# Patent

## Title
Use of human small leucine zipper protein in adipocyte differentiation procedure

## Abstract

The present invention relates to a use of a human small leucine zipper protein in the adipocyte differentiation procedure. More specifically, sLZIP binds with PPARγ2 to induce the formation of a complex of HDAC3 and PPARγ2, thereby functioning as a corepressor to negatively inhibit the transcriptional activity of PPARγ2 and suppress the differentiation to adipocytes, and thus can be used as a marker for treating diabetes and obesity and developing new medicines therefor.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Phase of International Application No. PCT/KR2014/003864, filed Apr. 30, 2014, which claims priority to Korean Application No. 10-2013-0048133, filed Apr. 30, 2013, the disclosures of which are all incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to a use of human small leucine-zipper proteins in differentiation of mesenchymal stem cells into adipocytes.
BACKGROUND ART
Cellular therapy is a hopeful new approach to address unmet medical needs in patients. Currently, mesenchymal stem cells (MSCs) are used in multiple human clinical trials. However, it has become necessary to address problems such as differentiation regulation of mesenchymal stem cells for medical treatment.
Adipocytes and osteoblasts are differentiated from mesenchymal stem cells, and such differentiation is regulated by a transcription factor. The balance between adipogenesis and osteogenesis in mesenchymal stem cells is very important to repair/regenerate and maintain homeostasis. The disruption of controlling the balance of these processes during MSC differentiation leads to the disorders such as osteoarthritis and osteoporosis. PPARγ2 is expressed when mesenchymal stem cells are differentiated into adipocytes and involved in expression regulation of adipogenic genes. Also, Runx2 is expressed when osteoblasts are differentiated and involved in expression of osteogenic genes. Therefore, understanding the regulatory mechanism of transcription factors in osteoblast and adipocyte differentiation is very important.
PPARγ is a member of the PPAR family of transcription factors that includes PPARα, PPARγ, and PPARγ. PPARγ is a master regulator in adipogenesis, lipid biosynthesis, inflammation, and glucose metabolism. Alternative splicing produces PPARγ variants, including two major forms of the protein, PPARγ1 and PPARγ2. PPARγ2 differs from PPARγ1 by 30 additional amino acids on its N-terminus, and is expressed mainly in macrophages and adipogenic cells and partially expressed in bone marrow stromal cells. PPARγ1 is expressed in a wide range of tissues, including skeletal muscle, adipose tissue and bone. Binding of PPARγ to specific DNA sequences, including peroxisome proliferator-activated response element (PPRE) which consists of 2 direct repeats of the consensus nuclear receptor half-site separated by 1 base pair, requires heterodimerization with a second member of the nuclear receptor family, retinoic X receptor (RXR). This element is found in ap2 related to lipid storage and a CD36 promoter involved in cholesterol transport. The heterologous complex of PPARγ and RXR is associated with the nuclear receptor corepressor complex, including histone deacetylase (HDAC), nuclear receptor corepressor (NCoR) and silencing mediator for retinoid and thyroid receptors (SMRT) in the absence of PPAR ligand. Ligand binding to PPARγ triggers a conformational change and the corepressor complex is replaced by coactivators such as the p160/steroid receptor coactivator (p160/SRC) family, the mediator complex including PPARγ binding protein (PBP), PGC-1 (PPARγ coactivator-1) and CREB binding protein (CBP), and p300, leading to transcriptional initiation of target genes by a conformational change. Many transcription factors and ligands are involved in expression and function regulation of PPARγ. CCAAT/enhancer-binding protein (C/EBP) is directly bound to a PPARγ promoter to promote transcription. Prostaglandin J2, which is a natural PPARγ ligand, and thiazolidinediones (TZD), which is a synthetic reagent, for example, rosiglitazone and pioglitazone also increase a transcriptional activity of PPARγ. Retinoblastoma gene (RB) and cyclin D1 inhibit the transcriptional activity of PPARγ as a negative regulator.
Leucine zipper protein (LZIP) is a member of the large family of bZIP that belongs to the CREB/ATF gene family. LZIP includes a basic DNA-binding domain and a leucine-zipper domain that binds to a consensus cAMP-responsive element (CRE) and an AP-1 element. A human LZIP was identified as a host cell factor 1 (HCF-1) interacting protein that promotes cell proliferation and cellular transformation. N-terminal 92 amino acids of LZIP are a potent transactivation domain that consists of two LxxLL-transcriptional coactivator interaction motifs. LZIP includes five members, CREB3 (LZIP, Luman), CREB3L1 (OASIS), CREB3L2 (BBF2H7), CREB3L3 (CREB-H), and CREB3L4 (AIbZIP), which have a homology and different functions of transcription factors. Function of LZIP has been reported that LZIP binds to CCR1 and participates in regulation of Lkn-1-dependent cell migration. Also, LZIP binds to the CCR2 promoter, enhances expression of CCR2 and increases monocyte migration.
In recent years, a small LZIP, which is an isoform of LZIP, has been identified, and includes 354 amino acids having no transmembrane domain. sLZIP is not involved in LKN-1-dependent cell migration, activates HDACs, and thereby inhibits a transcriptional activity of a glucocorticoid receptor.
Accordingly, the inventors studied the regulation mechanism of sLZIP that regulates transcriptional activities of PPARγ and Runx2 in connection with differentiation of mesenchymal stem cells into osteoblasts and adipocytes, and thereby completed the invention.
DISCLOSURE
Technical Problem
The present invention provides a use of stem cells as a differentiation regulator by identifying roles of a human small leucine-zipper protein (abbreviation: sLZIP) in differentiation of mesenchymal stem cells into adipocytes.
The present invention also provides a use of the sLZIP for preventing or treating diabetes or obesity.
The present invention also provides a screening use of the sLZIP for a medicine for preventing or treating diabetes or obesity.
Technical Solution
In order to achieve the above objects, the present invention provides a composition for inhibiting differentiation of mesenchymal stem cells into adipocytes comprising human small leucine-zipper proteins as a differentiation regulator.
The present invention also provides a composition for preventing or treating diabetes comprising human small leucine-zipper proteins.
The present invention provides a use of human small leucine-zipper proteins for preparing a composition for preventing or treating diabetes.
The present invention also provides a method of treating diabetes of an animal, including administering a composition for preventing or treating diabetes comprising a pharmaceutically effective dose of human small leucine-zipper proteins to a subject.
The present invention also provides a composition for preventing or treating obesity comprising human small leucine-zipper proteins.
The present invention also provides a use of human small leucine-zipper proteins for preparing a composition for preventing or treating obesity.
The present invention also provides a method of treating obesity of an animal, including administering a composition for preventing or treating obesity comprising a pharmaceutically effective dose of human small leucine-zipper proteins to a subject.
The present invention also provides a screening method of a medicine for preventing or treating diabetes, including bringing genes of human small leucine-zipper proteins in contact with a candidate material outside a human body and determining whether the candidate material promotes or inhibits expression of the genes.
The present invention also provides a screening method of a medicine for preventing or treating diabetes, including bringing human small leucine-zipper proteins in contact with a candidate material outside a human body and determining whether the candidate material promotes or inhibits a function or an activity of the protein.
The present invention also provides a screening method of a medicine for preventing or treating obesity, including bringing genes of human small leucine-zipper proteins in contact with a candidate material outside a human body and determining whether the candidate material promotes or inhibits expression of the genes.
The present invention also provides a screening method of a medicine for preventing or treating obesity, including bringing human small leucine-zipper proteins in contact with a candidate material outside a human body and determining whether the candidate material promotes or inhibits a function or an activity of the protein.
Advantageous Effects
According to the present invention, human small leucine-zipper proteins (sLZIPs) are bound to PPARγ2 to induce the formation of an HDAC3 and PPARγ2 complex, and serve as a corepressor of PPARγ2 to negatively inhibit the transcriptional activity of PPARγ2 and inhibit differentiation into adipocytes. Therefore, sLZIP serve as a regulator that regulates a balance of differentiation of adipocytes and osteoblasts in mesenchymal stem cells.
Therefore, sLZIP can be used for a therapeutic use for diabetes or obesity, or used as a marker for development of a new therapeutic medicine.



