JP2003014738A - Fat cell differentiation control and/or screening method of anti-inflammatory substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the screening method of a fat cell differentiation inhibition substance that can inhibit differentiation to the fat cell of a non-differentiated cell safely and effectively as compared with growth factors, cytokine, or the like, and to provide the screening method of an anti-inflammatory substance with less side effect. SOLUTION: NFκB and PPARγ are brought into contact under the present or non-present of a substance to be tested, and the combination between both of them is compared under both conditions in the fat cell differentiation control and/or the screening method of the anti-inflammatory substance.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for screening adipocyte differentiation control and / or anti-inflammatory substances. More specifically, the present invention uses the degree of binding between NFκB and PPARγ as an index to control adipocyte differentiation and / or
Or, it relates to a screening method for an anti-inflammatory substance.

[0002]

BACKGROUND ART Pluripotent mesenchymal cells in the bone marrow are
Differentiate into cells such as adipocytes, osteoblasts, chondrocytes and fibroblasts [J. Cell. Biochem., 58, 393-402 (1995);
Science, 289, 1501-1504 (2000)]. Balanced differentiation of stem cells is essential for maintaining the proper state of the bone marrow environment. However, this balance adjustment mechanism is hardly known to date.

On the other hand, it is known that bone marrow increases in fat content as it ages. This is a phenomenon called "lipid myelination", and it has been reported that the number of osteoblast lineages decreases with this lipid myelination, and bone formation in old age compared to young age. Is thought to be one of the causes of decreased bone mass and decreased bone mass [BIO Clinica 16
(3), 227-32 (2001)]. That is, it means that adipocyte differentiation in bone marrow is greatly involved in the pathological conditions of aging-related bone / cartilage diseases such as osteoporosis, osteoarthritis, and delayed healing of fractures.

Peroxisome proliferator-activated receptor γ (PPARγ) is considered to play an important role in promoting the differentiation of adipocytes [Genes Dev. 14, 1293-.
307 (2000), Mol. Cell 4, 597-609 (1999), Mol. Cell
4, 611-7 (1999)]. Regarding the adipocyte differentiation promoting action of PPARγ, there is a cis element (PPRE) capable of being recognized and bound by PPARγ in the expression regulatory region of a marker gene in adipocyte differentiation, and PPARγ is a retinoid X receptor α (RXRα). Directly promoting the expression of the marker gene by forming a heterodimer with and binding to this sequence [Cell Biochem. Biophys. 32 Spring,
187-204 (2000)], overexpressing PPARγ in a fibroblast cell line, and adding a PPARγ agonist to differentiate into adipocytes [Cell 79 (7), 1147-56 (1994)] It has been reported that a differentiated cell line differentiates into adipocytes [J. Biol. Chem. 270 (22), 12953-6 (1995)] and the like, and PPARγ is a transcription factor that directly controls adipocyte differentiation. Is shown.

On the other hand, growth factors such as IGF, EGF and TGFβ and IL-
1, TNFα and other cytokines were known to inhibit adipocyte differentiation [J. Clin. Invest., 89, 223-3
3 (1992); J. Cell. Biol., 108, 1105-13 (1989).
etc]. Among these, IGF and EGF phosphorylate the A / B region existing at the N-terminal of PPARγ by MAP kinase (MAPK) activated by their actions, thereby causing ligand-dependent transcriptional activation of PPARγ. Indirect suppression has been reported [Science 274, 2100-3 (1996)]. However,
MAPK is not activated by IL-1, TNFα, TGFβ, etc., suggesting the existence of a differentiation inhibitory mechanism different from IGF and EGF.

[0006] Therefore, the present inventors attempted to identify a signaling factor located downstream of the negative regulatory mechanism of IL-1, TNFα, TGFβ and the like for the adipocyte differentiation promoting action of PPARγ. As a result, IL-1 and TNFα activate NFκB by activating TAK1 / TAB1, NFκB-inducible kinase (NIK), and IκB kinase (IKK), and subsequent kinase cascades, and PPARγ-induced adipocyte-specific It was revealed that it suppresses the ligand-dependent transcriptional activation of the gene and thereby suppresses the differentiation of adipocytes.

[0007]

[Problems to be Solved by the Invention] However, NFκB
It remains unclear as to what mechanism the activation of the protein suppresses transcriptional activation by PPARγ, and whether there is a signaling factor located further downstream of NFκB.

[0008] A substance that suppresses the differentiation of undifferentiated cells into adipocytes by negatively regulating the differentiation promoting action of PPARγ is used for the prevention of bone diseases such as osteoporosis and osteoarthritis, which are caused by fat myelination. Expected to be effective in treatment. In addition, fat accumulates systemically with aging, and it is expected that it can be prevented or treated. However, growth factors such as IGF and EGF and factors that activate them are not suitable for long-term use because they have a risk of inducing carcinogenesis. In addition, IGF and EGF suppress the transcriptional activation of PPARγ through phosphorylation of PPARγ by MAPK, but since this suppression mechanism does not suppress the binding of PPARγ to target DNA, other stimuli coexist. In that case, transcriptional activation may occur, and it may not be possible to expect a regulatory action in the strict sense.

On the other hand, IL-1 and TNFα are known to have receptors on the cell membrane and regulate various intracellular signal cascades. Like cell growth factors, it has few drawbacks of increasing the risk of carcinogenesis, but there is concern about its influence on other factors such as inflammation.

As described above, when a factor located upstream of the signal cascade is used, there arise problems such as side effects due to activation of factors other than the target and diminished effects due to dilution of the action on the target factor. Therefore, identifying a factor that directly suppresses the binding of PPARγ to a target gene can provide a means for searching a safer adipocyte differentiation regulator that has few side effects and can exert efficacy at a low dose. In that respect, it is extremely important.

