
  • the present invention has a amino acid sequence of SEQ ID NO: 1 to 3 and the prevention or treatment of osteoporosis containing an inhibitor of a protein having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5 as an active ingredient It provides a pharmaceutical composition.
  • the present invention is a pharmaceutical for inhibiting osteoclast fusion containing an amino acid sequence of SEQ ID NO: 1 to 3 and containing an inhibitor of a protein having an amino acid sequence of SEQ ID NO: 4 or 5 or more than 80% homology as an active ingredient To provide a composition.
  • It provides a method for measuring the protein expression level to provide information of the diagnosis, treatment results or prognosis of osteoporosis comprising the step of selecting the individual of the protein mRNA or the expression level of the protein increased compared to the normal control group; .
  • the present invention is an antibody binding to a protein having an amino acid sequence of SEQ ID NO: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5, a nucleic acid complementary to the gene encoding the protein, and Provided are a kit for diagnosing, treating, or prognosing osteoporosis comprising any one selected from the group consisting of primers or probes specific for the gene.
  • test composition or compound to a protein expressing cell line having the amino acid sequence of SEQ ID NOs: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5;
  • test composition or compound to a protein expressing cell line having the amino acid sequence of SEQ ID NOs: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5;
  • the present invention provides a composition for inducing osteoclast fusion comprising a protein having an amino acid sequence of SEQ ID NO: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5.
  • PIRO forms an important axis in RANK signaling system and is a direct inducer of progranulin as a late stage factor of osteoclast fusion and bone resorption, so that PIRO is used as a biomarker to prevent or prevent new osteoporosis. It can be useful for developing therapeutics, diagnosing osteoporosis, evaluating treatment results or prognosis, and using PIRO inhibitors can effectively prevent or treat osteoporosis.
  • MBMC mouse bone marrow cells
  • HBMC human bone marrow cells
  • C human leukocytes
  • Figure 5 is a result of analyzing the effect down and down PGRN of rat bone marrow cells.
  • FIG. 6 is a result of analyzing the expression patterns of c-Fos, NFATc1, TRAP, OSCAR and PGRN according to PGRN knockdown in inducing osteoclast differentiation of rat bone marrow cells.
  • Figure 7 shows the results of serum PGRN concentration in LPS-treated or OVX mouse model, human osteoporosis patients and long bed patients.
  • Figure 9 shows the amino acid sequence of the murine PIRO protein (mPIRO-1, GM10800) and the structure in the biofilm.
  • Figure 10 shows the cDNA sequence of the human PIRO gene, the amino acid sequence of the PIRO protein and the structure in the biofilm.
  • Figure 11 shows the alignment of the phylogeny tree and amino acid sequence of the mouse PIRO gene group and human PIRO gene.
  • FIG. 12 is a result of analyzing the expression of PIRO according to PGRN knockdown in the process of inducing osteoclast differentiation between rat bone marrow cells and human bone marrow cells.
  • Figure 13 shows the results of analyzing MNC formation according to PIRO knockdown in the process of inducing osteoclast differentiation of rat bone marrow cells.
  • 15 is a schematic diagram showing the functions of PGRN and PIRO in the RANKL / RANK axis predicted in the present invention.
  • PIRO genes and proteins were found to be raised (approximately 20-fold) by progranulin (PGRN), an osteoclast differentiation hormone and an important biomarker of osteoporosis. It has been shown to be a very important factor in cell fusion and bone resorption. Accordingly, inhibition of PIRO can inhibit osteoclast fusion and bone resorption, prevent or treat osteoporosis, and confirm the diagnosis of osteoporosis, evaluation of treatment results or prognosis by confirming PIRO expression levels, and PIRO. By investigating whether or not inhibition is possible, an agent for preventing or treating osteoporosis and an osteoclast fusion inhibitor can be screened, and fusion of osteoclasts can be induced using PIRO as an active ingredient.
  • PGRN progranulin
  • the present invention provides a pharmaceutical composition for the prevention or treatment of osteoporosis, comprising an amino acid sequence of SEQ ID NO: 1 to 3 and containing an inhibitor of a protein having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5 as an active ingredient. .
  • the present invention also provides a pharmaceutical composition for inhibiting osteoclast fusion containing the inhibitor of the protein as an active ingredient.
  • the protein is called a PIRO protein
  • the gene encoding the PIRO protein is called a PIRO gene.
  • the PIRO protein is preferably composed of an amino acid sequence of SEQ ID NO: 4 or 5, but is not limited thereto, and may be composed of a sequence in which one or several amino acids are added, deleted or substituted in SEQ ID NO: 4 or 5.
  • the PIRO gene is preferably composed of the nucleotide sequence of SEQ ID NO: 6 or 7, but is not limited thereto, and may be composed of a sequence in which one or several bases are added, deleted or substituted in SEQ ID NO: 6 or 7.
  • the inhibitor may be an expression or activity inhibitor.
  • inhibitors of expression of the PIRO protein include antisense nucleotides that complementarily bind to the mRNA of the PIRO gene, and RNAi (short interfering RNA, short hairpin RNA and microRNA). It is preferably one selected from the group consisting of, wherein the inhibitor of the activity of the PIRO protein is preferably any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers and antibodies that complementarily bind to the PIRO protein. It is not limited. Also included are siPIRO2, which is a siRNA of mouse PIRO (mPIRO-1), and si-hPIRO2, which is a siRNA of human PIRO (hPIRO) used in an embodiment of the present invention.
  • RNA interference is a post-transcriptional gene silencing mechanism in which degradation of the corresponding mRNA occurs by introducing two stranded chain RNAs (dsRNAs) corresponding to the PIRO gene into a cell or organism.
  • dsRNAs two stranded chain RNAs
  • RNAi is a very powerful way to create targeted knockouts or 'knockdowns' at the RNA level, as multicellular division continues before the gene expression is restored by the RNAi effect (Elbashir et al. Nature). May 24; 411 (6836): 494-8, 2001).
  • RNAi techniques in gene silencing use standard molecular biology methods.
  • the dsRNA corresponding to the sequence of the target gene to be inactivated can be generated by standard methods, eg, both strands simultaneous transcription of template DNA using T7 RNA polymerase.
  • the production kit of dsRNA used for RNAi may use a commercially available product. Methods of transfection of plasmids treated to produce dsRNA or dsRNA are known in the art.
  • Nucleic acid molecules that are antisense to nucleic acids encoding PIRO can be used as inhibitors.
  • An 'antisense' nucleic acid comprises a nucleic acid sequence that is complementary to a 'sense' nucleic acid encoding a PIRO, eg, complementary to the coding strand of a two stranded chain cDNA molecule or complementary to an mRNA sequence.
  • the antisense nucleic acid can form hydrogen bonds with the sense nucleic acid.
  • the antisense nucleic acid may be complementary to the entire PIRO coding strand or only a portion thereof (eg, coding region).
  • the antisense nucleic acid molecule may be complementary to the entire coding region of the PIRO mRNA, but more preferably oligonucleotides that are antisense to only a portion of the coding or non-coding region of the PIRO mRNA (eg, translation initiation).
  • Antisense oligonucleotides can be, for example, about 5 to 50 nucleotides in length.
  • Antisense nucleic acids can be constructed using compound synthesis and enzyme binding reactions using known methods.
  • Mimetics eg, peptides or nonpeptidic agents
  • Mimetics that inhibit the protein binding domain of a PIRO polypeptide can be made to inhibit the original PIRO polypeptide from binding to VHL.
  • composition may further contain one or more active ingredients exhibiting the same or similar function in addition to the PIRO inhibitor.
  • the composition may contain 0.1 to 90 parts by weight of the PIRO inhibitor relative to 100 parts by weight of the total composition.
  • composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. And can be used in the form of general pharmaceutical formulations.
  • composition can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.
  • the daily dosage of the composition is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once to several times a day, but the weight, age, sex, health, diet of the patient The range varies depending on the time of administration, the method of administration, the rate of excretion and the severity of the disease.
  • compositions can be administered in various parenteral formulations during actual clinical administration, when formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like commonly used.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • It provides a method for measuring the protein expression level to provide information of the diagnosis, treatment results or prognosis of osteoporosis, comprising the step of: selecting the individual whose expression level of the mRNA or protein of PIRO increased compared to the normal control.
  • the mRNA expression level of PIRO is RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real time RT-PCR (Quentitative or semi-Quentitative real-).
  • time RT-PCR, Northern blot, and DNA or RNA chips can be measured by any one method selected from the group consisting of.
  • the protein expression level of PIRO can be measured by any one method selected from the group consisting of tissue immunostaining, enzyme immunoassay (ELISA) and Western blot.
  • the present invention provides a kit for diagnosing osteoporosis, treatment results or prognosis comprising any one selected from the group consisting of an antibody binding to the PIRO protein, a nucleic acid complementary to the PIRO gene, and a primer or probe specific for the PIRO gene. to provide.
  • test composition or compound 1) treating the test composition or compound to a PIRO protein expressing cell line;
  • the mRNA expression level of PIRO is RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real time RT-PCR (Quentitative or semi-Quentitative real-).
  • time RT-PCR, Northern blot, and DNA or RNA chips can be measured by any one method selected from the group consisting of.
  • the protein expression level of PIRO can be measured by any one method selected from the group consisting of tissue immunostaining, enzyme immunoassay (ELISA) and Western blot.
  • the present invention provides a composition for inducing osteoclast fusion containing a PIRO protein as an active ingredient.
  • M-CSF was isolated from mouse bone marrow cells (MBMC) (FIG. 1A), human bone marrow cells (HBMC) (FIG. 1B) and human leukocytes (FIG. 1C).
  • MBMC mouse bone marrow cells
  • HBMC human bone marrow cells
  • FIG. 1C human leukocytes
  • RANK receptor activator of NF ⁇ B ligand
  • PGRN was identified as a hormone with potent osteoclast differentiation in all cell types. Also, as in A of FIG. 1, osteoclast differentiation capacity of PGRN was dependent on RANKL, and TRAP + mutinucleated osteoclasts (MNCs) increased with increasing PGRN concentration.
  • MNCs TRAP + mutinucleated osteoclasts
  • PGRN Hydroxy apatite and dentin were used to determine bone resorption capacity, which is the osteoclast function of PGRN. As shown in FIG. 2, PGRN markedly promoted F-actin expression for osteoclast actin ring composition and increased Pit production. In other words, bone resorption significantly increased depending on the concentration of PGRN.
  • PGRN is a potent differentiation hormone of mouse and human osteoclasts, and has been shown to enhance osteoporosis of osteoclasts but not to osteoblast differentiation.
  • Example 2 Progranulin Forms an Important Axis of RANK Signaling
  • rat bone marrow cells (MBMC) were treated with M-CSF and RANKL to differentiate into osteoclasts, whereby PGRN was expressed in culture supernatant and total RNA. Measured.
  • PGRN expression was induced from 48 hours after RANKL treatment.
  • mRNAs of osteoclast differentiation related genes c-Fos (12 hours induction), NFATc1 (48 hours induction), TRAP (48 hours induction), OSCAR (48 hours induction) and PGRN (48 hours induction) Decreased significantly with shPGRN3 treatment.
  • PGRN is a component in RANK signaling, plays a critical role in the formation of MNCs, and affects the expression of c-Fos, NFATc1, TRAP and OSCAR involved in osteoclast differentiation.
  • Serum PGRN was measured after LPS, a potent inflammatory factor, was induced intraperitoneally (LPS-induced osteoporosis model).
  • PGRN expression was increased due to osteoporosis induction by LPS treatment.
  • Serum PGRN was measured in patients with osteoporosis and in bedding for a long time.