DESCRIPTION OF DRAWINGS
FIG. 1 shows an effect of sLZIP according to the present invention on transcriptional activities of PPARγ. FIGS. 1A to 1C show the results obtained by measuring a transcriptional activity of PPARγ2 due to sLZIP when rosiglitazone (RZD) (FIG. 1A), pioglitazone (PZD) (FIG. 1B) and troglitazone (TZD) (FIG. 1C), which are ligands of PPARγ2, are treated. FIG. 1D shows the result obtained by measuring a transcriptional activity of PPARγ2 due to si-sLZIP. FIG. 1E shows the result obtained by measuring a transcriptional activity of PPARγ2 when mesenchymal stem cells overexpressing sLZIP are differentiated.
FIG. 2 shows the result obtained by examining a binding ability of sLZIP according to the present invention with respect to PPARγ. FIGS. 2A and B show the results obtained by examining a binding ability between sLZIP and PPARγ2 in 293T cells transfected with GST-sLZIP and Myc-PPARγ2 (FIG. 2A) and GST-PPARγ2 and Flag-sLZIP (FIG. 2B). FIG. 2C shows the immunoblotting result obtained by measuring whether His-sLZIP and PPARγ2 bind using anti-PPARγ2 and anti-His antibodies. FIG. 2D shows the measurement results of localizations in the nucleus of GFP-PPARγ2 and Flag-sLZIP expressed in C3H10T1/2 cells.
FIG. 3 shows the analysis result of genetic maps and binding regions of sLZIP and PPARγ2 according to the present invention. FIG. 3A represents sLZIP and FIG. 3B represents PPARγ2.
FIG. 4 shows the identification result of roles of HDAC3 in transcriptional activity inhibition of PPARγ2 due to sLZIP according to the present invention. FIG. 4A shows a transcriptional activity effect of PPARγ2 after TSA serving as an inhibitor of HDAC is treated. FIG. 4B shows a transcriptional activity effect of PPARγ2 after expression is inhibited through HDAC si-RNA. FIG. 4C shows a transcriptional activity effect of PPARγ2 when HDAC3 is overexpressed. FIG. 4D shows the result obtained by measuring localizations of HDAC3 and sLZIP in the nucleus. FIG. 4E is the result showing binding of HDAC3 and sLZIP according to GST pull-down analysis.
FIG. 5 shows the result of corepressor inducement of PPARγ2 due to sLZIP according to the present invention. FIG. 5A shows interaction of PPARγ2 ligand-dependent sLZIP and PPARγ2. FIG. 5B shows a binding effect of sLZIP and PPARγ2 due to PPARγ2 ligand treatment according to time of day. FIG. 5C shows a binding effect of sLZIP and HDAC3, which is an inhibitor of PPARγ2, in the presence of a PPARγ2 ligand. FIG. 5D shows a binding effect of sLZIP and a PPARγ2 and HDAC3 complex. FIG. 5E shows a binding effect of sLZIP and a PPARγ2 and HDAC3 complex according to NCoR1 treatment. FIG. 5F shows a regulation mechanism of sLZIP for ligand-dependent binding with PPARγ2 using sLZIP deletion mutants and a relation examination result.
FIG. 6 shows the result obtained by examining an effect of sLZIP according to the present invention on interaction between PPARγ2 and HDAC3. FIG. 6A shows an effect of sLZIP on interaction between PPARγ2 and HDAC3. FIG. 6B shows the result of ubiquitination of HDAC3 due to sLZIP. FIG. 6C shows an effect of sLZIP on a PPARγ2 coactivator.
FIG. 7 shows the result of the found binding region of sLZIP according to the present invention in an FABP4 enhancer region. FIG. 7A shows the result obtained by analyzing FABP4 enhancer sequences by a transcription element search system (TESS) and AliBaba2. FIG. 7B shows the EMSA result obtained by examining a DNA binding ability of sLZIP with various elements in an FABP4 enhancer region. FIGS. 7C and 7D show the results of supershift analysis and cold competition analysis through which a specific DNA binding ability of sLZIP with a CREB element in an FABP4 enhancer region was identified. FIG. 7E shows the test result of a transcriptional activity of PPARγ2 using FABP4 enhancer-luciferase constructs. FIG. 7F shows the ChIP result.
FIG. 8 shows an effect of sLZIP according to the present invention in an adipogenesis. FIGS. 8A and 8B show an effect of sLZIP on adipogenesis in vivo. FIGS. 8C and 8D show an effect of a transcriptional activity and PPARγ2 expression in adipogenesis of homologous mouse LZIPs. FIG. 8E shows an effect of sLZIP on adipocyte differentiation in multipotential mesenchymal progenitor cells.
FIG. 9 shows an effect of sLZIP according to the present invention on adipocyte differentiation in vivo. FIGS. 9A and 9B show preadipocytes (FIG. 9A) isolated from epididymal adipose tissues of sLZIP TG mice, and adipocytes differentiated from mouse embryonic fibroblasts (FIG. 9B). FIGS. 9C and 9D show the results obtained by measuring an mRNA expression level of PPARγ2 target genes using RT-PCR (FIG. 9C) and real-time PCR (FIG. 9D).
FIG. 10 is a diagram schematically illustrating a differentiation regulation effect of sLZIP according to the present invention in mesenchymal stem cells.