Therefore, an object of the present invention is to target D of PPARγ.
A substance that directly controls binding to NA (that is, PPRE) was identified, and a substance that regulates the transcriptional activation action of PPARγ by utilizing the interaction between the substance and PPARγ, and thus the fat of undifferentiated cells It is intended to provide a system for screening a substance that controls differentiation into cells and dedifferentiation from adipocytes.

[0012]

[Means for Solving the Problems] As a result of intensive studies to achieve the above-mentioned object, the present inventors found that NFκB was found to be PPARγ.
It was found that the inhibition of adipocyte differentiation is suppressed by suppressing the binding of the protein to the target DNA, and that NFκB directly suppresses the DNA binding ability of PPARγ by directly binding to PPARγ. The present invention has been completed based on such knowledge.

That is, the present invention is characterized in that NFκB and PPARγ are brought into contact with each other in the presence and absence of a test substance, and the degree of binding between NFκB and PPARγ under both conditions is compared, which is characterized by adipocyte differentiation. A method for screening a regulatory substance is provided. The method can take various embodiments depending on the means for providing NFκB and PPARγ to the reaction system and the means for measuring the degree of binding between them.

That is, as means for providing NFκB and PPARγ to a reaction system, (i) a method of providing a cell extract from a host cell engineered to express them, (ii)
Isolate and purify them from host cells that have been engineered to express them (eg, isolate from host cells that have been engineered to express as fusion proteins with the appropriate tags, using a carrier that has an affinity for the tags). Purified), (iii) In vitro synthesis from an expression vector containing DNA encoding them using a cell-free translation system, (iv) Host cells engineered to express them And the like.

As means for measuring the degree of binding between NFκB and PPARγ, (i) a method of directly measuring the NFκB-PPARγ complex, (ii) a method of measuring the amount of PPARγ bound to PPRE as an index, ( iii) A method of measuring expression of a reporter gene under the control of a promoter region containing PPRE as an index, (iv) a method of measuring using a two-hybrid system, and the like. Specific aspects of each of these means are as follows.
This will be described in detail in the "Embodiment of the Invention" below.

The screening method for the adipocyte differentiation regulator of the present invention also has an aspect as a screening method for anti-inflammatory substances. PPARγ ligands such as thiazolidine derivatives and prostaglandin J ₂ suppress the activation of NFκB, resulting in cyclooxygenase-2 (CO2) induced by stimulation of inflammatory cytokines such as TNFα.
X-2) was known to exert an anti-inflammatory effect by inhibiting the expression [eg J. Biiol. Chem. 275
(36), 28173-9 (2000)], the mechanism of NFκB activation suppression remains unclear. According to the invention PPAR
Since it was revealed that γ and NFκB are directly bound, PPARγ ligand was shown to suppress the induction of NFκB expression of proinflammatory genes through the formation of NFκB-PPARγ complex. Therefore, the substance capable of enhancing the binding between NFκB and PPARγ selected by the screening method of the present invention may be a strong candidate compound for the anti-inflammatory substance.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention provides a method for screening adipocyte differentiation control and / or anti-inflammatory substances.
In the present invention, “adipocyte differentiation regulator” means PPARγ
It inhibits (or promotes) the binding of PPARγ to PPRE by enhancing (or inhibiting) the binding of NFκB to, and thereby inhibits (or enhances) the transcriptional activation of adipocyte-specific genes by PPARγ. It refers to a substance capable of suppressing (or promoting) the differentiation of undifferentiated cells into adipocytes or promoting (or suppressing) dedifferentiation from adipocytes. Further, in the present invention, the "anti-inflammatory substance" means
By enhancing the binding between NFκB and PPARγ, it means a substance that suppresses the induction of the expression of proinflammatory genes by NFκB and thus can exert an anti-inflammatory effect.

NFκB used in the present invention is PPAR.
The origin is not particularly limited as long as it can bind to γ and suppress the binding of PPARγ to PPRE, and may be NFκB derived from any mammal, or a natural or artificial mutant thereof. May be NFκB is known as a transcription factor that acts by forming homo- or heterodimers, and p100 and p105 function by being processed and p5.
0, p52 and C-Rel, V-Rel, p65, RelB, Dorsal, Dif, etc. are not processed. Except for RelB, it works by forming homo or heterodimer, but RelB is p50
Or it functions only by forming a heterodimer with p52. As shown in Examples below, since it is the LZ-RHD region of the p65 subunit that has the ability to bind to PPARγ, NFκB in the present invention may be the p65 subunit alone, or LZ-RHD. N-terminal and / or C as long as it has a region
It may be a fragment with the terminal deleted. Alternatively,
Factors with high homology with p65 (C-Rel, V-Rel, RelB, Dorsa
l, Dif, etc.), the factor of which the binding ability to PPARγ was confirmed, or a fragment thereof. Furthermore, the NF of the present invention
κB may be a fusion protein with a sequence such as His-tag or GST that enables rapid and simple separation and purification based on affinity. In the present specification, the term “NFκB (p65)” is abbreviated below, including p65 and a factor having high homology with p65 capable of binding to PPARγ and fragments thereof.

In the screening system of the present invention, NFκ
B is preferably provided from a cell containing a gene encoding NFκB, for example, as a cell extract. The gene encoding NFκB may be the NFκB gene that is endogenous to the cell, or may be the DNA that encodes NFκB that has been introduced into a predetermined host cell using a known method. In any case, it is preferable that the cells have an expression vector containing a DNA encoding PPARγ introduced therein.