  • PGRN is an important biomarker of osteoporosis.
  • a total of 23,000 transcriptome analyzes identified 54 genes upregulated and 108 genes downregulated.
  • the gene of GM10800 mice was increased 20 times (A in FIG. 8), and 20 times in PGRN treatment after 72 hours even in real time PCR (FIG. 8 B). Since the osteoclast marker TRAP was induced irrespective of PGRN, it can be seen that PIRO is a late factor induced at 72 hours of RANK signaling. At the same time, RANKL alone induced PIRO expression about five times higher, indicating that RANKL induced PGRN expression endogenously and acted.
  • GM10800 the P rogranulin- I nduced Receptor- l ike molecule during O steoclastogenesis in means (PIRO) and he named after the PIRO the rat is composed of 26 gene family (family) to found the first gene in mPIRO-1 Final named.
  • mPIRO-1 was a receptor protein having five transmembrane (TM) domains.
  • the mPIRO-1 protein was composed of two PIRO domains from the 24th amino acid (P) to the 98th amino acid (F) and the 99th amino acid (P) to the 179th amino acid (F) (FIG. 9).
  • NCBI blastp searches were performed using the amino acid sequence of mPIRO-1 to obtain the amino acid sequence of a partial human PIRO protein, and a portion of the human highthrouhput genome sequence (HTGS) database and EST database search, and human bone marrow cells-deduced osteoclast RNA.
  • Full-length cDNA sequences were obtained by uncertain gene sequencing using sanger sequencing via RT-PCR.
  • hPIRO protein contains three PIRO domains from 1st amino acid (M) to 78th amino acid (F), 79th amino acid (M) to 186th amino acid (S), and 187th amino acid (M) to 269th amino acid (W). (FIG. 10).
  • mPIRO-1 is on chromosome 2
  • hPIRO is on chromosome 18.
  • mPIRO-1 and hPIRO were most similar (FIG. 11).
  • HBMC was treated with M-CSF + RANKL or M-CSF + RANKL + PGRN, and total RNA was isolated to measure hPIRO mRNA by real time PCR.
  • MBMC was treated with M-CSF to differentiate into macrophages, mPIRO-1 siRNA was treated, and after 24 hours, culture medium was replaced and RANKL was treated to induce osteoclast differentiation, and then mPIRO-1 mRNA was real time PCR. Was measured and TRAP + MNC was measured.
  • siPIRO2 almost suppressed MNC formation by RANKL, and thus mPIRO-1 was shown to play a critical role in the fusion of osteoclasts .
  • HBMC was differentiated into osteoclasts and treated with hPIRO siRNA and control siRNA to observe MNC formation.
  • sequences of the primers, shRNAs and siRNAs used in the examples of the present invention are as follows.
  • PGRN and PIRO form an important axis of RANK signaling.
  • RANK signaling sequentially regulates PGRN expression (48 hours) and PIRO expression (72 hours).
  • PIRO is a gene directly induced by PGRN as a late-stage factor of osteoclast fusion and bone resorption.
  • Path I is an autocrine manner in which the PGRN is produced in the osteoclasts of differentiation itself and expresses the PIRO gene through the PGRN receptor.
  • Path II is another cell in the bone marrow by RANK signaling, eg osteoblasts.
  • PGRN is expressed in these cells and acts on stromal cells and mesenchymal stem cells, and signaling through the PGRN receptors of osteoclasts, which are differentiated in a paracrine manner, is involved in the production of multinucleated osteoclasts and bone resorption.
  • PIRO forms an important axis in RANK signaling system and is a direct inducer of progranulin as a late stage factor of osteoclast fusion and bone resorption, so that PIRO is used as a biomarker to prevent or prevent new osteoporosis. It can be useful for developing therapeutics, diagnosing osteoporosis, evaluating treatment results or prognosis, and using PIRO inhibitors can effectively prevent or treat osteoporosis.

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Abstract

The present invention relates to a progranulin induced receptor like gene during osteoclastogenesis (PIRO) which is a novel biomarker for osteoclast fusion. More specifically, the present invention relates to a composition for treating or preventing osteoporosis by comprising, as an active ingredient, an inhibitor of PIRO, which has been newly developed as a factor involved in osteoclast fusion causing osteoporosis, or for inducing osteoclast fusion by comprising the PIRO as an active ingredient. According to the present invention, since PIRO, which is a late phase factor of osteoclast fusion and bone resorption and a direct inducer gene of progranulin, plays an important role in the RANK signaling pathway, PIRO may be useful as a biomarker in the development of a novel agent for preventing or treating osteoporosis, the diagnosis of osteoporosis, or the determining of therapeutic results, or prognoses. Also, by using a PIRO inhibitor, osteoporosis may be effectively prevented or treated.

Description

파골세포 융합의 새로운 바이오 마커인 파이로Pyro, a new biomarker for osteoclast fusion
본 발명은 파골세포 융합의 새로운 바이오 마커인 PIRO(Progranulin induced receptor like gene during Osteoclastogenesis)에 관한 것으로, 구체적으로 골다공증을 유발하는 파골세포의 융합에 관여하는 인자로 PIRO를 새롭게 발굴함에 따라 이의 억제제를 유효성분으로 하여 골다공증을 예방하거나 치료하고, 이 PIRO를 유효성분으로 하여 파골세포의 융합을 유도하는 조성물에 관한 것이다.The present invention relates to a new biomarker of osteoclast fusion, PIRO (Progranulin induced receptor like gene during Osteoclastogenesis), and specifically, an inhibitor thereof is effective by newly discovering PIRO as a factor involved in the fusion of osteoclasts causing osteoporosis. The present invention relates to a composition for preventing or treating osteoporosis as a component and inducing fusion of osteoclasts using this PIRO as an active ingredient.
우리의 뼈는 조골세포(osteoblasts)와 파골세포(osteoclasts)의 분화와 양적인 균형, 및 미네랄의 축적으로 만들어진 견고한 골격(scaffold) 역할을 한다. 여성의 폐경 이후 에스트로젠의 양적 감소는 잘 알려진 것과 같이 파골세포의 분화를 촉진하여 골밀도의 감소를 가져와서 골다공증(osteoporosis)을 야기한다. 이런 기전은 생체내(in vivo) 및 시험관내(in vitro) 동물모델에서 재현이 잘 된다.Our bones act as a solid scaffold created by the differentiation and quantitative balance of osteoblasts and osteoclasts and the accumulation of minerals. The quantitative reduction of estrogen after menopause in women promotes osteoclast differentiation, leading to a decrease in bone mineral density, as is well known, leading to osteoporosis. This mechanism is well reproduced in in vivo and in vitro animal models.
성숙된 파골세포는 각 세포의 핵이 세포융합에 의해 다핵체 세포로 변화하고 엑틴고리(actin ring)로 융합된 세포들이 묶인 형태로 기존의 뼈 조직을 파괴하여 골다공증을 유발한다. 파골세포의 전구체(progenitors) 세포는 골수성 세포 계열(myeloid cell lineage)에 속하여 골수세포에 M-CSF(macrophage-colony stimulating factor)를 처리하면 전구세포(preosteoclast) 및 대식세포(macrophages)로 분화하고, 파골세포 분화의 마스터 조절자(master regulator)인 RANK-리간드(RANKL)를 처리하면 수용체인 RANK에 결합하여 다양한 신호전달을 만들어서 세포안의 칼슘 오실레이터(oscillator)의 변화로 인한 Ca2+-의존 키나아제(Ca2+-dependent kinase)의 활성화를 통한 전사인자 NFAT-c1을 활성화하여 파골세포 분화에 필요한 여러 단백질 발현의 완성에 이르게 한다. 또한 TGF-β, IGF, IFN-γ, TNF-α 및 SemaD와 같이 조골세포 또는 파골세포에서 발현하여 파골세포의 분화의 양과 속도를 조절하는 cell-communication(coupling) factors들도 RANKL과 같이 작용하여 항상성과 병적상태에서 파골세포의 분화와 사멸을 조절한다.In mature osteoclasts, the nucleus of each cell is changed into multinucleated cells by cell fusion, and the osteoblasts are destroyed by destroying existing bone tissue in a form in which cells fused by actin rings are bundled. The osteoclast progenitors cells belong to the myeloid cell lineage, and when treated with a macrophage-colony stimulating factor (M-CSF) on the bone marrow cells, they differentiate into preosteoclasts and macrophages. Treatment of RANK-ligand (RANKL), the master regulator of osteoclast differentiation, binds to RANK, a receptor, and produces a variety of signaling pathways, resulting in Ca 2+ -dependent kinases caused by changes in calcium oscillators in cells ( Activation of the transcription factor NFAT-c1 through activation of Ca 2+ -dependent kinase leads to the completion of expression of several proteins required for osteoclast differentiation. In addition, cell-communication (coupling) factors that express osteoblasts or osteoclasts such as TGF-β, IGF, IFN-γ, TNF-α and SemaD to regulate the amount and rate of osteoclast differentiation act like RANKL. Regulates osteoclast differentiation and death in homeostasis and pathology.
프로그래뉼린(Progranulin)은 12개의 "시스테인 풍부 모티프(cysteine-rich motif)"로 구성된 7개 반의 그래뉼린(granulin, Grn) 도메인으로 구성된 88kD의 당 단백질이다. 인간 프로테옴 지도(the human proteome atlas)에서는 혈청 또는 혈장에서 80kD 당화 단백질(glycosylated protein)로 존재한다. 몇 개의 그룹에서 프로에피셀린(proepithelin) 및 PC 세포-유래 성장 인자 등으로 발견되어 불렸지만 HUGO의 정식 명칭은 "GRN"이다.Progranulin is a 88kD glycoprotein consisting of seven half-granulin (Grn) domains consisting of twelve "cysteine-rich motifs". The human proteome atlas is present as 80 kD glycosylated protein in serum or plasma. Although it has been found and called proepithelin and PC cell-derived growth factor in some groups, the official name of HUGO is "GRN".
그래뉼린(granulin peptide)은 백혈구에서 분비되는 펩타이드로 처음 발견되었다(Bateman et.al., BBRC, 1990). 지금까지 그래뉼린의 기능에 대하여, 상처 치유 인자(wound-healing factor)로서 작용하는 것이 보고되었으며(Bateman et. al., Nature Medicine, 2003), 또한 신경 성장 인자(Neuronal growth factor)로서 작용하는 것이 보고되었다(Bateman et. al., BMC Neuroscience, 2009; 및 Van Damme et, al., JCB, 2008). 또한, 그래뉼린은 타우 부정적 계열의 전측두엽성치매(tau-negative familial FTD)의 원인 유전자(Baker & Cruts et. al., Nature, 2006)이고, 물질 대사 호르몬(Metabolic hormone)(Youn et. al., Diabetes, 2009)이며, 식욕 억제 호르몬(Kim et al., Endocrinology, 2011)이고, 인슐린 저항성 인자(Matsubara et. al., Cell Metabolism, 2012)라는 것이 보고되었다.Granulin peptides were first discovered as peptides secreted from white blood cells (Bateman et.al., BBRC, 1990). So far, it has been reported that it acts as a wound-healing factor on the function of granulin (Bateman et. Al., Nature Medicine, 2003), and also acts as a neuronal growth factor. Reported (Bateman et. Al., BMC Neuroscience, 2009; and Van Damme et, al., JCB, 2008). In addition, granulin is the causative gene of tau-negative familial FTD (Baker & Cruts et. Al., Nature, 2006) and metabolic hormone (Youn et. Al. , Diabetes, 2009), appetite suppressing hormone (Kim et al., Endocrinology, 2011), and insulin resistance factor (Matsubara et. Al., Cell Metabolism, 2012).