MODES OF THE INVENTION
Hereinafter, a configuration of the present invention will be described in detail.
The present invention relates to a composition for inhibiting differentiation of mesenchymal stem cells into adipocytes comprising human small leucine-zipper proteins as a differentiation regulator.
The inventors identified that sLZIP binds with PPARγ2 to induce the formation of a complex of HDAC3 and PPARγ2 on differentiation of mesenchymal stem cells into adipocytes, serves as a corepressor of PPARγ2 to negatively inhibit a transcriptional activity of PPARγ2 and inhibits differentiation into adipocytes, and adipocyte differentiation of sLZIP transgenic mice is inhibited in an experiment using the sLZIP transgenic mice, and thereby completed the invention.
In general, sLZIP is a protein that an isoform of LZIP, includes 354 amino acids having no transmembrane domain region, is not involved in LKN-1-dependent cell migration and activates HDACs, and thereby inhibits a transcriptional activity of a glucocorticoid receptor.
Based on the result, the present invention proposes for the first time the fact that sLZIP is a differentiation regulator of mesenchymal stem cells, which regulates a balance of differentiation of adipocytes and osteoblasts.
According to one embodiment of the present invention, sLZIP inhibits a transcriptional activity of PPARγ2 in dose-dependent manner. Inhibition of the transcriptional activity of PPARγ2 occurred when sLZIP was directly bound to PPARγ2. Binding of sLZIP and PPARγ2 was performed in the nucleus. In order to examine a domain of sLZIP that is necessary to bind with PPARγ2, various sLZIP deletion mutants, for example, N-terminal deletion mutants (1-228), C-terminal deletion mutants (229-354) and CC-terminal deletion mutants (297-354) were prepared, and a PPAR binding domain was analyzed. As a result, a wild type sLZIP and C and CC domains of sLZIP were bound to PPARγ2, but N domains of sLZIP were not bound to PPARγ2. It can be seen that a CC-terminal domain containing a proline-rich region of sLZIP is important for binding with PPARγ2. Also, domains of PPARγ2 necessary for binding with sLZIP were examined. As a result, a ligand binding domain (AF-2) of PPARγ2 was necessary for binding with sLZIP. The result proved that LxxLL motifs of sLZIP are not necessary for a binding ability although PPARγ2 interacts with sLZIP.
It has been known that the transcriptional activity of PPARγ is regulated by a coactivator and a corepressor, and PPARγ2 is bound to a corepressor complex, for example, HDAC3, SMRT and NCoR, in a resting state. Accordingly, an effect of sLZIP on a transcriptional activity of PPARγ2 due to HDACs was examined. As a result, when there was no HDAC inhibitor, sLZIP inhibited the transcriptional activity of PPARγ2, but sLZIP did not decrease the transcriptional activity of PPARγ2 in cells treated with the HDAC inhibitor. Such transcriptional activity inhibition of PPARγ2 was limited to only HDAC3 among class 1 HDACs. Also, sLZIP was bound to HDAC3, and thus it can be seen that sLZIP was bound to HDAC3 to negatively regulate the transcriptional activity of PPARγ2. Since the corepressor complex is replaced by a coactivator when ligand binding with a nuclear receptor is performed, binding between sLZIP and PPARγ2 according to PPARγ2 ligand treatment was examined. As a result, when there was no ligand, sLZIP was bound to PPARγ2, and when there was a ligand, sLZIP was isolated from PPARγ2 in a time-dependent manner. Next, an effect of sLZIP for binding of PPARγ2 with the corepressor complex was examined. The result showed that, when there was no PPARγ2 ligand, sLZIP was bound to HDAC3 in a PPARγ2 corepressor complex. Also, it has been reported that LZIP includes an N-terminal activity domain (1-220) and is involved in a transcriptional activity of cAMP-response elements (CREs)-containing reporter genes. sLZIP, which is an isoform of the LZIP, is bound to PPARγ2, and is considered to be bound to a FABP4 promoter region and regulate a transcriptional activity thereof. In order to understand such a regulation mechanism, an effect of sLZIP when PPARγ2 and HDAC3 bind was examined. As a result, when there was a ligand, sLZIP increased binding between PPARγ2 and HDAC3, and when there was no ligand, PPARγ2 was bound to a corepressor such as HDAC3. When a PPARγ2 ligand was added, the PPARγ-corepressor complex was isolated and degradation according to an ubiquitin and proteasome pathway was induced. Also, sLZIP inhibited HDAC3 ubiquitination when the ligand was treated. Further, sLZIP inhibited the complementing of a coactivator PGC-1α for PPARγ2. That is, it can be seen that sLZIP is bound to PPARγ2 to regulate the transcriptional activity of PPARγ2, and increases the formation of the corepressor complex.
PPARγ2 serves as a master regulator of adipogenesis, is highly expressed during adipocyte differentiation, and regulates expression of genes involved in adipogenesis. Therefore, an effect of sLZIP on adipogenesis in vivo was examined. As a result, the transcriptional activity of PPARγ2 decreased in adipocytes of sLZIP TG mice more than in wild type (WT) mice. It was examined whether homologous mouse LZIPs have an influence on a transcriptional activity and expression of PPARγ2 during adipogenesis. As a result, mouse LZIPs also decreased the transcriptional activity of PPARγ2 compared to the control group. Interestingly, mouse LZIP expression decreased during adipogenesis. sLZIP inhibited adipocyte differentiation in differentiation of multipotent mesenchymal progenitor cells into adipocytes. Also, in adipocyte differentiation in vivo, differentiation of primary preadipocytes isolated from sLZIP TG mice and mouse embryonic fibroblasts (MEFs) was inhibited compared to wild type mice. Expression of FABP4 and other PPARγ2 target genes, for example, C/EBPα and LPL, was also down-regulated.
Based on the result, it can be seen that sLZIP inhibits the transcriptional activity of PPARγ2, and thereby serves as a negative regulator on differentiation of adipocytes in vitro and in vivo.
A composition for promoting differentiation of the present invention may include sLZIP such as natural or recombinant sLZIP or sLZIP having a substantially equivalent physiological activity thereto. Proteins having a substantially equivalent physiological activity include natural or recombinant sLZIP, a functional equivalent thereof, and a functional derivative thereof.
The term “functional equivalent” refers to an amino acid sequence variant in which some or all of amino acids of natural proteins are substituted or some of the amino acids are deleted or added, and that has a substantially equivalent physiological activity to that of natural sLZIP.
The term “functional derivative” refers to a protein that has been modified to increase or decrease physical and chemical properties of the sLZIP, and has a substantially equivalent physiological activity to that of natural sLZIP.
sLZIP of the present invention is a protein originating from a mammal, and preferably a human, and refers to a protein having a known sequence, for example, human-derived GenBank accession no. FJ263669, and more specifically, a protein represented by an amino acid sequence listed in SEQ ID NO: 1.
According to a detailed example, sLZIP used in the present invention may be prepared by genetic engineering methods that are known to those skilled in the art from GenBank accession no. FJ263669 and the like.
When proteins are prepared by a gene recombination method for natural sLZIP, if mammal cells are used instead of E. coli or insect cells, it is considered to be more similar to a natural type in terms of a degree of activity or solubility of proteins.
The recombinant sLZIP may be isolated using a typical column chromatography method and the like. Also, a degree of purification of proteins may be determined by sodium dodecyl sulfate-polyacrylamide-polyacrylamide gel electrophoresis (SDS-PAGE) and the like.
The composition for inhibiting differentiation of the present invention may be added as a differentiation regulating factor when mesenchymal stem cells are cultured in vitro. For example, when a differentiation-inducing culture of mesenchymal stem cells is performed, natural or recombinant sLZIPs are added so that the number of osteoblasts can be regulated through a quantitative change thereof.
The composition for inhibiting differentiation of the present invention may further include a known differentiation-inducing factor that induces differentiation of mesenchymal stem cells in addition to the sLZIP. For example, a ciliary neurotrophic factor (CNTF), bone morphogenetic proteins (BMPs), a transforming growth factor (TGFα), or a neuregulin-1 (Nrg1)/glia1 growth factor-2 (GGF2) may be used.
The present invention also relates to a composition for preventing or treating diabetes comprising human small leucine-zipper proteins.
The present invention also provides a use of human small leucine-zipper proteins for preparing a composition for preventing or treating diabetes.
The present invention also relates to a composition for preventing or treating obesity comprising human small leucine-zipper proteins.
The present invention also provides a use of human small leucine-zipper proteins for preparing a composition for preventing or treating obesity.
Since the sLZIP inhibits differentiation of mesenchymal stem cells into adipocytes, it can be used as an agent for preventing or treating diabetes or obesity.
sLZIP used in the composition for preventing or treating diabetes or obesity of the present invention is a protein originating from a mammal, and preferably a human, and refers to a protein having a known sequence, for example, human-derived GenBank accession no. FJ263669, and more specifically, a protein represented by an amino acid sequence listed in SEQ ID NO: 1.
The sLZIP may be included as a natural or recombinant protein type or a transformed stem cell type that overexpresses sLZIP.
The transformed stem cells that overexpress natural or recombinant sLZIP may be prepared by introducing a vector that expresses natural or recombinant sLZIP into stem cells using known methods.
The present invention also provides a method of treating diabetes of an animal, including administering a composition for preventing or treating diabetes comprising a pharmaceutically effective dose of sLZIP to subject.
The present invention also provides a method of treating obesity of an animal, including administering a composition for preventing or treating obesity comprising a pharmaceutically effective dose of sLZIP to a subject.
Since the pharmaceutical composition and the administration method used in the method of treating diabetes or obesity have already been described above, redundant description will not be provided in order to avoid excessive complexity in the present specification.
Meanwhile, a subject to which the pharmaceutical composition for preventing or treating diabetes or obesity can be administered includes all animals, for example, non-human animals such as dogs, cats, and rats.
Also, a pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier includes a carrier and a vehicle that are commonly used in the field of medicine, and specifically, includes an ion exchange resin, alumina, aluminum stearate, lecithin, a serum protein (for example, human serum albumin), a buffer material (for example, various phosphates, glycine, sorbic acid, potassium sorbate, and a partial glyceride mixture of saturated vegetable fatty acids), water, salts or electrolytes (for example, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, a cellulosic substrate, a polyethylene glycol, sodium carboxymethyl cellulose, polyarylates, waxes, a polyethylene glycol, lanolin, and the like, but the carrier is not limited thereto.
Also, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.
As an aspect, the composition according to the present invention may be prepared as an aqueous solution for parenteral administration. Preferably, Hank's solution, Ringer's solution, or a buffer solution such as a physically buffered saline, may be used. An aqueous injection suspension may include a substrate that may increase a viscosity of the suspension such as sodium carboxymethyl cellulose, sorbitol, or dextran.
The pharmaceutical composition of the present invention may be systemically or topically administered, and may be formulated in an appropriate formulation using a known technique for administration. For example, when the composition is administered orally, the composition may be mixed with an inert diluent or an edible carrier, sealed in a hard or soft gelatin capsule, or compressed into a tablet, and then administered. In oral administration, an activity compound may be mixed with an excipient and used in the form of an intake tablet, a buccal tablet, a troche, a capsule, an elixir, a suspension, syrup, a wafer, and the like.
Various formulations for injection, parenteral administration, and the like injection, parenteral administration, and the like may be prepared using commonly used methods or techniques. Since sLZIP is very soluble in a saline or a buffer solution, sLZIP is stored in a freeze-dried state, and then an effective dose of sLZIP may be formulated in a saline or a buffer solution for administration in an appropriate form for intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, percutaneous administration, and the like immediately before administration.
An effective dose of an active ingredient of the pharmaceutical composition of the present invention refers to an amount that is necessary to prevent, inhibit, or alleviate disease.
Therefore, the effective dose may be regulated according to various factors such as type of disease, severity of disease, an active ingredient contained in the composition and type and content of other components, types of formulation, a patient's age, weight, general health condition, and gender, diet, an administration time, an administration route, a secretion rate of the composition, a treatment period, and medicine used at the same time. For example, when administration is performed once or several times a day in adults, a dose of 0.1 ng/kg to 10 g/kg of sLZIP of the present invention may be administered.
The present invention also provides a screening method of a medicine for preventing or treating diabetes, including bringing genes of human small leucine-zipper proteins in contact with a candidate material outside a human body and determining whether the candidate material promotes or inhibits expression of the genes.
The present invention also provides a screening method of a medicine for preventing or treating diabetes, including bringing human small leucine-zipper proteins in contact with a candidate material outside a human body and determining whether the candidate material promotes or inhibits a function or an activity of the protein.
The present invention also provides a screening method of a medicine for preventing or treating obesity, including bringing genes of human small leucine-zipper proteins in contact with a candidate material outside a human body and determining whether the candidate material promotes or inhibits expression of the genes.
The present invention also provides a screening method of a medicine for preventing or treating obesity, including bringing human small leucine-zipper proteins in contact with a candidate material outside a human body and determining whether the candidate material promotes or inhibits a function or an activity of the protein.
According to the screening method of the present invention, first, a candidate material to be analyzed may be in contact with diabetes or mast cells including the gene or protein.
The candidate material may include a material promoting or inhibiting transcription into mRNA and translation into proteins in sLZIP gene sequences and a material estimated to have a possibility of a medicine promoting or inhibiting a function or an activity of sLZIP proteins according to a general selecting method, or randomly selected individual nucleic acids, proteins, peptides, other extracts, natural products, compounds, and the like.
Then, an amount of expression of the gene, an amount of proteins, or an activity of proteins may be measured in candidate material-treated cells. In the measurement result, when an increase or a decrease of the amount of expression of the gene, the amount of proteins, or the activity of the proteins is measured, the candidate material may be determined as a material capable of preventing or treating diabetes or obesity.
In the above description, measurement of the amount of expression of the gene, the amount of proteins, or the activity of proteins may be performed by various methods known in the related art, for example, RT-PCR, real time polymerase chain reaction, a Western blot, a Northern blot, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay analysis (RIA), radioimmunodiffusion, an immunoprecipitation assay, and the like, but the method is not limited thereto.
A candidate material exhibiting an activity of promoting gene expression or promoting a function of proteins obtained through the screening method of the present invention can be a candidate material of a therapeutic agent for diabetes or obesity.
Such a candidate material of a therapeutic agent for diabetes or obesity serves as a leading compound in the later development process of a therapeutic agent for diabetes or obesity. When the leading compound modifies and optimizes a structure thereof such that functions of sLZIP genes or proteins expressed therefrom may be promoted or inhibited, a novel therapeutic agent for diabetes or obesity can be developed.
Hereinafter, examples of the present invention will be described in detail. However, the following examples are only examples of the present invention, and the scope of the present invention is not limited to the following examples.
Preparation Example
A Dulbecco's modified Eagle's medium (DMEM) was commercially available from GIBCO technologies, Inc (Gaithersburg), and fetal bovine serum was commercially available from HyClone Laboratory (Logan, Utah). Anti-PPARγ2, anti-HDAC1, 2, 3, 4, 6, 8 and 9, anti-β-actin and anti-GST antibodies were commercially available from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-mouse and anti-rabbit peroxidase-bound secondary antibodies were commercially available from Pierce (Madison, Wis.). β-glycerophosphate and ascorbic acid were commercially available from Sigma (St. Louis, Mo.).
(Cell Culture and Differentiation)
Primary human mesenchymal stem cells (MSCs), C3H10T1/2, 293T and MEF cells were cultured in a DMEM to which thermally inactivated 10% FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL) were added. All cell types were cultured in a humidified incubator containing CO2 5% under a temperature condition of 37° C. In order to induce osteoblast differentiation, the medium was exchanged with a DMEM in which 50 μg/mL of ascorbic acid, 10 mM of β-glycerophosphate and 10% FBS were contained for 8 days. The differentiation medium was changed once every two days.
(Transient Expression and Viral Infection)
C3H10T1/2 and 293T cells were plated in a 12-well culture dish at a density of 2×105 cells/well and incubated for 24 hours. Then, according to instructions of the manufacturer, the cells were transfected with a 0.2 μg of reporter genes and 0.1 to 0.5 μg of a laboratory plasmid using Lipofectamine 2000 or Genefectine. After 24 hours, the cells were cultured in a serum-free DMEM with or without 10 nM of rosiglitazone. siRNAs were prepared using sLZIP and HDAC target sequences (Table 1). For an RNA interference experiment, according to instructions of the manufacturer, the cells were transfected with scrambled control group RNA and appropriate siRNAs using Lipofectamine 2000. Human MSC and C3H10T/1/2 cells were infected with an adenovirus vector containing cDNA of human sLZIP or an empty vector. The infected medium was exchanged with a fresh medium after two hours.