NFκB may also be provided from an expression vector containing a DNA encoding NFκB by in vitro synthesis using a cell-free translation system derived from rabbit reticulocyte extract or wheat germ extract. . In such a case, labeled NFκB can be easily prepared using a labeled amino acid such as ³⁵ S-Met.

PPARγ used in the present invention is NFκ
As long as it can bind to B, its origin is not particularly limited, and may be PPARγ derived from any mammal,
Alternatively, it may be a natural or artificial mutant thereof. PPARγ is classified into the AF region structure in order from the N-terminal, but the region essential for binding to NFκB is the N-terminal A / B region or C region.
Rγ may be a fragment in which a part or all of the other region is deleted as long as it has any of the regions. When the screening system of the present invention uses the binding of PPARγ to PPRE as an index, PPARγ must necessarily include the C region that is a site that recognizes and binds PPRE. Further, when the screening system of the present invention uses the expression of a reporter gene under the control of a promoter region containing PPRE as an index, PPARγ is a C-terminal E / F region which is a region that promotes transcription in a ligand binding-dependent manner. Must be included. Furthermore, the PPARγ of the present invention may be a fusion protein with a sequence such as His-tag or GST that enables rapid and simple separation and purification based on affinity.

In the screening system of the present invention, PPAR
γ is preferably from a host cell into which an expression vector containing DNA encoding PPARγ is introduced by a known method,
For example, it is provided as a cell extract. The host cell is
It is preferably a mammalian cell that endogenously expresses NFκB, or a cell into which an expression vector containing a foreign NFκB-encoding DNA is further introduced.

PPARγ is also a DNA encoding PPARγ
May be provided by in vitro synthesis using a cell-free translation system derived from a rabbit reticulocyte extract or a wheat germ extract. In such a case, labeled PPAR should be labeled with a labeled amino acid such as ³⁵ S-Met.
γ can be easily prepared.

NFκB and PPARγ may also be provided as host cells engineered to express them,
In this case, the host cell itself constitutes the reaction system.

The method for screening an adipocyte differentiation control and / or anti-inflammatory substance of the present invention comprises contacting NFκB with PPARγ in the presence and absence of a test substance to determine the binding of NFκB and PPARγ under both conditions. Characterized by comparing the degree. If the binding of NFκB and PPARγ is significantly enhanced (or inhibited) in the presence of the test substance, the transcriptional activation of adipocyte-specific gene by PPARγ is significantly suppressed (or promoted). It becomes a strong candidate for a cell differentiation inhibitor (or promoter). If the binding of NFκB and PPARγ is significantly enhanced in the presence of the test substance, N
Since the transcriptional activation of proinflammatory genes by FκB is significantly suppressed, the substance is also a strong candidate for anti-inflammatory agent.

The method for screening an adipocyte differentiation regulator of the present invention takes various embodiments by changing the means for providing NFκB and PPARγ to the reaction system and the means for measuring the degree of binding between NFκB and PPARγ. be able to.

The first aspect of the screening method of the present invention is NFκB-PPARγ using an anti-NFκB antibody and / or an anti-PPARγ antibody or labeling NFκB or PPARγ itself.
The binding of NFκB and PPARγ is investigated by measuring the complex directly. In an assay system for B / F (solid-liquid) separation, when NFκB or PPARγ is provided as a fusion protein having a tag, a carrier having an affinity for the tag is used instead of the immobilized antibody. You can also Examples of the carrier include magnetic nickel beads in the case of a fusion protein with His-tag, and glutathione-immobilized resin in the case of a GST fusion protein. The use of such a tag is also useful for enabling rapid and simple separation when NFκB or PPARγ is provided as a cell extract as a cell extract as it is separated and purified, and then provided.

Specifically, the following method is exemplified. (1) NFκB and PPARγ in the presence and absence of the test substance
After contacting with and incubating for a certain time, NFκB-
PPARγ complex, free NFκB and free PPARγ are separated by electrophoresis, and labeled anti-NFκB (p65) antibody and labeled anti-PPARγ
An antibody is used to measure the band intensity corresponding to the NFκB-PPARγ complex. For example, an extract of mammalian cells into which an expression vector containing DNA encoding PPARγ has been introduced, and endogenous NFκB has been activated by stimulation with IL-1, TNFα, or exogenous NFκB (p65) is encoded. Non-denaturing gel electrophoresis of each reaction solution after incubation with or without addition of a test substance to the extract of host cells into which an expression vector containing DNA and an expression vector containing DNA encoding PPARγ were introduced Then, perform Western blotting with each antibody labeled with RI or non-RI (enzyme, fluorescence, etc.), and compare the intensities of the bands that cross-react with both antibodies. When NFκB and / or PPARγ is synthesized using a cell-free translation system as described above, if either NFκB or PPARγ is labeled with ³⁵ S-Met or the like, the labeled antibody serves as a probe. Instead of performing the Western blotting described above, autoradiography of the electrophoretic gel may be performed and the intensities of the bands on the higher molecular weight side may be compared.

(2) Alternatively, in the above (1), anti-NFκB (p65) antibody or anti-PPARγ antibody is added to the reaction solution for immunoprecipitation, and the precipitate is electrophoresed to give RI or non-RI ( (Enzyme, fluorescence, etc.) labeled anti-PPARγ antibody or anti-NFκ
Western blotting can also be performed using the B (p65) antibody to compare the intensities of the detected bands.
Immunoprecipitation can be preferably performed using an antibody immobilized on an insoluble carrier such as plastic beads or Sephadex. When NFκB and / or PPARγ is synthesized using a cell-free translation system as described above, NFκB or PPARγ
If one of PARγ is labeled with ³⁵ S-Met, etc., the antibody against the other will be added to the reaction solution for immunoprecipitation, and the precipitate will be electrophoresed for autoradiography of the electrophoretic gel. Then, the intensities of the detected bands may be compared.