최근 본 발명자들은 실험을 통해 프로그래뉼린이 RANK-의존적인 세포 신호 인자(cell-communication factor)로 작용하고 LPS 같은 염증유발을 통한 골다공증에서 매개 인자로 작용하며, 인간의 경우 전립선암 환자, 골다공증 환자 및 침대에 오래 누워 있어서 골밀도가 떨어진 환자의 혈청에서 대조군에 비해 현저히 높은 농도로 존재하는 것을 확인함으로써, 프로그래뉼린이 파골세포 분화 인자(Osteoclast Differentiation Factor)이며, 골다공증 바이오마커 또는 전립선 암의 혈청 바이오마커인 것을 밝혔으며, 이를 통해 프로그래뉼린 억제제를 유효성분으로 함유하는 골다공증 예방 또는 치료용 조성물에 관해 특허출원한 바 있다.In recent years, we have experimented with progranulin as a RANK-dependent cell-communication factor and as a mediator in osteoporosis through provoked inflammation such as LPS, and in humans with prostate cancer, osteoporosis and Progenullin is an osteoclast differentiation factor and is a osteoporosis biomarker or a serum biomarker of prostate cancer. Through this, a patent application for a composition for preventing or treating osteoporosis containing a progranulin inhibitor as an active ingredient has been disclosed.
이에, 본 발명자들은 프로그래뉼린 이외에 다른 새로운 골다공증 바이오마커를 발굴하고자 하였으며, 파골세포 분화과정에서 프로그래뉼린에 의해 발현 수준이 크게 상승(약 20배)하는 유전자(Progranulin-Induced Receptor-like molecule during Osteoclastogenesis, PIRO)를 발굴하였다. 사람과 쥐의 세포를 대상으로 한 생체내 및 생체외 실험을 통해 PIRO가 RANK 신호전달계에서 중요한 축을 이루고, 파골세포의 융합과 골흡수(bone resorption)의 후기 단계 인자로서 프로그래뉼린의 직접적인 유도유전자임을 확인하였으며, PIRO가 파골세포 융합 인자로써 골다공증 바이오마커인 것을 밝힘으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have tried to discover other osteoporosis biomarkers in addition to progranulin, and genes (progranulin-induced receptor-like molecules during Osteoclastogenesis (PIRO) was excavated. In vivo and ex vivo experiments in human and rat cells, PIRO is an important axis of RANK signaling and is a direct inducer of progranulin as a late-stage factor in osteoclast fusion and bone resorption. The present invention was completed by revealing that PIRO is an osteoporosis biomarker as an osteoclast fusion factor.
본 발명의 주된 목적은 PIRO를 골다공증에 대한 바이오마커로 사용하고, 파골세포 융합 인자로 사용하는 용도를 제공하는데 있다.It is a main object of the present invention to provide a use of PIRO as a biomarker for osteoporosis and as an osteoclast fusion factor.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질의 억제제를 유효성분으로 함유하는 골다공증의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention has a amino acid sequence of SEQ ID NO: 1 to 3 and the prevention or treatment of osteoporosis containing an inhibitor of a protein having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5 as an active ingredient It provides a pharmaceutical composition.
[서열번호 1] PFSVF LAIFH VLKCV FLIFR DFQFS RHIPG PSVCI SHFSR FLVIS SFFKS[SEQ ID NO 1] PFSVF LAIFH VLKCV FLIFR DFQFS RHIPG PSVCI SHFSR FLVIS SFFKS
[서열번호 2] FSLXF SFLAI XHVLQ WTFLN FPPFS VFLAI FXVLK CVFLI FRDFQ XSRHI PGPSV CISHF XRFLV ISXFF KSSSX CFSFS MIFSF LAIFH VLQWT FLNXP PF[SEQ ID NO 2] FSLXF SFLAI XHVLQ WTFLN FPPFS VFLAI FXVLK CVFLI FRDFQ XSRHI PGPSV CISHF XRFLV ISXFF KSSSX CFSFS MIFSF LAIFH VLQWT FLNXP PF
[서열번호 3] FXVVK WMFLI FHDFQ FS[SEQ ID NO 3] FXVVK WMFLI FHDFQ FS
(X = 모든 종류의 아미노산)(X = all kinds of amino acids)
또한, 본 발명은 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질의 억제제를 유효성분으로 함유하는 파골세포(osteoclasts) 융합 억제용 약학적 조성물을 제공한다.In addition, the present invention is a pharmaceutical for inhibiting osteoclast fusion containing an amino acid sequence of SEQ ID NO: 1 to 3 and containing an inhibitor of a protein having an amino acid sequence of SEQ ID NO: 4 or 5 or more than 80% homology as an active ingredient To provide a composition.
또한, 본 발명은 In addition, the present invention
1) 피검 개체로부터 분리된 혈액, 혈장 또는 혈청으로부터 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질의 mRNA 또는 상기 단백질의 발현 수준을 측정하는 단계; 및1) measuring the mRNA level of the protein or the expression level of the protein having an amino acid sequence of SEQ ID NO: 1 to 3 and at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5 from blood, plasma or serum isolated from the test subject Doing; And
2) 상기 단백질의 mRNA 또는 상기 단백질의 발현 수준이 정상 대조군에 비해 증가한 개체를 선별하는 단계;를 포함하는 골다공증 진단, 치료 결과 또는 예후 평가의 정보를 제공하기 위한 단백질 발현 수준의 측정 방법을 제공한다.It provides a method for measuring the protein expression level to provide information of the diagnosis, treatment results or prognosis of osteoporosis comprising the step of selecting the individual of the protein mRNA or the expression level of the protein increased compared to the normal control group; .
또한, 본 발명은 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질에 결합하는 항체, 상기 단백질을 암호화하는 유전자에 상보적인 핵산, 및 상기 유전자에 특이적인 프라이머 또는 프로브로 구성된 군으로부터 선택되는 어느 하나를 포함하는 골다공증 진단, 치료 결과 또는 예후 평가용 키트를 제공한다.In addition, the present invention is an antibody binding to a protein having an amino acid sequence of SEQ ID NO: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5, a nucleic acid complementary to the gene encoding the protein, and Provided are a kit for diagnosing, treating, or prognosing osteoporosis comprising any one selected from the group consisting of primers or probes specific for the gene.
또한, 본 발명은 In addition, the present invention
1) 피검 조성물 또는 화합물을 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질 발현 세포주에 처리하는 단계;1) treating the test composition or compound to a protein expressing cell line having the amino acid sequence of SEQ ID NOs: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5;
2) 상기 세포주의 상기 단백질의 mRNA 또는 상기 단백질의 발현 수준을 측정하는 단계; 및2) measuring the mRNA level of the protein or the expression level of the protein of the cell line; And
3) 상기 단백질의 mRNA 또는 단백질의 발현 수준이 무처리 대조군에 비해 감소된 피검 조성물 또는 화합물을 선별하는 단계;를 포함하는 골다공증 예방 또는 치료제의 스크리닝 방법을 제공한다.3) selecting a test composition or compound in which the expression level of the mRNA or the protein of the protein is reduced compared to the untreated control group; provides a screening method for preventing or treating osteoporosis.
또한, 본 발명은 In addition, the present invention
1) 피검 조성물 또는 화합물을 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질 발현 세포주에 처리하는 단계;1) treating the test composition or compound to a protein expressing cell line having the amino acid sequence of SEQ ID NOs: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5;
2) 상기 세포주의 상기 단백질의 mRNA 또는 상기 단백질의 발현 수준을 측정하는 단계; 및2) measuring the mRNA level of the protein or the expression level of the protein of the cell line; And
3) 상기 단백질의 mRNA 또는 상기 단백질의 발현 수준이 무처리 대조군에 비해 감소된 피검 조성물 또는 화합물을 선별하는 단계;를 포함하는 파골세포 융합 억제제의 스크리닝 방법을 제공한다.3) selecting a test composition or compound in which the mRNA level of the protein or the expression level of the protein is reduced compared to the untreated control group; provides a method for screening an osteoclast fusion inhibitor comprising a.
아울러, 본 발명은 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질을 유효성분으로 함유하는 파골세포 융합 유도용 조성물을 제공한다.In addition, the present invention provides a composition for inducing osteoclast fusion comprising a protein having an amino acid sequence of SEQ ID NO: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5.
본 발명에 따르면 PIRO는 RANK 신호전달계에서 중요한 축을 이루고, 파골세포의 융합과 골흡수(bone resorption)의 후기 단계 인자로서 프로그래뉼린의 직접적인 유도유전자이므로, PIRO를 바이오마커로 사용하여 새로운 골다공증 예방 또는 치료제 개발, 또는 골다공증 진단, 치료 결과 또는 예후 평가에 유용하게 사용할 수 있으며, PIRO 억제제를 사용하면 골다공증을 효과적으로 예방 또는 치료할 수 있다.According to the present invention, PIRO forms an important axis in RANK signaling system and is a direct inducer of progranulin as a late stage factor of osteoclast fusion and bone resorption, so that PIRO is used as a biomarker to prevent or prevent new osteoporosis. It can be useful for developing therapeutics, diagnosing osteoporosis, evaluating treatment results or prognosis, and using PIRO inhibitors can effectively prevent or treat osteoporosis.
도 1은 쥐 골수세포(mouse bone marrow cells, MBMC)(A), 인간 골수세포 (human bone marrow cells, HBMC)(B) 및 인간 백혈구(C)의 파골세포 분화를 유도하는 과정에서 프로그래뉼린(PGRN)의 처리에 따른 파골세포의 형성 정도를 분석한 결과이다.1 shows progranulin in the process of inducing osteoclast differentiation of mouse bone marrow cells (MBMC) (A), human bone marrow cells (HBMC) (B) and human leukocytes (C). This is a result of analyzing the degree of formation of osteoclasts according to the treatment of (PGRN).
도 2는 PGRN의 처리에 따른 F-actin 유도 및 actin-ring 형성 촉진과 골흡수(bone-resorption) 양상을 분석한 결과이다.2 is a result of analyzing the F-actin induction, actin-ring formation and bone-resorption pattern according to the treatment of PGRN.
도 3은 조골세포 분화에서 PGRN의 효과를 분석한 결과이다.3 shows the results of analyzing the effect of PGRN on osteoblast differentiation.
도 4는 쥐 골수세포의 파골세포 분화를 유도하는 과정에서 내생성(endogenous) PGRN의 양상을 분석한 결과이다.4 is a result of analyzing the pattern of endogenous PGRN in the process of inducing osteoclast differentiation of rat bone marrow cells.
도 5는 쥐 골수세포의 PGRN을 낙다운하고 이에 따른 효과를 분석한 결과이다.Figure 5 is a result of analyzing the effect down and down PGRN of rat bone marrow cells.
도 6은 쥐 골수세포의 파골세포 분화를 유도하는 과정에서 PGRN 낙다운에 따른 c-Fos, NFATc1, TRAP, OSCAR 및 PGRN의 발현 양상을 분석한 결과이다.6 is a result of analyzing the expression patterns of c-Fos, NFATc1, TRAP, OSCAR and PGRN according to PGRN knockdown in inducing osteoclast differentiation of rat bone marrow cells.
도 7은 LPS 처리 또는 OVX 생쥐 모델, 인간 골다공증환자 및 오랜 침대환자에서 serum PGRN 농도를 분석한 결과이다.Figure 7 shows the results of serum PGRN concentration in LPS-treated or OVX mouse model, human osteoporosis patients and long bed patients.
도 8은 쥐 골수세포의 파골세포 분화를 유도하는 과정에서 PGRN에 따른 유전자의 발현 양상을 분석한 결과이다.8 is a result of analyzing the expression of genes according to PGRN in the process of inducing osteoclast differentiation of rat bone marrow cells.