TABLE 1







siRNA sequence (+dTdT)










Forward
Reverse





Control
CCUACGCCACCAAUUUGGU
ACGAAAUUGGUGGCGUAGG


group
(SEQ ID NO: 3)
(SEQ ID NO: 4)


 


sLZIP
GGACCCAGAUGACUCCACA
AUAUGCUGUGGAGUCAUCU



GCAUAU(SEQ ID NO: 5)
GGGUCC(SEQ ID NO: 6)


 


HDAC1
CGACUGUUUGAGAACCUUA
UAAGGUUCUCAAACAGUCGCU



(SEQ ID NO: 7)
(SEQ ID NO: 8)


 


HDAC2
GGUCAAUAAGACCAGAUA
UUAUCUGGUCUUAUUGACCG



A(SEQ ID NO: 9)
(SEQ ID NO: 10)


 


HDAC3
GCCGGUUAUCAACCAGGUA
UACCUGGUUGAUAACCGGC



(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


HDAC8
CAUUCAGGAUGGCAUACAA
UUGUAUGCCAUCCUGAAUGGG



(SEQ ID NO: 13)
(SEQ ID NO: 14)









(Semi-Quantitative RT-PCR and Real-Time PCR)

According to instructions of the manufacturer, a TRIzol reagent (Invitrogen, Carlsbad, Calif.) was added to directly lyse cells in a culture dish. Accupower RT PreMix (BioNeer, Daejeon, Korea) was used to synthesize cDNA from 2 μg of total RNA. A reaction was performed for 60 minutes at 42° C. and for 5 minutes at 94° C. PCR amplification was performed using oligomers listed in Table 2 and a Hipi PCR Mix Kit (ELPIS). As an internal control group, GAPDH was amplified. A PCR product was subjected to electrophoresis in 1-2% (w/v) agarose gel containing 0.5 μg/mL ethidium bromide. A size of the PCR product was measured by comparing it with 1 kb DNA ladder marker (Invitrogen). An intensity of bands amplified according to RT-PCR was analyzed using MultiImageTM Light Cabinet (version 5.5, Alpha Innotech Corp., San Leandro, Calif.).
Real-time PCR was performed in LightCycler 480 using a SYBR Green Master Mix (Roche, Mannheim, Germany). β-actin was used as an internal control group. A target gene expression level rate with respect to β-actin was calculated using CT method. A Ct value is defined as a PCR cycle number at which a fluorescence signal reaches a fixed target threshold. Experiments were technically repeated three times for each experiment.







TABLE 2







Primer sequences for semi-quantitative RT-PCR










Forward
Reverse





sLZIP
AGCAGCAGCATGTACTCCT
CTAGCCTGAGTATCTGTC



CT (SEQ ID NO: 15)
CT (SEQ ID NO: 16)


 


GAPDH
CCATCACCATCTTCCAGGA
CCAGGAAATCATGTGCAA



G (SEQ ID NO: 17)
TC (SEQ ID NO: 18)


 


FABP4
GTGGGAACCTGGAAGCTTG
CTTCACCTTCCTGTCGTC



TC (SEQ ID NO: 19)
TGC 




(SEQ ID NO: 20)


 


mLZIP
ATGGATCCTGGTGGTCAG
CTAACCTGAATACCTGCC



(SEQ ID NO: 21)
(SEQ ID NO: 22)


 


PPARγ2
ATGGGTGAAACTCTGGGAG
CTAATACAAGTCCTTGTA



A (SEQ ID NO: 23)
GA (SEQ ID NO: 24)


 


TG mice
GGACGATGATGACAAGGAC
GTCAGAGGAGTACATGCT


genotype
T (SEQ ID NO: 25)
GCT




(SEQ ID NO: 26)
















TABLE 3







Primer sequences for real-time RT-PCR










Forward
Reverse





FABP4
CATCAGCGTAAATGGGGAT
TCGACTTTCCATCCCACTTC



T(SEQ ID NO: 27)
(SEQ ID NO: 28)


 


C/EBPα
TGGACAAGAACAGCAACG
TCACTGGTCAACTCCAGCAC



AG(SEQ ID NO: 29)
(SEQ ID NO: 30)