(3) The measurement of the NFκB-PPARγ complex can also be carried out using a conventional immunoassay system. For example, anti-NFκB (p65) antibody or anti-PPARγ antibody is added to the reaction solution for immunoprecipitation, and the liquid phase is removed, followed by RI or non-RI
(Enzyme, fluorescence, etc.) labeled anti-PPARγ antibody or anti-NFκB (p
65) You may perform RIA, EIA, FIA, etc. using an antibody and compare the detected signal intensities. Further, when NFκB and / or PPARγ is synthesized using a cell-free translation system as described above, if either NFκB or PPARγ is labeled with ³⁵ S-Met or the like, an antibody against the other reacts. The NFκB-PPARγ complex can also be measured by performing RIA after removing the liquid phase by performing immunoprecipitation in addition to the liquid.

(4) Alternatively, if a homogeneous immunoassay that does not require B / F separation, such as surface plasmon resonance, fluorescence polarization immunoassay, fluorescence quenching immunoassay, and fluorescence energy transfer immunoassay, is used, it can be directly used in the reaction solution.
The NFκB-PPARγ complex can be measured.

The second aspect of the screening method of the present invention is that the degree of binding between NFκB and PPARγ is PPA that binds to PPRE.
The amount of Rγ is measured as an index. Binds to PPRE in the presence of the test substance as compared to the absence
If the amount of PPARγ decreases (or increases) significantly, NFκB
The binding of PPARγ with PPARγ is enhanced (or inhibited) by the substance
It has been done.

PPARγ forms a heterodimer with RXR to form P
Since it binds to PRE, in this embodiment, NFκB and PP
RXR must also coexist in the ARγ reaction mixture. Three subtypes of α, β and γ are known for RXR, and any of them may be used in the present invention. Also, PPAR
There is no particular limitation as long as it can form a heterodimer with γ and bind to PPRE, and any mutant other than naturally occurring one can be used. RXR can be preferably provided as a cell extract, for example, from a host cell into which an expression vector containing a DNA encoding RXR has been introduced. Preferably, the cells are simultaneously introduced with an expression vector containing DNA encoding PPARγ.

In the present invention, the term "PPRE" means a DNA sequence which is present in the promoter region of an adipocyte-specific gene (differentiation marker gene) in which PPARγ is transcriptionally activated and which is recognized and bound by PPARγ, or in the sequence, It means a DNA sequence in which one or several bases have been deleted, substituted or added, and which can recognize and bind to PPARγ. For example,
An example is PPRE derived from acyl CoA oxidase. The PPRE used in the present invention may have tandemly linked two or more unit sequences in order to enhance the affinity for PPARγ.

The amount of PPARγ bound to PPRE can be measured using a labeled anti-PPARγ antibody or by labeling PPARγ itself, as in the above-mentioned first embodiment. Specifically, it is prepared as described above.
PPRE immobilized on an insoluble carrier such as plastic beads or Sephadex in the reaction mixture of NFκB and PPARγ / RXR
After adding and incubating and B / F separating, PPARγ bound to the solid phase can be measured by an immunoassay using an anti-PPARγ antibody labeled with RI or non-RI (enzyme, fluorescence, etc.). Further, when PPARγ is synthesized using a cell-free translation system, if PPARγ is labeled with ³⁵ S-Met or the like, by measuring the RI labeling amount bound to the solid phase after B / F separation, , The amount of PPARγ bound to PPRE can be measured.

Alternatively, by using homogeneous immunoassays such as surface plasmon resonance, fluorescence polarization immunoassay, fluorescence quenching immunoassay, and fluorescence energy transfer immunoassay that do not require B / F separation, PPARγ bound to PPRE directly from the reaction solution can be measured. can do.

In a preferred embodiment, the amount of PPARγ binding to PPRE can be measured by gel shift assay. That is, NFκ prepared as described above
After adding RI or non-RI (enzyme, fluorescence, etc.) labeled PPRE to the reaction mixture of B and PPARγ / RXR and incubating, the reaction mixture is electrophoresed to separate PPRE-PPARγ / RXR complex and free PPRE. It can be investigated by separating and measuring the signal intensity of the band detected in the high molecular weight region.

The third aspect of the screening method of the present invention is to measure the degree of binding between NFκB and PPARγ using the expression of a reporter gene under the control of a promoter region containing PPRE as an index. If the expression of the reporter gene is significantly decreased (or increased) in the presence of the test substance as compared with the absence thereof, the transcriptional activation by the PPARγ / RXR heterodimer indicates that the test substance causes NFκB
Suppressed (or promoted) by enhancing the binding between PPARγ / RXR
Indicates that it will be done.

[0039] In this embodiment, a mammalian cell into which an expression vector containing a DNA encoding PPARγ, an expression vector containing a DNA encoding RXR and a reporter gene under the control of a promoter region containing PPRE is introduced, or And DNA encoding NFκB (p65)
A host cell into which an expression vector containing is introduced is cultured in the presence or absence of a test substance, the expression of the reporter gene in the resulting culture is measured, and the expression levels are compared. In addition, since the transcriptional activation of target genes by PPARγ is ligand-dependent, culture should be performed with thiazolidine derivatives such as troglitazone and PPARγ such as prostaglandin J _2.
It must be done in the presence of an agonist.