도 9는 쥐 PIRO 단백질(mPIRO-1, GM10800)의 아미노산 서열과 생체막에서의 구조를 나타낸 것이다.Figure 9 shows the amino acid sequence of the murine PIRO protein (mPIRO-1, GM10800) and the structure in the biofilm.
도 10은 인간 PIRO 유전자의 cDNA 서열과 PIRO 단백질의 아미노산 서열 및 생체막에서의 구조를 나타낸 것이다.Figure 10 shows the cDNA sequence of the human PIRO gene, the amino acid sequence of the PIRO protein and the structure in the biofilm.
도 11은 쥐의 PIRO 유전자군과 인간 PIRO 유전자의 phylogeny tree 및 아미노산 서열의 alignment 결과를 나타낸 것이다.Figure 11 shows the alignment of the phylogeny tree and amino acid sequence of the mouse PIRO gene group and human PIRO gene.
도 12는 쥐 골수세포와 인간 골수세포의 파골세포 분화를 유도하는 과정에서 PGRN 낙다운에 따른 PIRO의 발현 양상을 분석한 결과이다.12 is a result of analyzing the expression of PIRO according to PGRN knockdown in the process of inducing osteoclast differentiation between rat bone marrow cells and human bone marrow cells.
도 13은 쥐 골수세포의 파골세포 분화를 유도하는 과정에서 PIRO 낙다운에 따른 MNC 형성을 분석한 결과이다.Figure 13 shows the results of analyzing MNC formation according to PIRO knockdown in the process of inducing osteoclast differentiation of rat bone marrow cells.
도 14는 인간 골수세포의 파골세포 분화를 유도하는 과정에서 PIRO 낙다운에 따른 MNC 형성을 분석한 결과이다.14 shows the results of analyzing MNC formation according to PIRO down in the process of inducing osteoclast differentiation of human bone marrow cells.
도 15는 본 발명에서 예측된 RANKL/RANK axis에서 PGRN과 PIRO의 기능을 나타내는 모식도이다.15 is a schematic diagram showing the functions of PGRN and PIRO in the RANKL / RANK axis predicted in the present invention.
이하, 본 발명을 상세하게 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.
본 발명에서는 파골세포의 분화 호르몬(hormone)이며 골다공증의 중요한 바이오마커인 프로그래뉼린(Progranulin, PGRN)에 의해 발현 수준이 상승(약 20배)하는 PIRO 유전자 및 단백질을 발굴하였으며, 이 PIRO가 파골세포의 융합과 골흡수(bone resorption)의 매우 중요한 인자임을 밝혔다. 이에 따라 PIRO를 억제하면 파골세포의 융합과 골흡수를 억제할 수 있고, 골다공증의 예방 또는 치료가 가능하게 되며, PIRO의 발현 수준을 확인함으로써 골다공증 진단, 치료 결과 또는 예후를 평가할 수 있고, PIRO를 억제하는지의 여부를 조사함으로써 골다공증 예방 또는 치료제, 및 파골세포 융합 억제제를 스크리닝할 수 있으며, PIRO를 유효성분으로 하여 파골세포의 융합을 유도할 수 있는 것이다.In the present invention, PIRO genes and proteins were found to be raised (approximately 20-fold) by progranulin (PGRN), an osteoclast differentiation hormone and an important biomarker of osteoporosis. It has been shown to be a very important factor in cell fusion and bone resorption. Accordingly, inhibition of PIRO can inhibit osteoclast fusion and bone resorption, prevent or treat osteoporosis, and confirm the diagnosis of osteoporosis, evaluation of treatment results or prognosis by confirming PIRO expression levels, and PIRO. By investigating whether or not inhibition is possible, an agent for preventing or treating osteoporosis and an osteoclast fusion inhibitor can be screened, and fusion of osteoclasts can be induced using PIRO as an active ingredient.
본 발명은 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질의 억제제를 유효성분으로 함유하는 골다공증의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention or treatment of osteoporosis, comprising an amino acid sequence of SEQ ID NO: 1 to 3 and containing an inhibitor of a protein having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5 as an active ingredient. .
또한, 본 발명은 상기 단백질의 억제제를 유효성분으로 함유하는 파골세포 융합 억제용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for inhibiting osteoclast fusion containing the inhibitor of the protein as an active ingredient.
이하, 본 발명에서 상기 단백질은 PIRO 단백질이라 하며, PIRO 단백질을 암호화하는 유전자는 PIRO 유전자라 한다.Hereinafter, in the present invention, the protein is called a PIRO protein, and the gene encoding the PIRO protein is called a PIRO gene.
PIRO 단백질은 서열번호 4 또는 5의 아미노산 서열로 구성되는 것이 바람직하나 이에 한정되지는 않으며, 서열번호 4 또는 5에서 하나 또는 몇 개의 아미노산이 첨가, 결실 또는 치환된 서열로 구성될 수 있다.The PIRO protein is preferably composed of an amino acid sequence of SEQ ID NO: 4 or 5, but is not limited thereto, and may be composed of a sequence in which one or several amino acids are added, deleted or substituted in SEQ ID NO: 4 or 5.
PIRO 유전자는 서열번호 6 또는 7의 염기서열로 구성되는 것이 바람직하나 이에 한정되지는 않으며, 서열번호 6 또는 7에서 하나 또는 몇 개의 염기가 첨가, 결실 또는 치환된 서열로 구성될 수 있다.The PIRO gene is preferably composed of the nucleotide sequence of SEQ ID NO: 6 or 7, but is not limited thereto, and may be composed of a sequence in which one or several bases are added, deleted or substituted in SEQ ID NO: 6 or 7.
상기 억제제는 발현 또는 활성 억제제일 수 있다. 구체적으로, PIRO 단백질의 발현 억제제는 PIRO 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오티드, 및 RNAi[작은 간섭 RNA(short interfering RNA), 짧은 헤어핀 RNA(short hairpin RNA) 및 마이크로 RNA(miRNA)]로 이루어진 군으로부터 선택된 어느 하나인 것이 바람직하며, PIRO 단백질의 활성 억제제는 PIRO 단백질에 상보적으로 결합하는 화합물, 펩티드, 펩티드 미메틱스, 앱타머 및 항체로 이루어진 군으로부터 선택된 어느 하나인 것이 바람직하나 이에 한정되지 않는다. 본 발명의 실시예에서 사용한 쥐 PIRO(mPIRO-1)의 siRNA인 siPIRO2와 인간 PIRO(hPIRO)의 siRNA인 si-hPIRO2도 여기에 포함된다.The inhibitor may be an expression or activity inhibitor. Specifically, inhibitors of expression of the PIRO protein include antisense nucleotides that complementarily bind to the mRNA of the PIRO gene, and RNAi (short interfering RNA, short hairpin RNA and microRNA). It is preferably one selected from the group consisting of, wherein the inhibitor of the activity of the PIRO protein is preferably any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers and antibodies that complementarily bind to the PIRO protein. It is not limited. Also included are siPIRO2, which is a siRNA of mouse PIRO (mPIRO-1), and si-hPIRO2, which is a siRNA of human PIRO (hPIRO) used in an embodiment of the present invention.
이를 구체적으로 살펴보면 하기와 같다.Looking at this in detail.
1) RNAi1) RNAi
RNA 간섭(RNAi)은 PIRO 유전자에 대응하는 두 가닥 사슬 RNA(dsRNA)를 세포 또는 유기체에 도입함으로써 대응하는 mRNA의 분해가 일어나는 전사 후 유전자 사일런싱 메카니즘(post-transcriptional gene silencing mechanism)이다. 상기 RNAi 효과에 의해 유전자 발현이 복귀되기 전에 다중 세포 분열이 지속되므로 RNAi는 RNA 레벨에서 목표로 하는 낙아웃(knockout) 또는 '낙다운(knockdown)'을 만드는 매우 강력한 방법이다(Elbashir et al. Nature May 24;411(6836):494-8, 2001). 유전자 사일런싱에서의 RNAi 기술은 표준 분자 생물학 방법을 이용한다. 불활성화시킬 표적 유전자의 서열에 대응하는 dsRNA는 표준 방법, 예를 들면 T7 RNA 중합효소를 이용한 주형 DNA의 양 가닥 동시 전사에 의해 생성할 수 있다. RNAi에 사용되는 dsRNA의 생성 키트는 상업적으로 판매되는 제품을 사용할 수 있다. dsRNA 또는 dsRNA를 제조하도록 처리된 플라스미드의 트랜스펙션 방법은 공지의 기술이다.RNA interference (RNAi) is a post-transcriptional gene silencing mechanism in which degradation of the corresponding mRNA occurs by introducing two stranded chain RNAs (dsRNAs) corresponding to the PIRO gene into a cell or organism. RNAi is a very powerful way to create targeted knockouts or 'knockdowns' at the RNA level, as multicellular division continues before the gene expression is restored by the RNAi effect (Elbashir et al. Nature). May 24; 411 (6836): 494-8, 2001). RNAi techniques in gene silencing use standard molecular biology methods. The dsRNA corresponding to the sequence of the target gene to be inactivated can be generated by standard methods, eg, both strands simultaneous transcription of template DNA using T7 RNA polymerase. The production kit of dsRNA used for RNAi may use a commercially available product. Methods of transfection of plasmids treated to produce dsRNA or dsRNA are known in the art.
2) 안티센스 핵산 서열2) antisense nucleic acid sequences
PIRO를 코딩하는 핵산에 대해 안티센스인 핵산 분자를 저해제로 사용할 수 있다. '안티센스' 핵산은 PIRO를 코딩하는 '센스' 핵산에 상보적인, 예를 들면 두 가닥 사슬 cDNA 분자의 코딩 가닥에 상보적이거나 mRNA 서열에 상보적인 핵산 서열을 포함한다. 따라서 안티센스 핵산은 센스 핵산과 수소 결합을 형성할 수 있다. 상기 안티센스 핵산은 전체 PIRO 코딩가닥 또는 단지 그들의 일부(예: 코딩영역)에 상보적일 수 있다. 상기 안티센스 핵산 분자는 PIRO mRNA의 전체 코딩 영역에 상보적일 수 있으나, PIRO mRNA의 코딩 또는 비코딩 영역의 단지 일부(예: 번역 개시부)에만 안티센스인 올리고뉴클레오티드가 더 바람직하다. 안티센스 올리고뉴클레오티드는 예를 들면 약 5 내지 50 뉴클레오티드의 길이일 수 있다. 안티센스 핵산은 공지의 방법을 이용한 화합 합성 및 효소 결합 반응을 이용하여 구성할 수 있다.Nucleic acid molecules that are antisense to nucleic acids encoding PIRO can be used as inhibitors. An 'antisense' nucleic acid comprises a nucleic acid sequence that is complementary to a 'sense' nucleic acid encoding a PIRO, eg, complementary to the coding strand of a two stranded chain cDNA molecule or complementary to an mRNA sequence. Thus, the antisense nucleic acid can form hydrogen bonds with the sense nucleic acid. The antisense nucleic acid may be complementary to the entire PIRO coding strand or only a portion thereof (eg, coding region). The antisense nucleic acid molecule may be complementary to the entire coding region of the PIRO mRNA, but more preferably oligonucleotides that are antisense to only a portion of the coding or non-coding region of the PIRO mRNA (eg, translation initiation). Antisense oligonucleotides can be, for example, about 5 to 50 nucleotides in length. Antisense nucleic acids can be constructed using compound synthesis and enzyme binding reactions using known methods.