 


LPL
GGGCTCTGCCTGAGTTGTA
CCATCCTCAGTCCCAGAAAA



G(SEQ ID NO: 31)
(SEQ ID NO: 32)


 


Sox9
CTGAAGGGCTACGACTGG
TACTGGTCTGCCAGCTTCCT



AC(SEQ ID NO: 33)
(SEQ ID NO: 34)


 


Col2A1
GCCAAGACCTGAAACTCTG
GCCATAGCTGAAGTGGAAGC



C(SEQ ID NO: 35)
(SEQ ID NO: 36)


 


OSCAR
CACACACACCTGGCACCTA 
GAGACCATCAAAGGCAGAGC



C(SEQ ID NO: 37)
(SEQ ID NO: 38)


 


CTSK
CCAGTGGGAGCTATGGAA
AAGTGGTTCATGGCCAGTTC



GA(SEQ ID NO: 39)
(SEQ ID NO: 40)


 


TARP
TCCTGGCTCAAAAAGCAGT
ACATAGCCCACACCGTTCTC



T(SEQ ID NO: 41)
(SEQ ID NO: 42)


 


TG mice
TCGATTCCAGGCTTATGGA
AGTCGCTCGGTACCTCAGAA


sLZIP
G(SEQ ID NO: 43)
(SEQ ID NO: 44)


 


hGAPDH
GACAAGCTTCCCGTTCTCA
GAGTCAACGGATTTGGTCGT



G(SEQ ID NO: 45)
(SEQ ID NO: 46)


 


mGAPDH
ACCCAGAAGACTGTGGAT
CACATTGGGGGTAGGAACAC



GG(SEQ ID NO: 47)
(SEQ ID NO: 48)









(Western Blot Analysis)

Cells were obtained and washed with ice-cold PBS twice. An RIPA buffer (10 mM of HEPES, 10 mM of NaCl, 0.1 mM of EDTA, 0.1 mM of EGTA, 1% NP-40, 0.5 mM of PMSF, 0.1 mM of DTT, 0.1 mM of Na3VO4, and a protease inhibitor) was used to prepare cell extracts. A suspension was centrifuged at 16,000×g for 20 minutes at 4° C. Supernatants were collected and mixed with a sample buffer. A protein sample was isolated in SDS-PAGE (8 to 15%), and transferred to nitrocellulose membranes. The membranes and appropriate antibodies were incubated overnight at 4° C. Then, each immunoblot was incubated in secondary antibodies labeled with horseradish peroxidase. Immune-labeled proteins were observed using ECL analysis (Amersham), and an ECL reaction was developed using an X-ray film. The blot was stripped and then anti-β-actin was reacted again and used as an internal control group.
(Activity Analysis of Luciferase Reporter Gene)
Appropriately transfected cells were washed with cold PBS twice, and a reporter lysis buffer (Promega) was used and lysed in a culture dish. Luciferase analysis was performed using a luciferase analysis system (Promega Corporation, Madison, Wis.). A luciferase activity was recorded in Luminometer 20/20n (Turner BioSystems, Sunnyvale, Calif.) according to instructions of the manufacturer. The luciferase activity was normalized to a β-galactosidase activity. For β-galactosidase analysis, CMV-β-galactosidase was transfected with luciferase reporter genes. All pieces of data were represented as mean±standard deviation of the results from at least three independent experiments.
(Electrophoretic Mobility Shift Assay, EMSA)
T4 polynucleotide kinase (Promega, Madison, Wis.) was used to label 5′-terminal of oligonucleotides with [γ-32P]ATP (Perkin Elmer, Waltham, Mass.). According to instructions of the manufacturer, unbound nucleotides were removed through tubes in a Bio-Gel P-6 spin column (Bio-Rad, Inc., Hercules, Calif.). His-sLZIP proteins were pre-incubated for 20 minutes at room temperature in a 5× binding buffer [10×GRAB, 20 mM of DTT, poly(deoxyinosinic-deoxycytidylic), Sigma, St. Louis] and radiolabeled probes. Reaction mixtures were loaded in wells of 4% nondenaturing polyacrylamide gels, and subjected to electrophoresis for 45 minutes at 180 V in a 0.5×TBE buffer. For competition experiments, a binding reaction was incubated for 30 minutes with 100-fold molar excess of unlabeled CREB binding oligonucleotides before radiolabeled oligonucleotides were added. For supershift analysis, anti-His antibodies were added to a reaction mixture on ice for additional 30 minutes. Probes used for EMSA are listed in Table 4.










EMSA probe sequences

















#1 C/EBPβ
GAATTCCAGCAGGAATCA
CCTGATTCCTGCTGGAA



GG (SEQ ID NO: 49)
TTC(SEQ ID NO: 50)


 


#2 CREB/
GAAGGGATTGATGTCAGC
TCCTGCTGACATCAATC


AP-1
AGGA 
CCTTC 



(SEQ ID NO: 51)
(SEQ ID NO: 52)


 


#3 AP-1
GCAGGAGTCACCACCCAG
CTCTGGGTGGTGACTCC



AG (SEQ ID NO: 53)
TGC (SEQ ID NO: 54)


 


#4 C/EBPβ
ATGGAGTTCCCAGATGCC
CAGGCATCTGGGAACTC



TG (SEQ ID NO: 55)
CAT (SEQ ID NO: 56)


 


#5 AP-1
GTGGAAGTGTCACAGCCC
TTGGGCTGTGACACTTC



AA (SEQ ID NO: 57)
CAC (SEQ ID NO: 58)


 


#6 C/EBPβ
TCTCTCTCTTGCTAAACCT
GGAGGTTTAGCAAGAGA



CC (SEQ ID NO: 59)
GAGA (SEQ ID NO: 60)


 


#7 C/EBPβ
GGTTTCATTTCTGAATCAT
AGTAGATGATTCAGAAA



CTACT 
TGAAAC 



(SEQ ID NO: 61)
(SEQ ID NO: 62)


 


CREB
GAAGGGATTGGGGTCAGC
TCCTGCTGACCCCAATC


Mutant
AGG A 
CCTTC 



(SEQ ID NO: 63)
(SEQ ID NO: 64)









(Chromatin Immunoprecipitation Assay)