The host mammalian cells include human, monkey,
It is preferably a cell of mouse, rat, hamster or the like, or a cell of human origin. Specifically, COP, L, C127,
Mouse-derived cells such as Sp2 / 0, NS-1, NIH3T3, ST2, rat-derived cells, hamster-derived cells such as BHK and CHO, COS1, CO
Monkey-derived cells such as S3, COS7, CV1, Vero, and HeLa, 2
Examples include human-derived cells such as 93T.

Examples of the reporter gene include, but are not limited to, DNAs encoding luciferase, β-galactosidase, β-glucuronidase, chloramphenicol acetyltransferase, peroxidase and the like. The promoter is not particularly limited as long as it functions in mammalian cells and contains a PPRE sequence in its vicinity.

Gene transfer can be carried out by using a conventional liposome method or electroporation method.

The gene-introduced host cell is, for example, a minimum essential medium (MEM) containing about 5 to 20% fetal bovine serum (desirably, serum from which fat-soluble hormones have been removed from the serum by treatment with activated charcoal). Science, 122, 501 (1952)],
Dulbecco's Modified Minimum Essential Medium (DMEM) [Virology, 8, 3
96 (1959)], RPMI1640 medium [J. Am. Med. Assoc., 19
9, 519 (1967)], 199 medium [Proc. Soc. Exp. Biol. Me
d., 73, 1 (1950)] and the like.
The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to about 40 ° C. for about 3 to about 72 hours after the addition of the PPARγ agonist.

After the completion of the culture, the cells may be collected and disrupted in an appropriate lysis buffer, and the supernatant may be obtained by centrifugation to obtain a cell extract, which may be used to assay the expression of the reporter gene.

The fourth aspect of the screening method of the present invention is to determine the degree of binding between NFκB and PPARγ by the so-called two-hybri.
The d system is used to measure the expression of the reporter gene and the activation of the cytoplasmic signal transduction system as indicators.

In the present embodiment, expression containing one of the DNA encoding NFκB (p65) and the DNA encoding PPARγ so as to be expressed as a fusion protein with the DNA binding domain (BD) of a transcription factor such as GAL4. Vector and
An expression vector containing the other DNA so as to be expressed as a fusion protein with a transcriptional activation domain (AD) of a transcription factor such as VP16 is introduced into a host cell, and the resulting transformant is used in the presence of a test substance. After culturing in the absence, the expression level of the reporter gene in the resulting culture is measured and compared. The host cell carries a reporter gene containing a cis element (BD response element) capable of being recognized and bound by BD in the promoter region. Intracellularly expressed BD fusion protein binds to BD response element, but NFκ
If B and PPARγ interact, BD on the BD response element
And AD hybrid is formed, and the transcription of the reporter gene located downstream thereof is activated.

Examples of the host cell are the same mammalian cells as in the third embodiment, and examples of the reporter gene expression vector are the above reporter gene expression vector in which PPRE is replaced with a BD response element. It

Since both PPARγ and NFκB are transcriptional regulators themselves, there is a possibility that false positive or false negative may occur in the measurement system using the expression of the reporter gene as an index. Therefore, instead of BD and AD, for example, by using SOS protein and myristylation signal, by using a yeast temperature sensitive mutant strain such as cdc25H strain as a host cell, NFκB and P without using the expression of the reporter gene as an index.
It is also possible to detect the interaction of PARγ.

[0049]

The present invention will be described in more detail with reference to the following examples, which are merely examples and do not limit the scope of the present invention.

In the following experiment, the mouse bone marrow undifferentiated cell line ST2 was cultured in αMEM containing 10% FCS. Treatment of cells with various factors includes TNFα (1 ng / ml; GIBCOBRL), IL-1 (10 n
g / ml; Pepro tech, Inc).

The vector used was obtained as follows. NIK, NIK (629-947), IKKα, IKKα (K44
A), IKKβ, NFκB, and IκB expression vectors used were those provided by Professor Kunihiro Matsumoto of the Graduate School of Science, Nagoya University. For the p65 and p50 expression vectors, after obtaining cDNA from the human HeLa library, FLAG pcDNA3, Myc pcDNA3, pM, pVP
Subcloning into 16 expression vectors was used. pcDN
A-human PPARγ2 used was that provided by Yamanouchi Pharmaceutical. GST-PPARγ expression vector is pcDNA-human PPARγ
GST-PPARγ (full), GST-PPARγ (1-
138), GST-PPARγ (1-203), GST-PPARγ (1-311), GST-PP
ARγ (204-506), GST-PPARγ (312-506), GST-PPARγ (139
-311) incorporated into GST4T-1 (Amersham Pharmacia Biotech) was used. The GST-p65 expression vector is GST-p65 (ful
l), GST-p65 (1-48), GST-p65 (49-298), GST-p65 (299-53
We created 4 types of 8). As a reporter vector for examining the transcriptional activity of PPAR, 3XPPRE (PPRE derived from mouse acyl CoA oxidase gene; 5'-GTCGACAGGGGACCAGGACA
AAGGTCACGTTCGGGAGT -3 '; Sequence Listing SEQ ID NO: 1) DNA, p
The one incorporated in the GL3 Basic Luciferase vector (Promega) was used. This vector uses tk as a promoter
(Thymidine kinase) is used.