3) 펩티드 미메틱스(Peptide Mimetics)3) Peptide Mimetics
PIRO 폴리펩티드의 단백질 결합 도메인을 억제한 미메틱스(예, 펩티드 또는 비펩티드성 약제)를 제작하여 원래의 PIRO 폴리펩타이드가 VHL에 결합하는 것을 억제할 수 있다.Mimetics (eg, peptides or nonpeptidic agents) that inhibit the protein binding domain of a PIRO polypeptide can be made to inhibit the original PIRO polypeptide from binding to VHL.
상기 조성물은 PIRO 억제제에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상을 함유할 수 있다.The composition may further contain one or more active ingredients exhibiting the same or similar function in addition to the PIRO inhibitor.
상기 조성물은 전체 조성물 100 중량부 대비 PIRO 억제제를 0.1 중량부 내지 90 중량부로 함유할 수 있다.The composition may contain 0.1 to 90 parts by weight of the PIRO inhibitor relative to 100 parts by weight of the total composition.
상기 조성물은 임상 투여 시에 경구 또는 비경구로 투여가 가능하며 비경구 투여시 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내주사, 자궁내 경막주사, 뇌혈관내 주사 또는 흉부내 주사에 의해 투여될 수 있고, 일반적인 의약품 제제의 형태로 사용될 수 있다.The composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. And can be used in the form of general pharmaceutical formulations.
상기 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.
상기 조성물의 일일 투여량은 약 0.0001 내지 100㎎/㎏이고, 바람직하게는 0.001 내지 10㎎/㎏이며, 하루 1회 내지 수회 나누어 투여하는 것이 바람직하나 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다.The daily dosage of the composition is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once to several times a day, but the weight, age, sex, health, diet of the patient The range varies depending on the time of administration, the method of administration, the rate of excretion and the severity of the disease.
상기 조성물은 실제 임상 투여 시에 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The composition can be administered in various parenteral formulations during actual clinical administration, when formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like commonly used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
또한, 본 발명은 In addition, the present invention
1) 피검 개체로부터 분리된 혈액, 혈장 또는 혈청으로부터 PIRO의 mRNA 또는 단백질의 발현 수준을 측정하는 단계; 및1) measuring the expression level of mRNA or protein of PIRO from blood, plasma or serum isolated from the subject; And
2) PIRO의 mRNA 또는 단백질의 발현 수준이 정상 대조군에 비해 증가한 개체를 선별하는 단계;를 포함하는 골다공증 진단, 치료 결과 또는 예후 평가의 정보를 제공하기 위한 단백질 발현 수준의 측정 방법을 제공한다.It provides a method for measuring the protein expression level to provide information of the diagnosis, treatment results or prognosis of osteoporosis, comprising the step of: selecting the individual whose expression level of the mRNA or protein of PIRO increased compared to the normal control.
상기 방법에 있어서, PIRO의 mRNA 발현 수준은 RT-PCR, 정량적 또는 반정량적 RT-PCR(Quentitative or semi-Quentitative RT-PCR), 정량적 또는 반정량적 리얼 타임 RT-PCR(Quentitative or semi-Quentitative real-time RT-PCR), 노던 블롯(northern blot), 및 DNA 또는 RNA 칩(chip)으로 구성된 군으로부터 선택되는 어느 하나의 방법으로 측정할 수 있다.In the method, the mRNA expression level of PIRO is RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real time RT-PCR (Quentitative or semi-Quentitative real-). time RT-PCR, Northern blot, and DNA or RNA chips can be measured by any one method selected from the group consisting of.
상기 방법에 있어서, PIRO의 단백질 발현 수준은 조직면역염색법, 효소면역분석법(ELISA) 및 웨스턴 블롯(Western Blot)으로 구성된 군으로부터 선택되는 어느 하나의 방법으로 측정할 수 있다.In this method, the protein expression level of PIRO can be measured by any one method selected from the group consisting of tissue immunostaining, enzyme immunoassay (ELISA) and Western blot.
또한, 본 발명은 PIRO 단백질에 결합하는 항체, PIRO 유전자에 상보적인 핵산, 및 PIRO 유전자에 특이적인 프라이머 또는 프로브로 구성된 군으로부터 선택되는 어느 하나를 포함하는 골다공증 진단, 치료 결과 또는 예후 평가용 키트를 제공한다.In addition, the present invention provides a kit for diagnosing osteoporosis, treatment results or prognosis comprising any one selected from the group consisting of an antibody binding to the PIRO protein, a nucleic acid complementary to the PIRO gene, and a primer or probe specific for the PIRO gene. to provide.
또한, 본 발명은 In addition, the present invention
1) 피검 조성물 또는 화합물을 PIRO 단백질 발현 세포주에 처리하는 단계;1) treating the test composition or compound to a PIRO protein expressing cell line;
2) 상기 세포주의 PIRO의 mRNA 또는 단백질의 발현 수준을 측정하는 단계; 및2) measuring the expression level of mRNA or protein of the PIRO of the cell line; And
3) PIRO의 mRNA 또는 단백질의 발현 수준이 무처리 대조군에 비해 감소된 피검 조성물 또는 화합물을 선별하는 단계;를 포함하는 골다공증 예방 또는 치료제, 또는 파골세포 융합 억제제의 스크리닝 방법을 제공한다.3) screening a test composition or compound in which the expression level of mRNA or protein of PIRO is reduced compared to an untreated control group; and a method for screening an osteoporosis prevention or osteoclast fusion inhibitor comprising a.
상기 방법에 있어서, PIRO의 mRNA 발현 수준은 RT-PCR, 정량적 또는 반정량적 RT-PCR(Quentitative or semi-Quentitative RT-PCR), 정량적 또는 반정량적 리얼 타임 RT-PCR(Quentitative or semi-Quentitative real-time RT-PCR), 노던 블롯(northern blot), 및 DNA 또는 RNA 칩(chip)으로 구성된 군으로부터 선택되는 어느 하나의 방법으로 측정할 수 있다.In the method, the mRNA expression level of PIRO is RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real time RT-PCR (Quentitative or semi-Quentitative real-). time RT-PCR, Northern blot, and DNA or RNA chips can be measured by any one method selected from the group consisting of.
상기 방법에 있어서, PIRO의 단백질 발현 수준은 조직면역염색법, 효소면역분석법(ELISA) 및 웨스턴 블롯(Western Blot)으로 구성된 군으로부터 선택되는 어느 하나의 방법으로 측정할 수 있다.In this method, the protein expression level of PIRO can be measured by any one method selected from the group consisting of tissue immunostaining, enzyme immunoassay (ELISA) and Western blot.
또한, 본 발명은 PIRO 단백질을 유효성분으로 함유하는 파골세포 융합 유도용 조성물을 제공한다.In addition, the present invention provides a composition for inducing osteoclast fusion containing a PIRO protein as an active ingredient.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 새로운 파골세포 분화 호르몬인 프로그래뉼린(Progranulin, PGRN)Example 1.Progranulin (PGRN), a novel osteoclast differentiation hormone
1-1. 쥐 골수세포(mouse bone marrow cells, MBMC)(도 1의 A), 인간 골수세포 (human bone marrow cells, HBMC)(도 1의 B) 및 인간 백혈구(도 1의 C)를 분리하여 M-CSF(macrophage-colony stimulating factor)와 RANK(receptor activator of NFκB) 리간드(RANKL)를 처리하고 파골세포로 분화시키면서 여기에 재조합 인간 또는 생쥐 PGRN을 처리하여 파골세포의 형성을 관찰하였다.1-1. M-CSF was isolated from mouse bone marrow cells (MBMC) (FIG. 1A), human bone marrow cells (HBMC) (FIG. 1B) and human leukocytes (FIG. 1C). (macrophage-colony stimulating factor) and RANK (receptor activator of NFκB) ligand (RANKL) was treated with differentiation into osteoclasts and treated with recombinant human or mouse PGRN and observed the formation of osteoclasts.
도 1에서와 같이 모든 세포종류에서 PGRN은 강력한 파골세포 분화능이 있는 호르몬으로 확인되었다. 또한, 도 1의 A에서와 같이 PGRN의 파골세포 분화능은 RANKL에 의존적(dependent)이며, PGRN 농도의 증가에 따라 TRAP+ mutinucleated osteoclasts(MNCs)가 증가하는 것으로 나타났다.As shown in FIG. 1, PGRN was identified as a hormone with potent osteoclast differentiation in all cell types. Also, as in A of FIG. 1, osteoclast differentiation capacity of PGRN was dependent on RANKL, and TRAP + mutinucleated osteoclasts (MNCs) increased with increasing PGRN concentration.
1-2. PGRN의 파골세포 기능인 골흡수(bone resorption)능 측정을 위해 hydroxyl apatite와 dentin을 사용하였다. 도 2에서와 같이 PGRN은 파골세포 actin ring 조성을 위한 F-actin의 발현을 뚜렷이 촉진하였고, Pit 생성을 증가시켰다. 즉 골흡수가 PGRN의 처리 농도에 의존적으로 현저하게 증가하였다.1-2. Hydroxy apatite and dentin were used to determine bone resorption capacity, which is the osteoclast function of PGRN. As shown in FIG. 2, PGRN markedly promoted F-actin expression for osteoclast actin ring composition and increased Pit production. In other words, bone resorption significantly increased depending on the concentration of PGRN.
1-3. Alkaline phosphatase(ALP) 측정과 Alizarin res staining으로 PGRN이 조골세포(osteoblasts)에 미치는 영향을 확인한 결과, 도 3에서와 같이 PGRN은 조골세포의 분화에 영향을 미치지 않았다.1-3. As a result of confirming the effect of PGRN on osteoblasts by Alkaline phosphatase (ALP) measurement and Alizarin res staining, PGRN did not affect osteoblast differentiation as shown in FIG.
1-4. 소결1-4. Sintered
PGRN은 생쥐 및 인간 파골세포의 강력한 분화 호르몬이고, 파골세포의 골흡수능을 향상시키나 조골세포 분화에는 관여하지 않는 것으로 나타났다.PGRN is a potent differentiation hormone of mouse and human osteoclasts, and has been shown to enhance osteoporosis of osteoclasts but not to osteoblast differentiation.
실시예 2. RANK 신호전달의 중요한 축을 이루는 프로그래뉼린Example 2 Progranulin Forms an Important Axis of RANK Signaling
2-1. 지금까지의 결과는 외부에서 재조합 PGRN의 형태로 넣어주었을 때 PGRN이 파골세포 분화 및 성숙에 관여한다는 것을 보여준다.2-1. The results so far show that PGRN is involved in osteoclast differentiation and maturation when put in the form of recombinant PGRN from outside.
RANK 신호전달에서 내생(endogenous) PGRN의 영향을 알아보기 위해 쥐 골수세포(MBMC)에 M-CSF와 RANKL을 처리하여 파골세포로 분화시키고, 이때 배양상층액(culture supernatant)과 총 RNA에서 PGRN을 측정하였다.To investigate the effect of endogenous PGRN on RANK signaling, rat bone marrow cells (MBMC) were treated with M-CSF and RANKL to differentiate into osteoclasts, whereby PGRN was expressed in culture supernatant and total RNA. Measured.
도 4에서와 같이 RANKL 처리 후 48시간부터 PGRN의 발현이 유도되는 것으로 나타났다.As shown in FIG. 4, PGRN expression was induced from 48 hours after RANKL treatment.
2-2. PGRN의 생성이 RANK 신호전달의 중요한 요소임을 직접 증명하기 위해 shRNA를 이용한 유전자 낙다운(knock-down, KD) 실험을 실시하였다. 이때 사용한 mouse PGRN shRNA 서열은 표 1과 같다.2-2. To directly demonstrate that PGRN production is an important component of RANK signaling, gene knock-down (KD) experiments using shRNA were performed. The mouse PGRN shRNA sequence used at this time is shown in Table 1.