C3H10T1/2 cells grown in a 100-mm culture dish were infected with adenovirus HA-sLZIP. After the infection, the cells remained in a growth medium until the cells reaches 100% confluence, and the medium was exchanged with a differentiation medium. The cells were washed with 1×PBS, the medium was treated with 1% formaldehyde for 10 minutes at room temperature, and then glycine was added for 5 minutes at a final concentration of 0.125 M. The cells were strapped with PBS, and centrifuged at 1000×g for 5 minutes at 4° C. ChIP analysis was performed such that anti-HA antibodies and anti-rabbit IgG serving as an internal control group were used and precipitated together with a DNA-protein complex. An enhancer region of FABP4 including a functional CREs was amplified from the prepared DNA.
(Co-Immunoprecipitation Analysis)
293T cells were obtained and washed with cold PBS. The cells were re-suspended in an IP lysis buffer [25 mM of Tris-HCl (pH 7.4), 150 mM of NaCl, 1 mM of EDTA, 1% NP-40 and 5% glycerol, and a protease inhibitor]. A suspension was centrifuged at 16,000×g for 20 minutes at 4° C. Supernatants were collected and incubated with 0.5 μg of appropriate antibodies and 25 μl of protein A/G-agarose or a GST Sepharose 4B bead for 24 hours at 4° C. A protein complex was centrifuged at 1,000×g with a cold IP lysis buffer for 1 minute and washed five times. The final pellet was re-suspended in 50 μl of an SDS-sample buffer containing 5% β-mercaptoethanol and heated for 10 minutes at 100° C. A protein sample was isolated in SDS-PAGE (8 to 10%) and transferred to nitrocellulose membranes. Co-precipitated proteins were detected according to western blotting using specific antibodies.
(Fluorescence Microscopic Analysis)
C3H10T1/2 cells were transiently ci-transfected with Flag-sLZIP, GFP-PPARγ2 or GFP-sLZIP, and HDAC3 was grown on cover slip. After 24 hours, the cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized 0.2% Triton-X 100 for 5 minutes. The cells were incubated with 1% BSA for one hour, and then incubated with anti-Flag and anti-HDAC3 antibodies overnight at 4° C. The cells were washed with PBS, and then were incubated with Texas Red-labeled antibodies for 2 hours. A cover slide was washed with PBS, and mounted on and examined using a LSM 510 META confocal microscopy (Carl Zeiss, Jena, Germany).
(Purification of his-sLZIP Protein)
sLZIP was cloned into a pET-28a(+) vector. His-sLZIP proteins were expressed in Escherichia coli (E. coli) BL21 cells using a T7 isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible system. IPTG-induced cells were disrupted by sonification and cell lysates were clarified by centrifugation. His-sLZIP proteins were applied onto a nickel-nitrilotriacetic acid bead column (Bio-Rad, Richmond, Calif.). The column was washed with a great volume of a lysis buffer and 10 mM of imidazole, and eluted in a Ni-NTA elution buffer (Bio-Rad Laboratories, Hercules, Calif.). A fraction containing His-sLZIP proteins was dialyzed against 10% glycerol, and stored at −80° C. Protein purification was evaluated according to 10% SDS-PAGE/Coomassie blue staining and a purity of >98% in general was shown.
(sLZIP Transgenic Mouse Generation)
In order to generate human sLZIP transgenic (TG) mice, sLZIP genes were cloned into a pCMV-flag expression vector. The sLZIP TG mice were generated by Macrogen, Inc (Seoul, Korea). Transgenic founders were mated with wild type C57BL/6 mice to produce F1 heterozygotes. F1-F4 generations were genetically screened for transgenes at an age of 2 weeks old. The following two primers were used to amplify genomic DNA in order to identify wild type and TG mice: 5′-GGA CGA TGA TGA CAA GGA CT-3′ and 5′-GTC AGA GGA GTA CAT GCT GCT-3.′
(Statistical Analysis)
Data were represented as mean±standard deviation. Statistical evaluation was performed using one-way ANOVA. Data was considered to be statistically significant when p<0.05 is satisfied. All statistical analyses were performed using a computer program Prism (GraphPad Software, La Jolla, Calif.).
Example 1
Effect of sLZIP on Transcriptional Activity of PPARγ
A nuclear receptor PPARγ is an important positive regulator of adipocyte differentiation in MSCs, and serves as a negative regulator in osteoblast development. In previous studies, sLZIP was known to be related to many types of nuclear receptor transcriptional activities, for example, a GR, an estrogen receptor (ER) and an androgen receptor (AR). Also, human sLZIP includes two LxxLL motifs that are necessary and sufficient for interaction with nuclear receptors. Therefore, the inventors focused on a relation between sLZIP and PPARγ2.
In order to examine an effect of sLZIP on the transcriptional activity of PPARγ2, 293T cells were temporarily transfected with PPARγ2 (0.5 μg), β-galactosidase (0.1 μg), FABP4-Luc reporter genes (0.2 μg) and sLZIP at different contents (0.1 to 0.5 μg). After the transfection, the cells were stimulated with or without 10 nM of rosiglitazone (RZD) (FIG. 1A), pioglitazone (PZD) (FIG. 1B), and troglitazone (TZD) (FIG. 1C). A relative luciferase activity was analyzed with or without cell ligand treatment 24 hours after transfection. Also, the 293T cells were transiently transfected with si-sLZIP at different contents (50 and 100 nM), and the cells were treated or not treated with RZD (FIG. 1D). FABP4-Luc stable C3H10T1/2 cells were infected with a sLZIP-expressing adenovirus, and then differentiated into adipocytes for 4 days (FIG. 1E). A luciferase activity was normalized to a β-galactosidase activity and represented as a relative activity (fold change). All experiments were repeated three times, and bars in the graph represent mean±standard deviation. *, P<0.05; **, P<0.01
As shown in FIGS. 1A to 1C, sLZIP inhibits the transcriptional activity of PPARγ2 in a concentration-dependent manner. The transcriptional activity of PPARγ2 was down-regulated due to sLZIP according to the presence of various PPARγ2 ligands compared to the control group.
In order to determine the effect of sLZIP on the transcriptional activity of PPARγ2, sLZIP expression was knocked-down using siRNA (si-sLZIP) for sLZIP. As a result, sLZIP expression inhibition due to si-sLZIP increased the transcriptional activity of PPARγ2 (FIG. 1D).
Also, in order to examine whether the transcriptional activity of PPARγ2 in the cell is regulated by sLZIP, C3H10T1/2 cells stably expressing FABP4 reporter genes were infected with a sLZIP-expressing adenovirus and then differentiated into adipocytes. As a result, as shown in FIG. 1E, the transcriptional activity of PPARγ2 decreased in adipocytes due to sLZIP.
Example 2
Examination of Interaction of sLZIP and PPARγ2
In order to examine whether the transcriptional activity of PPARγ2 is regulated by interaction with sLZIP, interaction between sLZIP and PPARγ2 was examined. For this purpose, 293T cells were transfected with GST-sLZIP and Myc-PPARγ2. Cell lysates were obtained and GST pull-down analysis was performed using a glutathione 4B bead. A protein complex was analyzed in SDS-PAGE and then immunoblotted with specific antibodies (FIG. 2A). The 293T cells were transfected with GST-PPARγ2 and Flag-sLZIP, and identified according to GST-pull-down analysis (FIG. 2B). PPARγ2 was translated in vitro by a TNT translation system (Promega). His-sLZIP proteins were expressed in BL21 cells using an IPTG-inducement system. Purified His-sLZIP proteins were subjected to His pull-down analysis. Proteins bound to the bead were analyzed in SDS-PAGE, and then immunoblotted with anti-PPARγ2 and anti-His antibodies (FIG. 2C). GFP-PPARγ2 and Flag-sLZIP constructs were expressed in C3H10T1/2 cells, and analyzed using mouse anti-Flag and Texas Red-labeled anti-mouse antibodies under a fluorescence microscope. The nucleus was stained with DAPI (FIG. 2D).
As shown in FIG. 2A, sLZIP interacted with PPARγ2.
In order to verify the interaction, 293T cells were transfected with GST-PPARγ2 and Flag-sLZIP, and GST pull-down analysis was performed. As a result, PPARγ2 interacted with sLZIP (FIG. 2B).
It was examined whether sLZIP directly interacts with PPARγ2. As a result, as shown in FIG. 2C, PPARγ2 was directly bound to sLZIP.
Also, in order to characterize interaction between sLZIP and PPARγ2, subcellular localization of sLZIP and PPARγ2 were examined. sLZIP was localized in the nucleus together with PPARγ2 (FIG. 2D).
Based on the result, it can be seen that sLZIP interacted directly with PPARγ2 to negatively regulate the transcriptional activity of PPARγ2.
Activation of many nuclear receptors depends on a supplement of coactivators. Therefore, a domain of sLZIP necessary for binding with PPARγ2 was examined. For this purpose, 293T cells were transfected with GST-full length sLZIP (1-354), N-terminal deletion mutant sLZIP (1-228), C-terminal deletion mutant sLZIP (229-354), CC-terminal deletion mutant sLZIP (297-354) and PPARγ2. Cell lysates were obtained and GST pull-down analysis was performed using a glutathione 4B bead. A protein complex was analyzed in SDS-PAGE, and then immunoblotted with specific antibodies (FIG. 3A). 293T cells were transfected with Flag-full length PPARγ2, full length PPARγ2, N-terminal deletion mutant PPARγ2 (1-310), C-terminal deletion mutants PPARγ2 (139-505) and GST-sLZIP. Cell lysates were obtained and GST pull-down analysis was performed. A protein complex was analyzed in SDS-PAGE, and then immunoblotted with anti-GST and anti-Flag antibodies (FIG. 3B).