Reference Example 1 Suppression of transcriptional activation of PPARγ by enhancing NFκB activity PPARγ and RXRα expression vector, and a reporter vector into which PPRE was inserted at the same time, and troglitazone-induced PPARγ / RXRα heterodimer The effect of NFκB and the like on the reporter gene-activating ability mediated by NF was examined. 0.25 μg / well of 3XPPRE reporter plasmid, 0.2 μg of PPAR / pcDNA3.1, 0.01-
0.25 μg was added, and the total amount was adjusted to 1 μg with pBluescript M13. ST2 cells were subcultured in a 12-well dish the day before so that the confluence was 40-50%, and used for transfection. OPTI-MEM I Reduced Serum Medium (GIBCOBRL)
(Hereinafter referred to as OPTI-MEM) plus reagent (GIBCOBRL)
Dilute the DNA with the added solution and let it stand at room temperature for 15 minutes.
ution A, Lipofectamine diluted in OPTI-MEM
This was designated as Solution B, and Solution A and B were mixed and then left at room temperature for 15 minutes. Meanwhile, wash the cells with OPTI-MEM and add 0.5 ml O
The medium was exchanged with PTI-MEM. The solution mixture of Solution A and B was added dropwise, and the gene was introduced by incubating at 37 ° C. for 3 hours, and then the medium was replaced with the test ligand. After further incubating for 16 hours, 150 μl of lysis buffer was added per well and shaken for 5 minutes at room temperature to prepare a cell lysate. To measure luciferase activity, Dual-Luciferase reporter assay system (Promega)
Was used. Aliquot 10-25 μl of the prepared cell lysate into a plate, and use this for the Micro Lumat LB96P (Wallac Bertho).
ld). The efficiency of gene transfer was determined by the CMV-Renilla Luciferase vector (Promega, 25
The amount of luminescence from (ng / well) was measured as an index, and the transcription activation ability was expressed as a relative value corrected for the gene transfer efficiency. The result is shown in FIG.

IKK known to activate NFκB
Overexpression of α or IKKβ (lanes 3 and 4), or H
₂ O ₂ treatment (lane 8) and further overexpression of NFκB (p50, p65) (lane 6) showed the same inhibitory effect as NIK (lane 7). Inactive IKKα showed no suppressive effect (lane 5). From the above, it was revealed that the inhibitory action on the transcriptional activation ability of PPARγ is exerted by enhancing the NFκB activity.

Example 1 Investigation of Interaction between PPARγ and NFκB Expression vector V encoding a fusion protein of the DNA binding domain of transcription factor GAL4 and the N-terminal region (A / BC) of PPARγ, V
An expression vector encoding a fusion protein of the transcription activation domain of P16 and p65 or p50, and a reporter plasmid containing a luciferase gene downstream of a promoter region containing a GAL4 response element are transfected by the same method as in Reference Example 1, Luciferase activity was measured. The results are shown in Figure 2A.

PPARγ N-terminal region (GAL4-PPARγ (A / BC))
Since co-expression of p65 and p65 (pVP16-p65) increased transcriptional activity (lane 6), pPARγN-terminal region and NFκB p65
It is presumed that there is an interaction in. PPARγ N-terminal region and N
It was presumed that the FκB p50 had no interaction (lane 7).

An expression vector encoding a fusion protein of the DNA binding domain of GAL4 and the C-terminal region (DE / F) of PPARγ, an expression vector encoding a fusion protein of the transcriptional activation domain of VP16 and p65 or p50, and A reporter plasmid containing the luciferase gene downstream of the promoter region containing the GAL4 response element was transfected in the presence and absence of troglitazone (Tro) by the same method as in Reference Example 1, and the luciferase activity was measured. The results are shown in Figure 2B.

In the presence of Tro, the PPARγ C-terminal region (GAL4-PPA
Co-expression of Rγ (DE / F)) with p65 (pVP16-p65) increased transcriptional activity (lane 7), but this increased activity was clearly higher than when p65 was not overexpressed (lane 6). From this, it was suggested that there is an interaction between the PPARγ C-terminal region and p65. It was presumed that there was no interaction between the PPARγ C-terminal region and p50 of NFκB (lane 8).

From the results of this experiment, p65 interacts with both N-terminal and C-terminal regions of PPARγ, and no transcriptional inhibition by NFκB was observed in either N-terminal or C-terminal region. It was speculated that the fact that NFκB directly suppresses the function of the transcriptional activation region of PPARγ is unlikely to contribute to the suppression of the transcriptional activation ability of PPARγ by NFκB.

Example 2 PPARγ by GST-pull down method
Of direct interaction between NFκB and NFκB Interaction between various partial regions of PPARγ and p65 and p50, and
The direct interaction between various partial regions of p65 and PPARγ was examined as follows.

(Confirmation of expression and purification of GST fusion protein) p
E. coli containing the fusion protein expression vector cloned in GEX (Amersham Pharmacia Biotech) was transferred to 3-5 ml of LB (Am
(p +) medium, and 1 ml of this preculture liquid was added to 400 ml of LB (Amp +)
The cells were inoculated into and cultured at 37 ° C. When OD600 = 0.4-0.5, IPTG was added to a final concentration of 0.1 mM, and the cells were cultured at 30 ° C. thereafter. 90 minutes later, Escherichia coli was collected by centrifugation at 6000 rpm, 2 min, 4 ° C., and suspended in 20 ml of chilled 1 × PBS. Sonicator
After crushing the E. coli by (level 9, 10 seconds 3 times), 1 ml of 20% Triton-X 100 was added to the crushed solution, and the mixture was shaken at 4 ° C. for 30 minutes. The supernatant after centrifugation at 7,500 rpm for 5 minutes was transferred to another Falcon tube, washed twice with 1X PBS and equilibrated.
Add athione-Sepharose 4B (Amarsham Pharmacia Biotech)
200 ml was added and shaken at 4 ° C. for 30 minutes. Glutathione-Sepharose 4B was collected by centrifugation at 1,500 rpm for 2 minutes.
After discarding the supernatant, transfer Glutathione-Sepharose to a 1.5 ml Eppendorf tube and stir in 1 ml of 1xPBS, 1,500 rpm, 2
Centrifugation was repeated 5 times to wash Glutathione-Sepharose 4B. The expression level of the GST fusion protein was confirmed by SDS-PAGE.