도 5에서와 같이, 대조군(control)과 비교 시 sh-PGRN3가 가장 강한 낙다운 효과를 보였다. PGRN mRNA와 배양상층액의 PGRN 발현을 감소시켰고, 동시에 multinucleated osteoclasts(MNC) 생성도 현저하게 감소시켰다.As shown in Figure 5, compared with the control (control) sh-PGRN3 showed the strongest knockdown effect. PGRN mRNA and PGRN expression in culture supernatants were reduced, while at the same time, the production of multinucleated osteoclasts (MNC) was significantly reduced.
2-3. shPGRN3로 PGRN 유전자를 낙다운시켰을 때 파골세포 분화와 관련된 유전자의 발현 양상을 조사하였다.2-3. The expression patterns of genes related to osteoclast differentiation were investigated when shPGRN3 knocked down the PGRN gene.
도 6에서와 같이, 파골세포 분화 관련 유전자인 c-Fos(12시간 유도), NFATc1(48시간 유도), TRAP(48시간 유도), OSCAR(48시간 유도) 및 PGRN(48시간 유도)의 mRNA가 shPGRN3 처리로 현저하게 감소하였다.As shown in FIG. 6, mRNAs of osteoclast differentiation related genes c-Fos (12 hours induction), NFATc1 (48 hours induction), TRAP (48 hours induction), OSCAR (48 hours induction) and PGRN (48 hours induction) Decreased significantly with shPGRN3 treatment.
2-4. 소결2-4. Sintered
PGRN은 RANK 신호전달에서 하나의 구성요소(component)이고, MNCs 형성에 결정적인 역할을 하며, 파골세포 분화에 관여하는 c-Fos, NFATc1, TRAP 및 OSCAR의 발현에 영향을 미친다.PGRN is a component in RANK signaling, plays a critical role in the formation of MNCs, and affects the expression of c-Fos, NFATc1, TRAP and OSCAR involved in osteoclast differentiation.
실시예 3. 골다공증 생쥐 모델과 사람에서 혈중 PGRN의 농도변화Example 3 Serum PGRN Concentration Changes in Osteoporosis Mouse Model and Human
3-1. 강력한 염증 인자인 LPS를 복강으로 투여하여 골다공증을 유발한 후(LPS-induced osteoporosis model) serum PGRN을 측정하였다.3-1. Serum PGRN was measured after LPS, a potent inflammatory factor, was induced intraperitoneally (LPS-induced osteoporosis model).
도 7의 A에서와 같이 LPS 처리에 의한 골다공증 유발로 PGRN의 발현이 증가하였다.As shown in FIG. 7A, PGRN expression was increased due to osteoporosis induction by LPS treatment.
3-2. 난소제거 후 골다공증 유발한 생쥐(ovariectomized model, OVX model)에서 serum PGRN을 측정하였다.3-2. After ovarian removal, serum PGRN was measured in osteoporosis-induced mice (ovariectomized model, OVX model).
도 7의 B에서와 같이 난소제거에 의한 골다공증 유발로 PGRN의 발현이 증가하였다.As in B of FIG. 7, the expression of PGRN was increased due to osteoporosis induced by ovarian removal.
3-3. 골다공증 환자와 오랫동안 누워 침대 생활을 한 환자의 serum PGRN을 측정하였다.3-3. Serum PGRN was measured in patients with osteoporosis and in bedding for a long time.
도 7의 C에서와 같이 정상인과 비교할 때 PGRN의 발현이 유의하게 높았다.The expression of PGRN was significantly higher when compared to normal subjects as in C of FIG. 7.
3-4. 소결3-4. Sintered
PGRN은 골다골증의 중요한 바이오마커이다.PGRN is an important biomarker of osteoporosis.
실시예 4. PIRO(Example 4. PIRO ( PP rogranulin-rogranulin- II nduced Receptor-nduced Receptor- ll ike molecule during ike molecule during OO steoclastogenesis) 유전자 발굴steoclastogenesis) gene discovery
4-1. RNA-염기서열 분석을 통한 genome-wide expression profiling4-1. Genome-wide expression profiling through RNA-sequence analysis
MCSF + RANLK 또는 MCSF + RANKL + PGRN을 각각 처리하고 60시간 후에 총 RNA를 분리한 다음, poly(A+) RNA를 재정제하여 Illumina hi-seq 2500 platform으로 deep sequencing하여 gene ontology를 만들었다.After 60 hours of treatment with MCSF + RANLK or MCSF + RANKL + PGRN, total RNA was isolated, and then poly (A + ) RNA was reconstituted to deep sequencing with Illumina hi-seq 2500 platform to create gene ontology.
총 23,000 여 transcriptome 분석에서 상위조절(upregulated)된 54개의 유전자와 하향조절(downregulated)된 108개의 유전자를 발견하였다. 이중에서 GM10800이라는 쥐의 유전자는 20여배가 증가되었고(도 8의 A), real time PCR에서도 72시간 후 PGRN 처리 시 20배 증가하였다(도 8의 B). 파골세포 마커인 TRAP은 PGRN에 관계없이 유도되었으므로 PIRO는 RANK 신호전달 72시간에 유도되는 후기 인자(later factor)임을 알 수 있다. 동시에 RANKL 단독으로도 PIRO의 발현이 5배 정도 높게 유도되는 것으로 보아 RANKL에 의해 내생적으로 PGRN의 발현이 유도되고 이것이 작용하였을 가능성을 나타냈다. GM10800을 Progranulin-Induced Receptor-like molecule during Osteoclastogenesis(PIRO)라고 명명하고, 이후에 쥐의 PIRO가 26개의 유전자 패밀리(family)로 구성되어 있어서 가장 먼저 발견된 유전자라는 뜻에서 mPIRO-1으로 최종 명명하였다.A total of 23,000 transcriptome analyzes identified 54 genes upregulated and 108 genes downregulated. Among them, the gene of GM10800 mice was increased 20 times (A in FIG. 8), and 20 times in PGRN treatment after 72 hours even in real time PCR (FIG. 8 B). Since the osteoclast marker TRAP was induced irrespective of PGRN, it can be seen that PIRO is a late factor induced at 72 hours of RANK signaling. At the same time, RANKL alone induced PIRO expression about five times higher, indicating that RANKL induced PGRN expression endogenously and acted. GM10800 the P rogranulin- I nduced Receptor- l ike molecule during O steoclastogenesis in means (PIRO) and he named after the PIRO the rat is composed of 26 gene family (family) to found the first gene in mPIRO-1 Final named.
4-2. mPIRO-1의 염기서열 및 아미노산 서열을 분석한 결과, 5개의 transmembrane(TM) domain을 갖는 수용체 단백질인 것으로 나타났다. mPIRO-1 단백질은 24번째 아미노산(P)부터 98번째 아미노산(F)까지와 99번째 아미노산(P)부터 179번째 아미노산(F)까지의 두 개의 PIRO domain으로 구성되어 있었다(도 9).4-2. Analysis of the nucleotide sequence and amino acid sequence of mPIRO-1 revealed that it was a receptor protein having five transmembrane (TM) domains. The mPIRO-1 protein was composed of two PIRO domains from the 24th amino acid (P) to the 98th amino acid (F) and the 99th amino acid (P) to the 179th amino acid (F) (FIG. 9).
4-3. 인간의 PIRO 유전자(hPIRO) 발굴4-3. Discovery of human PIRO gene (hPIRO)
mPIRO-1의 아미노산 서열을 이용한 NCBI blastp 검색을 수행하여 부분적인 인간 PIRO 단백질의 아미노산 서열을 얻었고, human highthrouhput genome sequence(HTGS) 데이터베이스 및 EST 데이터베이스 써치, 및 human bone marrow cells-deduced 파골세포 RNA에서 부분적인 RT-PCR을 통하여 sanger sequencing을 사용하여 불확실한 유전자 서열 확인을 통해 full length cDNA 서열을 얻었다. hPIRO 단백질은 1번째 아미노산(M)부터 78번째 아미노산(F), 79번째 아미노산(M)부터 186번째 아미노산(S), 187번째 아미노산(M)부터 269번째 아미노산(W)까지 3개의 PIRO domain을 갖고 있었다(도 10).NCBI blastp searches were performed using the amino acid sequence of mPIRO-1 to obtain the amino acid sequence of a partial human PIRO protein, and a portion of the human highthrouhput genome sequence (HTGS) database and EST database search, and human bone marrow cells-deduced osteoclast RNA. Full-length cDNA sequences were obtained by uncertain gene sequencing using sanger sequencing via RT-PCR. hPIRO protein contains three PIRO domains from 1st amino acid (M) to 78th amino acid (F), 79th amino acid (M) to 186th amino acid (S), and 187th amino acid (M) to 269th amino acid (W). (FIG. 10).
4-4. 쥐의 PIRO 유전자군과 인간 PIRO(hPIRO)와의 계통발생적 비교4-4. Phylogenetic comparison of rat PIRO gene group and human PIRO (hPIRO)
쥐의 PIRO 유전자들은 염색체 2, 9, 12 등에 26개로 산재해 있으며, 여기에는 variants와 non-coding RNA가 포함된다. mPIRO-1은 염색체 2번에 존재하며 hPIRO는 염색체 18번에 존재한다. mPIRO-1과 hPIRO가 가장 유사하였다(도 11).There are 26 mouse PIRO genes scattered on chromosomes 2, 9, and 12, including variants and non-coding RNA. mPIRO-1 is on chromosome 2 and hPIRO is on chromosome 18. mPIRO-1 and hPIRO were most similar (FIG. 11).
실시예 5. PGRN에 의한 mPIRO-1과 hPIRO의 영향Example 5 Effect of mPIRO-1 and hPIRO by PGRN
MBMC에 M-CSF와 RANKL을 처리하여 파골세포로 분화한 후 그리고 shPGRN3로 낙다운시킨 후 날짜별로 총 RNA를 분리하여 real time PCR로 PIRO의 mRNA를 측정하였다.After treating M-CSF and RANKL in MBMC, they differentiated into osteoclasts and downgraded to shPGRN3. Total RNA was isolated by date and PIRO mRNA was measured by real time PCR.
또한, HBMC에 M-CSF + RANKL 또는 M-CSF + RANKL + PGRN을 처리한 후 총 RNA를 분리하여 real time PCR로 hPIRO의 mRNA를 측정하였다.In addition, HBMC was treated with M-CSF + RANKL or M-CSF + RANKL + PGRN, and total RNA was isolated to measure hPIRO mRNA by real time PCR.
도 12에서와 같이, mPIRO-1과 hPIRO의 발현이 PGRN에 의해 유도되는 것으로 나타났다.As shown in Figure 12, it was shown that the expression of mPIRO-1 and hPIRO is induced by PGRN .
실시예 6. mPIRO-1 낙다운에 따른 MNC 형성의 영향Example 6 Effect of MNC Formation by mPIRO-1 Knockdown
MBMC에 M-CSF를 처리하여 대식세포로 분화한 후 mPIRO-1 siRNA를 처리하고, 24시간 후 배양액을 교체하고 RANKL을 처리하여 파골세포 분화를 유도한 다음, mPIRO-1의 mRNA를 real time PCR로 측정하고 TRAP+ MNC를 측정하였다.MBMC was treated with M-CSF to differentiate into macrophages, mPIRO-1 siRNA was treated, and after 24 hours, culture medium was replaced and RANKL was treated to induce osteoclast differentiation, and then mPIRO-1 mRNA was real time PCR. Was measured and TRAP + MNC was measured.