As shown in FIG. 3A, a wild type sLZIP and C and CC domains of sLZIP were interacted with PPARγ2, but N domains of sLZIP were not interacted with PPARγ2. It can be seen that a CC-terminal domain containing a proline-rich region of sLZIP is important for binding with PPARγ2.
Also, domains of PPARγ2 necessary for binding with sLZIP were examined. As a result, as shown in FIG. 3B, a ligand binding domain (AF-2) of PPARγ2 was necessary for binding with sLZIP.
That is, it is proved that LxxLL motifs of sLZIP are not necessary for a binding ability while PPARγ2 is bound to sLZIP.
Example 3
Identification of Roles of HDAC3 in Inhibition of PPARγ2 Transcriptional Activity by sLZIP
The transcriptional activity of PPARγ is regulated by a coactivator and a corepressor. In general, in a resting state, PPARγ2 interacts with a corepressor complex, for example, HDAC3, SMRT and NCoR. However, when activation due to a ligand is performed, the corepressor complex is replaced with a coactivator, for example, p300/CBP, p160, and PGC-1, and leads transcription initiation of a target gene. In previous studies, it has been reported that sLZIP collects and activates HDACs, and thus decreases a GR transcriptional activity. HDAC3 was also specifically bound to LZIP among all class 1 HDACs. Therefore, in order to understand an effect of sLZIP on the transcriptional activity of PPARγ2 due to HDACs, trichostain A (TSA), which is an HDAC inhibitor, was used to examine whether HDACs are involved in the transcriptional activity inhibition of PPARγ2 due to sLZIP.
For this purpose, 293T cells were transiently transfected with PPARγ2 (0.5 μg), β-galactosidase (0.1 μg), FABP4-Luc reporter genes (0.2 μg) and sLZIP (0.1 μg). After the transfection, the cells were stimulated with or without 10 nM of RZD and 10 nM of TSA (FIG. 4A). A promoter activity was measured according to luciferase analysis. In the 293T cells, FABP4-Luc reporter genes, sLZIP, PPAR, β-galactosidase, and 100 nM of si-RNAs and HDAC1, 2, 3 and 8 for a control group were expressed. After the transfection, the cells were stimulated with or without 10 nM of RZD, and a promoter activity was measured (FIG. 4B). 293T cells were temporarily transfected with FABP4-Luc reporter genes, PPARγ2, β-galactosidase, HDAC3 (0.5 μg) and sLZIP (0.1 μg). After the transfection, the cells were stimulated with or without 10 nM of RZD, and luciferase analysis was performed (FIG. 4C). A luciferase activity was normalized to a β-galactosidase activity and represented as a relative intensity (fold change). All experiments were repeated three times, and bars in the graph represent mean±standard deviation. GFP-sLZIP expression constructs were expressed in C3H10T1/2 cells, rabbit anti-HDAC3 antibodies and Texas Red-labeled anti-rabbit antibodies were used and analysis was performed under a fluorescence microscope. The nucleus was stained with DAPI (FIG. 4D). 293T cells were transfected together with GST-HDAC3 and Flag-sLZIP. Cell lysates were pulled-down using a glutathione 4B bead. A protein complex was analyzed in SDS-PAGE, and anti-GST and anti-Flag antibodies were used for immunoblotting (FIG. 4E). *, P<0.05; **, P<0.01
As shown in FIG. 4A, while there is no TSA, sLZIP inhibits the transcriptional activity of PPARγ2, but sLZIP did not decrease the transcriptional activity of PPARγ2 in the cells treated with TSA.
Next, it was examined whether sLZIP-mediated transcriptional activity inhibition of PPARγ2 was limited to only HDAC3 among class 1 HDACs. As a result, as shown in FIG. 4B, siRNA for HDAC1, 2 and 8 was not involved in sLZIP-mediated PPARγ2 regulation. However, when HDAC3 was knocked-down, the sLZIP-mediated PPARγ2 transcriptional activity was significantly increased.
In order to verify the result, cells were transfected with sLZIP and HDAC3 expression constructs. As a result, inhibition of sLZIP-mediated PPARγ2 transcriptional activity was significantly down-regulated according to transfection of both sLZIP and HDAC3 (FIG. 4C).
Next, subcellular localization of sLZIP and HDAC3 was examined. As a result, sLZIP was localized in the nucleus together with HDAC3 (FIG. 4D).
Since it has been reported that HDAC3 specifically interacts with LZIP, interaction between sLZIP and HDAC3 was examined. The result showed that sLZIP interacted with HDAC3 (FIG. 4E).
That is, it can be seen that sLZIP was bound to HDAC3 to negatively regulate the PPARγ2 transcriptional activity.
Example 4
Effect of sLZIP on Corepressor Complex Formation of PPARγ2
The corepressor complex is replaced by a coactivator on ligand binding to a nuclear receptor. Therefore, interaction between sLZIP and PPARγ2 by ligand treatment was examined.
For this purpose, 293T cells were transfected with GST-PPARγ2 and Flag-sLZIP and were treated or not treated with 10 nM of RZD for 24 hours. Cell lysates were obtained and GST pull-down analysis (FIG. 5A) and immunoprecipitation (FIG. 5B) were performed using a glutathione 4B bead. A protein complex was analyzed in SDS-PAGE, and immunoblotted with anti-GST and anti-Flag antibodies. GST-PARγ2 and Flag-sLZIP were expressed in the 293T cells, and the cells were treated with 10 nM of RZD according to time of day. The cell lysates were GST pull-down analyzed (FIG. 5C). 293T cells were transfected with GST-HDAC3 and Flag-sLZIP, and treated or not treated with 10 nM of RZD for 24 hours. The cell lysates were GST pull-down analyzed (FIG. 5D). Flag-sLZIP was expressed in the 293T cells, and the cells were treated with 10 nM of RZD for 24 hours. Immunoprecipitation analysis of the cell lysates was performed using anti-NCoR1 antibodies. A protein complex was analyzed in SDS-PAGE, and then immunoblotted with anti-NCoR1 and anti-Flag antibodies (FIG. 5E). 293T cells were transiently transfected with PPARγ2 (0.5 μg), β-galactosidase (0.1 μg), FABP4-Luc reporter genes (0.2 μg) and various deletion mutants (0.1 μg) of sLZIP. After the transfection, the cells were stimulated with or without 10 nM of RZD (FIG. 5F). A luciferase activity was normalized to a β-galactosidase activity and represented as a relative activity (fold change). All experiments were repeated three times and data was represented as mean±standard deviation. *, P<0.05; **, P<0.01.
As a GST pull-down experiment result, when there was no stimulation with a ligand, sLZIP interacted with PPARγ2 (FIG. 5A). However, when the ligand was treated, sLZIP was dissociated from PPARγ2.
Immunoprecipitation analysis was used to examine interaction between sLZIP and PPARγ2 in cells expressing Flag-sLZIP and GST-PPARγ2. As a result, as shown in FIG. 5B, when the ligand was treated, PPARγ2 was dissociated from sLZIP.
In order to determine ligand-dependent dissociation, time dependence of interaction between sLZIP and PPARγ2 in a reaction with RZD was examined. As a result, sLZIP was dissociated from PPARγ2 in a time-dependent manner (FIG. 5C).
Next, an effect of sLZIP on interaction with the corepressor complex of PPARγ2 corepressor was examined. As a result, when no RZD was treated, sLZIP interacted with HDAC3 in a PPARγ2 corepressor complex (FIG. 5D). While PPARγ2 was dissociated from sLZIP due to RZD treatment, HDAC3 was remained as being interacted with sLZIP (FIG. 5D). However, when RZD was treated, NCoR1 was slightly dissociated from sLZIP (FIG. 5E).
Also, a regulation mechanism of sLZIP in ligand-dependent interaction with PPARγ2 and a relation thereof in regulation of the PPARγ2 transcriptional activity were examined. As a result, when RZD was treated, sLZIP was dissociated from PPARγ2, and still inhibited the PPARγ2 transcriptional activity (FIG. 5F). The transcriptional activity of PPARγ2 was inhibited by sLZIP deletion mutants (1-220) that were not interacted with PPARγ2 (FIG. 5F).
It has been reported that LZIP had an N-terminal activity domain (1-220) and was involved in a transcriptional activity of cAMP-response elements (CREs)-containing reporter genes. That is, it is considered that sLZIP interacts with PPARγ2, and probably bound to an FABP4 promoter region and regulates a transcriptional activity thereof.
Therefore, in order to understand a regulation mechanism of sLZIP in the PPARγ2 transcriptional activity by binding to the FABP4 promoter region, an effect of sLZIP on the interaction between PPARγ2 and HDAC3 was examined. For this purpose, 293T cells were transfected with GST-HDAC3, Myc-PPARγ2 and Flag-sLZIP, and were treated or not treated with 10 nM of RZD for 24 hours. Cell lysates were subjected to GST pull-down analysis using a glutathione 4B bead, and a protein complex was analyzed in SDS-PAGE, and then immunoblotted with anti-GST, anti-Myc and anti-Flag antibodies (FIG. 6A). GST-HDAC3, GST-PPARγ2, Myc-PPARγ2, Flag-sLZIP and Ha-UB were expressed in the 293T cells, and the cells were treated with 10 nM of RZD for 24 hours. Cell lysates were subjected to GST pull-down analysis (FIG. 6B). GST-PPARγ2 and Flag-sLZIP were expressed in the 293T cells and treated with 10 nM of RZD for 24 hours. Cell lysates were subjected to GST pull-down analysis, and a protein complex was analyzed in SDS-PAGE, and then immunoblotted with anti-PGC1α, anti-GST and anti-Flag antibodies (FIG. 6C).