(In vitro translation)
Promega TNT Coupled Reticulocyte Lysate SystemK
I used it. PPARγ / pcDNA3, p50 / pcDNA3 or p65 / p
cDNA3 1 μg, Rabbit reticulocyte lysate 25 μl, Reaction buffer 2 μl, T7 RNA polymerase 1 μl, RNase inhibitor 1 μl (TOYOBO), Amino acid mixture (Met-) 1 μl,
Add 4 μl of ³⁵ S-Met (Amersham Pharmacia) and add H ₂ O.
The total volume was adjusted to 50 μl with, and the above mixture was mixed, followed by incubation at 30 ° C. for 2 hours. The input sample was prepared by adding 2xSDS-PAGE sample buffer and boiling.

(GST-pull down assay) 25 μl of Glutathione-Sepharose 4B to which the GST fusion protein was bound was taken as one sample and weighed into a 1.5 ml Eppendorf tube. Add 10 μl of the in vitro translation reaction solution and 300 μl of NET-N + (50 mM Tris-C
l (pH7.5), 150mM NaCl, 5mM EDTA (pH8.0), 0.5% NP40,
1 mM PMSF, 1 mg / ml aprotinin) was added and mixed gently.
Place on ice with occasional mixing for 30 minutes, 1500rpm, 1min, 4 ℃
Centrifugation was performed and the supernatant was discarded. After adding 300-500 μl of NET-N + and mixing, the mixture was centrifuged again at 1500 rpm, 1 min, 4 ° C., and the supernatant was discarded.
The washing operation using 300-500 μl of NET-N + was repeated 5 times, 25 μl of 2xSDS-PAGE sample buffer was added, and the mixture was boiled and detected by SDS-PAGE. The results are shown in FIG. 3A and FIG.
Shown in B.

It was revealed that p65, not p50, directly binds to PPARγ, particularly NFκB strongly binds to the N-terminal portion of PPARγ. Furthermore, it was revealed that p65 binds to PPARγ in the LZ-RHD region, but not in the N-terminal and C-terminal regions.

Example 3 PPARγ / RXRα heterodimer PPR
Effect of NFκB activation on E-binding ability (gel shift assay) PP using calcium phosphate method
Vectors such as ARγ and RXRα were transfected into 293T cells, treated appropriately with cytokines, a nuclear extract of these cells was prepared, and gel shift assay was performed. That is, after gene transfer, the cells were washed with 1xPBS, and buffer A (10 mM HE
PES-KOH (pH7.8), 10mM KCl, 0.1mM EDTA (pH8.0), 1mM D
TT, 0.5 mM PMSF, 2 μg / ml aprotinin, 2 μg / ml peptastine, 2 μg / ml leupeptin) and mixed by vortex. After centrifuging at 6000 rpm, 1 min., 4 ° C, discard the supernatant and collect the precipitate with 100 μl of buffer C (50 mM Hepes-K
OH (pH7.8), 420mM KCl, 0.1mM EDTA (pH8.0), 5mM MgC
l ₂ , 20% Glycerol). The mixture was vortexed for 30 minutes at 4 ° C., centrifuged at 15000 rpm, 4 ° C., and the supernatant was collected as a nuclear extract. PPRE (AGGACAAAGGTCA; SEQ ID NO: 2) was used as a DNA probe, and end labeling with γ-ATP and T4 kinase was performed.
The reaction was carried out at 37 ° C for 1 hour. Then, it was left at 70 ° C. for 10 minutes and at room temperature for 1 hour. After purification by column, the probe was diluted to a concentration of approximately 1000 cpm / μl. Gel shift assay was performed using the above materials. That is, 1 μl of nuclear extract and poly dIdC (2 μg / μl) were added to the reaction solution (100 mM KC
l, 10mM HEPES (pH7.9), 1mM DTT, 0.1% Nonidet P-40,
7.5% glycerol) 1 μg BSA was mixed and left standing in ice for 15 minutes. Then mix 1 μl of labeled probe and leave it at room temperature for 20-30 minutes, add Dye and gel (1 x TBE, 6% acrylamide) and 1 x.
Electrophoresis was performed under TBE buffer under the condition of 130 V constant voltage. After the electrophoresis, the gel was dried and analyzed with BAS 2000 (Fuji film). Anti-PP for confirmation of supershift by antibody
ARγ2 antibody (ABR) was used. The results are shown in Fig. 4.

TNFα (0.1, 1 ng / ml ,: lanes 5 and 6), IL
-1 (1.0, 10 ng / ml: lanes 8, 9), H ₂ O ₂ (0.1, 1 mM:
(Lane 11, 12) Each treatment was performed on PPARγ / RXRα heterodimer P
Suppressed binding to PRE. In addition, it is clear from the supershift by the antibody that this binding includes PPARγ (lane 14). From the above, the suppressive action of PPARγ transcriptional activation by cytokines is
It was shown to be exerted by suppressing the binding of XRα heterodimer to PPRE.