도 13에서와 같이 siPIRO2가 RANKL에 의한 MNC 형성을 거의 억제하였고, 따라서 mPIRO-1은 파골세포의 융합에 결정적인 역할을 하는 것으로 나타났다.As shown in FIG. 13, siPIRO2 almost suppressed MNC formation by RANKL, and thus mPIRO-1 was shown to play a critical role in the fusion of osteoclasts .
실시예 7. hPIRO 낙다운에 따른 파골세포 융합의 영향Example 7 Effect of Osteoclast Fusion by hPIRO Knockdown
HBMC를 파골세포로 분화하고 hPIRO의 siRNA와 대조군 siRNA를 처리하여 MNC 형성을 관찰하였다.HBMC was differentiated into osteoclasts and treated with hPIRO siRNA and control siRNA to observe MNC formation.
도 14에서와 같이 si-hPIRO2가 MNC 형성을 상당히 억제하였고, 이에 따라 PIRO는 인간의 파골세포 융합에도 관여하는 것으로 나타났다.As shown in Figure 14 si-hPIRO2 significantly inhibited MNC formation, PIRO was also shown to be involved in human osteoclast fusion .
본 발명의 실시예에 사용된 프라이머, shRNA 및 siRNA의 서열은 다음과 같다.The sequences of the primers, shRNAs and siRNAs used in the examples of the present invention are as follows.
표 1
Primer sequences for Real-time RT-PCR
mPGRN Forward: 5′-TTC ACA CAC GAT GCG TTT CA-3′ (서열번호 8)Reverse: 5′-AGG GCA CAC AGA AAA AG-3′ (서열번호 9)
mPIRO Forward: 5′-CCT TTT TCA GTT TTC CTC GCC-3′(서열번호 10)Reverse: 5′-TGC ACA CTG AAG GAC CTG GAA-3′(서열번호 11)
mc-Fos Forward: 5′-GGT GAA GAC CGT GTC AGG AG-3′ (서열번호 12)Reverse: 5′-TAT TCC GTT CCC TTC GGA TT-3′ (서열번호 13)
mNFATc1 Forward: 5′-GAG TAC ACC TTC CAG CAC CTT-3′(서열번호 14)Reverse: 5′-TAT GAT GTC GGG GAA AGA GA-3′ (서열번호 15)
mTRAP Forward: 5′-TCA TGG GTG GTG CTG CT-3′ (서열번호 16)Reverse: 5′-GCC CAC AGC CAC AAA TCT-3′ (서열번호 17)
mOSCAR Forward: 5′-GGA ATG GTC CTC ATC TGC TT-3′ (서열번호 18)Reverse: 5′-GGA ATG GTC CTC ATC TGC TT-3′ (서열번호 19)
mGAPDH Forward: 5′-TCA AGA AGG TGG TGA AGC AG-3′ (서열번호 20)Reverse: 5′-AGT GGG AGT TGC TGT TGA AGT-3′(서열번호 21)
hPGRN Forward: 5′-TCC CCC TAA CCA AAT TCT CC-3′ (서열번호 22)Reverse: 5′-GGG ATG GCA GCT TGT AAT GT-3′ (서열번호 23)
hPIRO Forward: 5′-cct ttt tca gtt ttc ctc gcc-3′(서열번호 24)Reverse: 5′-tgc aca ctg aag gac ctg gaa-3′(서열번호 25)
hGAPDH Forward: 5′-TCA AGA AGG TGG TGA AGC AG-3′ (서열번호 26)Reverse: 5′-GGT GGA GGA GTG GGT GTC-3′ (서열번호 27)
siRNA sequences (Duplex)
mPIRO-siRNA 1 Sense: 5′-CUC CAU AUU CCA GGU CCU U(dTdT)-3′(서열번호 28)Antisense: 5′-AAG GAC CUG GAA UAU GGA G(dTdT)-3′(서열번호 29)
mPIRO-siRNA 2 Sense: 5′-CGU CCU AAA GUG UGU AUU U(dTdT)-3′(서열번호 30)Antisense: 5′-AAA UAC ACA CUU UAG GAC G(dTdT)-3′(서열번호 31)
mPIRO-siRNA 3 Sense: 5′-CGU CCU AAA GUG UGU AUU U(dTdT)-3′(서열번호 32)Antisense: 5′-AAA UAC ACA CUU UAG GAC G(dTdT)-3′(서열번호 33)
hPIRO-siRNA1 Sense: 5′-CAC UUA UGA GUG AGA ACA U(dTdT)-3′(서열번호 34)Antisense: 5′-AUG UUC UCA CUC AUA AGU G(dTdT)-3′(서열번호 35)
hPIRO-siRNA2 Sense: 5′-CUC CAU UUU UCA AGU CUC A(dTdT)-3′(서열번호 36)Antisense: 5′-UGA GAC UUG AAA AAU GAC G(dTdT)-3′(서열번호 37)
hPIRO-siRNA3 Sense: 5′-CUG UCU UCU UGC CAU AUU U(dTdT)-3′(서열번호 38)Antisense: 5′-AAA UAU GGC AAG ACA G(dTdT)-3′ (서열번호 39)
shRNA sequences (Vector: pMLP-GFP)
mPGRN-shRNA 1 5′-AAAGGAGGTGAAGTGCGACATA TAGTGAAGCCACAGATGTA TATGTCGCACTTCACCTCCTTC-3′(서열번호 40)
mPGRN-shRNA 2 5′-ATGACCTGATCCAGAGTAAGTA TAGTGAAGCCACAGATGTA TACTTACTCTGGATCAGGTCAC-3′(서열번호 41)
mPGRN-shRNA 3 5′-AGAGAAGGGCATTTCTGCCATA TAGTGAAGCCACAGATGTA TATGGCAGAAATGCCCTTCTCC-3′(서열번호 42)
hPGRN-shRNA 1 5′-ATGCCCTGATGGTTCTACCTGA TAGTGAAGCCACAGATGTA TCAGGTAGAACCATCAGGGCAC-3′(서열번호 43)
hPGRN-shRNA 2 5′-AAAGGACACTTCTGCCATGATA TAGTGAAGCCACAGATGTATATCATGGCAGAAGTGTCCTTC-3′(서열번호 44)
hPGRN-shRNA 3 5′-ACAACGTGAAGGCTCGATCCTA TAGTGAAGCCACAGATGTA TAGGATCGAGCCTTCACGTTGC-3′(서열번호 45)
hPGRN-shRNA 4 5′-ACCCAGGGCTACACGTGTGTAA TAGTGAAGCCACAGATGTA TTACACACGTGTAGCCCTGGGG-3′(서열번호 46)
Table 1
Primer sequences for Real-time RT-PCR
mPGRN Forward: 5′-TTC ACA CAC GAT GCG TTT CA-3 ′ (SEQ ID NO: 8) Reverse: 5′-AGG GCA CAC AGA AAA AG-3 ′ (SEQ ID NO: 9)
mPIRO Forward: 5'-CCT TTT TCA GTT TTC CTC GCC-3 '(SEQ ID NO: 10) Reverse: 5'-TGC ACA CTG AAG GAC CTG GAA-3' (SEQ ID NO: 11)
mc-Fos Forward: 5′-GGT GAA GAC CGT GTC AGG AG-3 ′ (SEQ ID NO: 12) Reverse: 5′-TAT TCC GTT CCC TTC GGA TT-3 ′ (SEQ ID NO: 13)
mNFATc1 Forward: 5′-GAG TAC ACC TTC CAG CAC CTT-3 ′ (SEQ ID NO: 14) Reverse: 5′-TAT GAT GTC GGG GAA AGA GA-3 ′ (SEQ ID NO: 15)
mTRAP Forward: 5'-TCA TGG GTG GTG CTG CT-3 '(SEQ ID NO: 16) Reverse: 5'-GCC CAC AGC CAC AAA TCT-3' (SEQ ID NO: 17)
mOSCAR Forward: 5'-GGA ATG GTC CTC ATC TGC TT-3 '(SEQ ID NO: 18) Reverse: 5'-GGA ATG GTC CTC ATC TGC TT-3' (SEQ ID NO: 19)
mGAPDH Forward: 5′-TCA AGA AGG TGG TGA AGC AG-3 ′ (SEQ ID NO: 20) Reverse: 5′-AGT GGG AGT TGC TGT TGA AGT-3 ′ (SEQ ID NO: 21)
hPGRN Forward: 5′-TCC CCC TAA CCA AAT TCT CC-3 ′ (SEQ ID NO: 22) Reverse: 5′-GGG ATG GCA GCT TGT AAT GT-3 ′ (SEQ ID NO: 23)
hPIRO Forward: 5′-cct ttt tca gtt ttc ctc gcc-3 ′ (SEQ ID NO: 24) Reverse: 5′-tgc aca ctg aag gac ctg gaa-3 ′ (SEQ ID NO: 25)
hGAPDH Forward: 5′-TCA AGA AGG TGG TGA AGC AG-3 ′ (SEQ ID NO 26) Reverse: 5′-GGT GGA GGA GTG GGT GTC-3 ′ (SEQ ID NO 27)
siRNA sequences (Duplex)
mPIRO-siRNA 1 Sense: 5′-CUC CAU AUU CCA GGU CCU U (dTdT) -3 ′ (SEQ ID NO: 28) Antisense: 5′-AAG GAC CUG GAA UAU GGA G (dTdT) -3 ′ (SEQ ID NO: 29)
mPIRO-siRNA 2 Sense: 5′-CGU CCU AAA GUG UGU AUU U (dTdT) -3 ′ (SEQ ID NO: 30) Antisense: 5′-AAA UAC ACA CUU UAG GAC G (dTdT) -3 ′ (SEQ ID NO: 31)
mPIRO-siRNA 3 Sense: 5′-CGU CCU AAA GUG UGU AUU U (dTdT) -3 ′ (SEQ ID NO: 32) Antisense: 5′-AAA UAC ACA CUU UAG GAC G (dTdT) -3 ′ (SEQ ID NO: 33)
hPIRO-siRNA1 Sense: 5'-CAC UUA UGA GUG AGA ACA U (dTdT) -3 '(SEQ ID NO: 34) Antisense: 5'-AUG UUC UCA CUC AUA AGU G (dTdT) -3' (SEQ ID NO 35)
hPIRO-siRNA2 Sense: 5′-CUC CAU UUU UCA AGU CUC A (dTdT) -3 ′ (SEQ ID NO: 36) Antisense: 5′-UGA GAC UUG AAA AAU GAC G (dTdT) -3 ′ (SEQ ID NO 37)
hPIRO-siRNA3 Sense: 5′-CUG UCU UCU UGC CAU AUU U (dTdT) -3 ′ (SEQ ID NO: 38) Antisense: 5′-AAA UAU GGC AAG ACA G (dTdT) -3 ′ (SEQ ID NO: 39)
shRNA sequences (Vector: pMLP-GFP)
mPGRN-shRNA 1 5′-AAAGGAGGTGAAGTGCGACATA TAGTGAAGCCACAGATGTA TATGTCGCACTTCACCTCCTTC-3 ′ (SEQ ID NO: 40)
mPGRN-shRNA 2 5′-ATGACCTGATCCAGAGTAAGTA TAGTGAAGCCACAGATGTA TACTTACTCTGGATCAGGTCAC-3 ′ (SEQ ID NO: 41)
mPGRN-shRNA 3 5′-AGAGAAGGGCATTTCTGCCATA TAGTGAAGCCACAGATGTA TATGGCAGAAATGCCCTTCTCC-3 ′ (SEQ ID NO: 42)
hPGRN-shRNA 1 5′-ATGCCCTGATGGTTCTACCTGA TAGTGAAGCCACAGATGTA TCAGGTAGAACCATCAGGGCAC-3 ′ (SEQ ID NO: 43)
hPGRN-shRNA 2 5′-AAAGGACACTTCTGCCATGATA TAGTGAAGCCACAGATGTATATCATGGCAGAAGTGTCCTTC-3 ′ (SEQ ID NO: 44)
hPGRN-shRNA 3 5′-ACAACGTGAAGGCTCGATCCTA TAGTGAAGCCACAGATGTA TAGGATCGAGCCTTCACGTTGC-3 ′ (SEQ ID NO 45)
hPGRN-shRNA 4 5′-ACCCAGGGCTACACGTGTGTAA TAGTGAAGCCACAGATGTA TTACACACGTGTAGCCCTGGGG-3 ′ (SEQ ID NO: 46)
상기 실시예의 결과를 종합하면 다음과 같다.The result of the above embodiment is summarized as follows.