As shown in FIG. 6A, as a GST pull-down analysis result, sLZIP enhanced interaction between PPARγ2 and HDAC3 in the presence of ligand.
When no ligand was treated, PPARγ2 interacted with a corepressor such as HDAC3, and when a PPARγ2 ligand, rosiglitazone, was added, dissociation of a PPARγ-corepressor complex occurred, and degradation by a ubiquitin and proteasome pathway was induced. It was measured whether sLZIP is involved in HDAC3 ubiquitination. As a result, sLZIP inhibited HDAC3 ubiquitination when the ligand was treated (FIG. 6B).
Also, sLZIP inhibited the complementing of a coactivator PGC-1α for PPARγ2 (FIG. 6C).
Based on the result, it can be seen that sLZIP interacts with PPARγ2 to regulate the PPARγ2 transcriptional activity, and enhances the formation of the corepressor complex.
Example 5
Analysis of Binding Region of sLZIP and FABP4 Enhancer Region
In order to understand a regulatory mechanism of a DNA-binding ability of sLZIP in an FABP4 promoter, a Transcription Element Search System (TESS) and AliBaba2, which is a program for predicting a binding region of transcription factors, were used to analyze an FABP4 promoter region. It has been reported that PPARγ2 binds with 518-bp enhancer in adipocyte P2(FABP4) genes including two PPARγ2 binding elements, ARE6 and ARE7. Therefore, it was tried to find an expected sLZIP binding region in an FABP4 enhancer region. FIG. 7A shows a potential sLZIP binding region in the FABP4 enhancer region.
A DNA binding ability of sLZIP with various elements in the FABP4 enhancer region was examined. For this purpose, recombinant His-sLZIP fusion proteins (3 μg) were incubated with γ-32P-labeled oligonucleotide probes containing expected CREB binding elements (FIG. 7B). The γ-32P-labeled oligonucleotide probes were incubated while an amount of purified His-sLZIPs was increased. For competition analysis, unlabeled CREB oligonucleotide probes of 100-fold excess were used and His antibodies were added to a reaction mixture on ice for additional 40 minutes (FIG. 7C). His-sLZIP proteins were subjected to EMSA using wild type and CREB mutant (TGATGTCA→TGGGGTCA) probes (FIG. 7D). 293T cells were temporarily transfected with PPARγ2 (0.5 μg), β-galactosidase (0.1 μg), FABP4 enhancer-luciferase reporter genes (0.2 μg) and sLZIP mutants at different contents (0.1 to 0.5 μg). After the transfection, the cells were stimulated with 10 nM of RZD (FIG. 7F). A luciferase activity was normalized to a β-galactosidase activity and represented as a relative activity (fold change). All experiments were repeated three times, and bars in the graph represent mean±standard deviation. ChIP analysis was performed using HA-sLZIP-specific antibodies and then PCR was performed. Input chromatins or immunoprecipitated chromatins were subjected to PCR and used as a control group for variables.
As shown in FIG. 7B, sLZIP was directly bound to a CREB region.
In order to determine a specific DNA binding ability of sLZIP with a CREB element in the FABP4 enhancer region, supershift analysis using unlabeled CREB probes of 100-fold excess and cold competition analysis using His-antibodies were performed. As a result, a DNA binding ability of sLZIP increased in a concentration-dependent manner (FIG. 7C). However, sLZIP was not bound to the CREB element when there were competitors. sLZIP was supershifted when His antibodies were treated (FIG. 7C). sLZIP was not bound to CREB mutant probes (TGATGTCA→TGGGGTCA) (FIG. 7D).
In order to confirm the EMSA result, FABP4 enhancer-luciferase constructs were used to examine a transcriptional activity of PPARγ2. As a result, as shown in FIG. 7E, sLZIP decreased the transcriptional activity of PPARγ2 in a concentration-dependent manner. When ChIP was performed, sLZIPs were gathered in the CREB element in the FABP4 enhancer when RZD was treated or not (FIG. 7F).
In summary, sLZIP is directly bound to the CREB element in the FABP4 enhancer, and induces an increase of the corepressor complex of PPARγ2.
Example 6
Effect of sLZIP in Adipogenesis
Adipocyte differentiation is a well-regulated procedure. PPARγ2 is a major regulator of adipogenesis, is highly expressed during adipocyte differentiation, and regulates expression of genes involved in adipogenesis. An effect of sLZIP on adipogenesis in vivo was examined. For this purpose, in order to produce human sLZIP transgenic mice, sLZIP genes were cloned in a pCMV-flag expression vector. The sLZIP TG mice were produced in Macrogen, Inc (Seoul, Korea) (FIG. 8A). Preadipocytes were isolated from epididymal adipose tissues of sLZIP TG mice. Preadipocytes were transfected with FABP4 promoter reporter genes (0.2 μg) and PPARγ2 (0.5 μg) containing PPAR binding elements and treated with or without 10 nM of RZD. A promoter activity was measured according to luciferase analysis (FIG. 8B). 293T cells were transiently transfected with PPARγ2 (0.5 μg), β-galactosidase (0.1 μg), FABP4 enhancer-luciferase reporter genes (0.2 μg) and mouse LZIP (0.5 μg). After the transfection, the cells were stimulated with 10 nM of RZD (FIG. 8C). A luciferase activity was normalized to a β-galactosidase activity and represented as a relative activity (fold change). All experiments were repeated three times, and bars in the graph represent mean±standard deviation. The medium of C3H10T1/2 and 3T3-L1 cells was exchanged with a differentiation medium in which 10% FBS, 5 μg/mL of insulin, 1 μM of dexamethasone, and 0.5 mM of IBMX were contained. Total RNA was extracted from the cells, and an mRNA expression level of mouse LZIPs was measured using RT-PCR analysis. GAPDH was used as an internal control group. An experiment was repeated three times (FIG. 8D). C3H10T1/2 and 3T3-L1 were stained with Oil Red O (FIG. 9A) and preadipocytes were isolated from epididymal adipose tissues of sLZIP TG mice (FIG. 9B). Preadipocytes isolated from epididymal adipose tissues of sLZIP TG mice were differentiated into adipocytes. Total RNA was isolated from the cells, and an mRNA expression level of mouse LZIPs was measured using RT-PCR (FIG. 9C) and real-time PCR (FIG. 9D).
The transcriptional activity of PPARγ2 was examined in adipocytes of sLZIP TG mice. As a result, the transcriptional activity of PPARγ2 was decreased in the adipocytes of the sLZIP TG mice more than in wild type mice (FIG. 8B).
It was examined whether homologous mouse LZIPs influence a transcriptional activity and expression of PPARγ2 during adipogenesis. As a result, mouse LZIPs also decreased the transcriptional activity of PPARγ2 compared to the control group (FIG. 8C). Interestingly, mouse LZIP expression decreased during adipocyte differentiation (FIG. 8D).
In order to confirm the luciferase analysis result, an effect of sLZIP on differentiation of multipotential mesenchymal progenitor cells into adipocytes was examined. As a result, in oil red O staining, sLZIP inhibited adipocyte differentiation (FIG. 8E).
In order to confirm in vitro results, an effect of sLZIP when adipocytes are differentiated in vivo was examined. As a result, as shown in FIGS. 9A and 9B, differentiation of primary preadipocytes and mouse embryonic fibroblasts (MEFs) isolated from sLZIP TG mice was inhibited more than in wild type mice. Similarly to the result, expression of FABP4 and other PPARγ2 target genes, for example, C/EBPα and LPL, was also down-regulated (FIGS. 9C and 9D).
In conclusion, as shown in FIG. 10, sLZIP inhibits the transcriptional activity of PPARγ2, and thereby serves as a negative regulator on adipocytes differentiation in vitro and in vivo.
The present invention can be used as a therapeutic agent for diabetes or obesity.

## Claims

1. A screening method of a medicine for preventing or treating diabetes, comprising:
contacting a cell transformed with human small leucine-zipper proteins (sLZIP) and Peroxisome proliferator-activated receptor γ2 (PPARγ2) with a candidate material, wherein either the human sLZIP or PPARγ2 is coupled to a detectable component; and
detecting an increase in formation of the complex of the human sLZIP and PPARγ2 in the cell, whereby said candidate material is determined to be a material capable of preventing or treating diabetes.

2. The screening method of claim 1, wherein the cell is an animal cell.

3. The screening method of claim 2, wherein the detectable component is selected from the group consisting of a fluorescent protein, Glutathione S transferase (GST), Flag-tag, Myc-tag and HA-tag.

4. The screening method of claim 1 or 2, wherein detecting is carried out by a reaction using the detectable component, or the human sLZIP or PPARγ2-specific antibody.