Example 4 PPARγ / RXRα heterodimer PPR
Effect of NFκB and NIK on E-binding ability A gel shift assay similar to that in Example 3 was performed by overexpressing various factors. Results are shown in FIG. PPARγ / RXR is affected by both p65 and p50, which are components of NFκB
Binding of α-heterodimer to PPRE was suppressed (lane
(3, 4), it was more strongly suppressed by the presence of both factors (lane
Five). DNA binding was also suppressed by NIK (lane
7), Blocking the NFκB signal by the presence of IκB (32A / 36A) canceled the DNA binding inhibition (lane 8).
From the above, the suppressive action of the transcriptional activation ability of PPARγ by the NFκB signal was observed in the PPRE of PPARγ / RXRα heterodimer.
It was shown that it is exerted by suppressing the binding to.

[0067]

Industrial Applicability The lipid cell differentiation inhibitor selected by the screening method of the present invention enhances the binding between PPARγ and NFκB, thereby directly blocking the transcriptional activation of adipocyte-specific gene by PPARγ. Therefore, it is possible to more safely and effectively suppress the differentiation of undifferentiated cells into adipocytes, or to dedifferentiate from adipocytes, as compared with growth factors, cytokines, etc., which have conventionally been known to have adipocyte differentiation inhibitory effects. Can be promoted. Therefore, the screening method of the present invention is, for example, a prophylactic and therapeutic agent for various bone / cartilage / connective tissue diseases caused by fat myelination, and further prevention of various diseases caused by fat accumulation in systemic tissues associated with aging. And is extremely useful in that it can provide a potent candidate compound for a therapeutic agent. On the other hand, the lipid cell differentiation promoting substance selected by the screening method of the present invention can be a potent candidate compound for the insulin sensitizer as well as the thiazolidine derivative already clinically applied. In addition, the anti-inflammatory substance selected by the screening method of the present invention enhances the binding between NFκB and PPARγ, resulting in COX.
Since it suppresses the induction of NFκB expression of proinflammatory genes such as -2, the screening method is a strong candidate for anti-inflammatory agents with a different mechanism of action from COX-2 selective inhibitory anti-inflammatory agents, which are said to have fewer side effects. A compound may be provided.

[0068]

[Sequence list]                           Sequence Listing <110> Sumitomo Pharmaceuticals <120> Method for screening of substance suppressing adipocyte       differentiation. <130> A4703 <160> 2 <210> 1 <211> 38 <212> DNA <213> Mus musculus <400> 1 gtcgacaggg gaccaggaca aaggtcacgt tcgggagt 38 <210> 2 <211> 13 <212> DNA <213> Mus musculus <400> 2 aggacaaagg tca 13

[Brief description of drawings]

FIG. 1 shows IKKα, IKKβ, inactivated IKK, NFκB, NIK and H ₂ O that affect Tro-induced transcriptional activation of PPARγ.
It is a figure which shows the effect of ₂ processing.

[Fig. 2] Interaction between PPARγ N-terminal region and p65 or p50 (A)
FIG. 2 is a diagram showing the interaction (B) between the PPARγ C-terminal region and p65 or p50.

FIG. 3 is a diagram showing the binding ability of various partial regions of PPARγ to p65 or p50 (A) and the binding ability of various partial regions of p65 to PPARγ (B).

FIG. 4 shows the effect of NFκB activation by TNFα, IL-1 and H ₂ O ₂ treatment on the binding ability of PPARγ / RXRα heterodimer to PPRE.

FIG. 5 is a diagram showing the inhibitory effect of NFκB on the binding ability of PPARγ / RXRα heterodimer to PPRE and the cancellation of the inhibitory effect of NFκB by inactivating NFκB by IκBα.

Continued front page    F term (reference) 2G045 BB10 BB29 BB46 BB50 BB51                       CB01 FB01 FB03 FB05 FB12                       GC15                 4B024 AA11 BA63 CA04 DA02 EA04                       FA02 FA10 GA11 HA01                 4B063 QA18 QQ08 QQ49 QR51 QR58                       QS31 QX02

Claims (7)

[Claims] 1. NFκB in the presence and absence of a test substance
And PPARγ are contacted, and NFκB and PPAR under both conditions
A method for screening an adipocyte differentiation regulatory and / or anti-inflammatory substance, which comprises comparing the degree of γ binding. 2. The method according to claim 1, wherein the means for measuring the degree of binding between NFκB and PPARγ is to measure the amount of NFκB-PPARγ complex. 3. The method according to claim 1, wherein the means for measuring the degree of binding between NFκB and PPARγ measures the amount of PPARγ bound to PPRE. 4. The method according to claim 3, wherein the means for measuring the amount of PPARγ bound to PPRE is a gel shift assay. 5. The method for measuring the degree of binding between NFκB and PPARγ uses the expression of a reporter gene under the control of a promoter region containing PPRE as an index.
The method described. 6. An expression vector containing a reporter gene under the control of a promoter region containing PPRE is introduced,
Moreover, mammalian cells engineered to express NFκB, PPARγ and RXR were treated with PPAR in the presence and absence of a test substance.
A method for screening an adipocyte differentiation control and / or anti-inflammatory substance, which comprises culturing in a medium containing an agonist of γ and comparing the expression of the reporter gene in the mammalian cells under both conditions . 7. A vector expressing one of NFκB (p65) or PPARγ as a fusion protein with the DNA binding domain of a transcription factor, a vector expressing the other as a fusion protein with the transcription activation domain of a transcription factor, and DNA
A host cell introduced with an expression vector containing a reporter gene under the control of a promoter region containing a cis element recognized and bound by a binding domain is cultured in a medium in the presence or absence of a test substance, A method for screening an adipocyte differentiation regulatory and / or anti-inflammatory substance, which comprises comparing the expression of the reporter gene in the host cell under conditions.