첫째, PGRN과 PIRO는 RANK 신호전달의 중요한 축을 이룬다.First, PGRN and PIRO form an important axis of RANK signaling.
둘째, RANK 신호전달은 PGRN의 발현(48시간) 및 PIRO의 발현(72시간)을 순차적으로 조절한다.Second, RANK signaling sequentially regulates PGRN expression (48 hours) and PIRO expression (72 hours).
셋째, PIRO는 파골세포 융합과 골흡수의 아주 중요한 후기 인자(late-stage factor)로서 PGRN에 의해 직접적으로 유도되는 유전자이다.Third, PIRO is a gene directly induced by PGRN as a late-stage factor of osteoclast fusion and bone resorption.
상기와 같은 연구결과를 바탕으로 도 15에서와 같은 RANKL/RANK axis에서 PGRN과 PIRO의 기능이 예상된다.Based on the above research results, the functions of PGRN and PIRO in the RANKL / RANK axis as shown in FIG. 15 are expected.
이에 따르면, RANKL이 RANK에 결합한 후 NFATc1의 활성화 신호전달에서 PGRN을 생성하는 두 가지 경로(pathway)가 예상된다. 경로 I은 autocrine manner로 분화과정의 파골세포 자체에서 PGRN이 생성되어 PGRN 수용체를 통해 PIRO 유전자를 발현하는 것이고, 경로 II는 RANK 신호전달에 의해서 골수에 존재하는 다른 세포, 예를 들어 조골세포(osteoblasts), stromal cells 및 mesenchymal stem cell에 작용하여 그들 세포에서 PGRN이 발현되고 paracrine manner로 분화 중인 파골세포의 PGRN 수용체를 통한 신호전달이 PIRO 유전자를 발현시켜 multinucleated osteoclasts 생성과 골흡수에 관여하는 것이다.According to this, two pathways are expected to generate PGRN in the activation signaling of NFATc1 after RANKL binds to RANK. Path I is an autocrine manner in which the PGRN is produced in the osteoclasts of differentiation itself and expresses the PIRO gene through the PGRN receptor. Path II is another cell in the bone marrow by RANK signaling, eg osteoblasts. PGRN is expressed in these cells and acts on stromal cells and mesenchymal stem cells, and signaling through the PGRN receptors of osteoclasts, which are differentiated in a paracrine manner, is involved in the production of multinucleated osteoclasts and bone resorption.
본 발명에 따르면 PIRO는 RANK 신호전달계에서 중요한 축을 이루고, 파골세포의 융합과 골흡수(bone resorption)의 후기 단계 인자로서 프로그래뉼린의 직접적인 유도유전자이므로, PIRO를 바이오마커로 사용하여 새로운 골다공증 예방 또는 치료제 개발, 또는 골다공증 진단, 치료 결과 또는 예후 평가에 유용하게 사용할 수 있으며, PIRO 억제제를 사용하면 골다공증을 효과적으로 예방 또는 치료할 수 있다.According to the present invention, PIRO forms an important axis in RANK signaling system and is a direct inducer of progranulin as a late stage factor of osteoclast fusion and bone resorption, so that PIRO is used as a biomarker to prevent or prevent new osteoporosis. It can be useful for developing therapeutics, diagnosing osteoporosis, evaluating treatment results or prognosis, and using PIRO inhibitors can effectively prevent or treat osteoporosis.

Claims (12)

  1. 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질의 억제제를 유효성분으로 함유하는 골다공증의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of osteoporosis, comprising an amino acid sequence of SEQ ID NO: 1 to 3 and containing an inhibitor of a protein having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5.
  2. 제 1항에 있어서,The method of claim 1,
    상기 단백질은 서열번호 4 또는 5의 아미노산 서열로 구성되는 것을 특징으로 하는 골다공증의 예방 또는 치료용 약학적 조성물.The protein is a pharmaceutical composition for the prevention or treatment of osteoporosis, characterized in that consisting of the amino acid sequence of SEQ ID NO: 4 or 5.
  3. 제 1항에 있어서,The method of claim 1,
    상기 억제제는 상기 단백질을 암호화하는 유전자의 mRNA에 상보적으로 결합하는 안티센스뉴클레오티드, 작은 간섭 RNA(short interfering RNA) 및 짧은 헤어핀 RNA(short hairpin RNA), 및 상기 단백질에 결합하는 앱타머(apatamer) 및 항체로 구성된 군으로부터 선택된 어느 하나인 것을 특징으로 하는 골다공증의 예방 또는 치료용 약학적 조성물.The inhibitor comprises antisense nucleotides complementarily binding to mRNA of the gene encoding the protein, short interfering RNA and short hairpin RNA, aptamer binding to the protein and A pharmaceutical composition for the prevention or treatment of osteoporosis, characterized in that any one selected from the group consisting of antibodies.
  4. 제 3항에 있어서,The method of claim 3, wherein
    상기 유전자는 서열번호 6 또는 7의 염기 서열로 구성되는 것을 특징으로 하는 골다공증의 예방 또는 치료용 약학적 조성물.The gene is a pharmaceutical composition for the prevention or treatment of osteoporosis, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 6 or 7.
  5. 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질의 억제제를 유효성분으로 함유하는 파골세포(osteoclasts) 융합 억제용 약학적 조성물.A pharmaceutical composition for inhibiting osteoclast fusion containing an amino acid sequence of SEQ ID NO: 1 to 3 and containing an inhibitor of a protein having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5.
  6. 1) 피검 개체로부터 분리된 혈액, 혈장 또는 혈청으로부터 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질의 mRNA 또는 상기 단백질의 발현 수준을 측정하는 단계; 및1) measuring the mRNA level of the protein or the expression level of the protein having an amino acid sequence of SEQ ID NO: 1 to 3 and at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5 from blood, plasma or serum isolated from the test subject Doing; And
    2) 상기 단백질의 mRNA 또는 상기 단백질의 발현 수준이 정상 대조군에 비해 증가한 개체를 선별하는 단계;를 포함하는 골다공증 진단, 치료 결과 또는 예후 평가의 정보를 제공하기 위한 단백질 발현 수준의 측정 방법.2) selecting the mRNA of the protein or the individual in which the expression level of the protein is increased compared to a normal control; measuring the protein expression level to provide information of osteoporosis diagnosis, treatment results or prognostic evaluation comprising a.
  7. 제 6항에 있어서,The method of claim 6,
    상기 단백질의 mRNA 발현 수준은 RT-PCR, 정량적 또는 반정량적 RT-PCR(quantitative or semi-quantitative RT-PCR), 정량적 또는 반정량적 리얼 타임 RT-PCR(quantitative or semi-quantitative real-time RT-PCR), 노던 블롯(northern blot), 및 DNA 또는 RNA 칩(chip)으로 구성된 군으로부터 선택되는 어느 하나의 방법으로 측정하는 것을 특징으로 하는 골다공증 진단, 치료 결과 또는 예후 평가의 정보를 제공하기 위한 단백질 발현 수준의 측정 방법.The mRNA expression level of the protein is RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real-time RT-PCR ), Northern blot, and protein expression for providing information of osteoporosis diagnosis, treatment outcome or prognostic assessment, characterized in that it is measured by any one method selected from the group consisting of DNA or RNA chips. How to measure the level.
  8. 제 6항에 있어서,The method of claim 6,
    상기 단백질의 발현 수준은 조직면역염색법, 효소면역분석법(ELISA) 및 웨스턴 블롯으로 구성된 군으로부터 선택되는 어느 하나의 방법으로 측정하는 것을 특징으로 하는 골다공증 진단, 치료 결과 또는 예후 평가의 정보를 제공하기 위한 단백질 발현 수준의 측정 방법.Expression level of the protein is measured by any one method selected from the group consisting of tissue immunostaining, enzyme immunoassay (ELISA) and Western blot to provide information on the diagnosis of osteoporosis, treatment results or prognosis Methods of Measuring Protein Expression Levels.
  9. 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질에 결합하는 항체, 상기 단백질을 암호화하는 유전자에 상보적인 핵산, 및 상기 유전자에 특이적인 프라이머 또는 프로브로 구성된 군으로부터 선택되는 어느 하나를 포함하는 골다공증 진단, 치료 결과 또는 예후 평가용 키트.An antibody binding to a protein having an amino acid sequence of SEQ ID NOs: 1 to 3 and having at least 80% homology with an amino acid sequence of SEQ ID NO: 4 or 5, a nucleic acid complementary to the gene encoding the protein, and a primer specific for the gene Or a kit for diagnosing osteoporosis, treatment results or prognosis comprising any one selected from the group consisting of probes.
  10. 1) 피검 조성물 또는 화합물을 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질 발현 세포주에 처리하는 단계;1) treating the test composition or compound to a protein expressing cell line having the amino acid sequence of SEQ ID NOs: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5;
    2) 상기 세포주의 상기 단백질의 mRNA 또는 상기 단백질의 발현 수준을 측정하는 단계; 및2) measuring the mRNA level of the protein or the expression level of the protein of the cell line; And
    3) 상기 단백질의 mRNA 또는 단백질의 발현 수준이 무처리 대조군에 비해 감소된 피검 조성물 또는 화합물을 선별하는 단계;를 포함하는 골다공증 예방 또는 치료제의 스크리닝 방법.3) selecting a test composition or compound in which the expression level of the mRNA or protein of the protein is reduced compared to the untreated control; screening method for preventing or treating osteoporosis.
  11. 1) 피검 조성물 또는 화합물을 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질 발현 세포주에 처리하는 단계;1) treating the test composition or compound to a protein expressing cell line having the amino acid sequence of SEQ ID NOs: 1 to 3 and having at least 80% homology with the amino acid sequence of SEQ ID NO: 4 or 5;
    2) 상기 세포주의 상기 단백질의 mRNA 또는 상기 단백질의 발현 수준을 측정하는 단계; 및2) measuring the mRNA level of the protein or the expression level of the protein of the cell line; And
    3) 상기 단백질의 mRNA 또는 상기 단백질의 발현 수준이 무처리 대조군에 비해 감소된 피검 조성물 또는 화합물을 선별하는 단계;를 포함하는 파골세포 융합 억제제의 스크리닝 방법.3) screening the test composition or compound wherein the mRNA level of the protein or the expression level of the protein is reduced compared to the untreated control; screening method for osteoclast fusion inhibitor comprising a.
  12. 서열번호 1 내지 3의 아미노산 서열을 갖고 서열번호 4 또는 5의 아미노산 서열과 80% 이상의 상동성을 갖는 단백질을 유효성분으로 함유하는 파골세포 융합 유도용 조성물.A composition for inducing osteoclast fusion comprising a protein having an amino acid sequence of SEQ ID NOs: 1 to 3 and having a homology of 80% or more with an amino acid sequence of SEQ ID NO: 4 or 5 as an active ingredient.
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