WO2014178427A1

WO2014178427A1 - Method for controlling expression of semaphorin 3a

Info

Publication number: WO2014178427A1
Application number: PCT/JP2014/062073
Authority: WO
Inventors: 弥生鎌田; 光俊冨永; 建二 ▲高▼森
Original assignee: 学校法人順天堂
Priority date: 2013-05-02
Filing date: 2014-05-01
Publication date: 2014-11-06
Also published as: JPWO2014178427A1; JP6385341B2

Abstract

As the results of intensive studies, the present inventors identified transcription factors that control the expression of semaphorin 3A gene. By inhibiting or promoting these transcription factors, the expression of semaphorin 3A can be controlled (suppressed or enhanced). Further, by screening compounds with the use of an activity of inhibiting or promoting such a transcription factor as an index, a compound capable of controlling the expression of semaphorin 3A can be obtained. The compound capable of controlling the expression of semaphorin 3A is useful in treating itch in skin diseases, allergic rhinitis, osteoporosis and nerve damage, in particular, for treating itch.

Description

Semaphorin 3A expression regulation method

The present invention relates to an agent that enhances or suppresses the expression of semaphorin 3A using a transcription factor, and a method for screening a compound that enhances or suppresses the expression of semaphorin 3A using the inhibition or promotion of a specific transcription factor as an index. .

Semaphorin (Semaphorin) is identified as a guidance factor that controls cell migration such as nerve axon elongation and is involved in various biological functions and pathologies such as immune response, organogenesis, bone metabolism control, and cancer progression It is known that it is a molecular group. Sema family proteins are classified into Sema1 to Sema7 classes based on the difference in domain structure, and further subclassified (Non-patent Document 1).

Semaphorin 3A (Sema3A), which is one of them, is known as a typical molecule of a nerve guidance factor that determines the elongation direction of nerve axons, and the presence of Sema3A inhibits nerve axon elongation. This is thought to lead to a two-sided pharmacological action. That is, it is to promote nerve regeneration by inhibiting Sema3A and to enhance Sema3A to suppress nerve elongation. In fact, for the former, a technique as a nerve regeneration promoting agent is disclosed (Patent Document 1), and for the latter, there is a report that it may be used to reduce itchiness, particularly in skin tissue (Non-Patent Document 2). .

In patients with atopic dermatitis and psoriasis with disruption of skin barrier function, intraepidermal nerve fibers become dense with the decrease in Sema3A expression in normal skin, and intractable itching that does not respond to antihistamines develops (Non-patent document 3), an attempt to significantly suppress itching and inflammation by externally applying an ointment containing recombinant Sema3A protein to NC / Nga mice with atopic dermatitis (Non-patent document 3) Document 4) and a technique for using Sema3A protein itself as a therapeutic agent for pruritic skin diseases such as atopic dermatitis have been developed (Patent Document 2).

Although this approach of supplementing Sema3A to suppress the induction of nerve axons in the skin and consequently suppress itching may be a promising technique for itching treatment, for practical application, Sema3A protein Preparation and stable formulation development are key. Sema3A is a secreted protein member of semaphorin and has three structural domains, a semaphorin (Sema) domain of 500 amino acid residues, a C-2 type immunoglobulin (Ig) domain, and a basic terminal domain. It has been shown that homodimerization is indispensable in order to exhibit physiological activity (Non-patent Document 4). When Sema3A is formulated as a pharmaceutical product, a high molecular weight protein with a molecular weight of over 100,000, which is a homodimer of a Sema domain consisting of 500 amino acid residues, is an active ingredient, but it can stably produce a high molecular weight dimeric protein. It is never easy to develop a preparation that can ensure reasonable cost and stable quality as a therapeutic agent.

As one of the techniques for solving such formulation problems, an approach of inducing a therapeutic effect with a substance that enhances the expression of Sema3A in vivo can be considered. As an example of this technique, it has been found that an extract of Asunalo (Thujopsisdolabrata), which is a cypress family, has its action, and its technique is disclosed (Patent Document 3).

Japanese Patent No. 4975231 JP 2008-297243 A JP 2011-111416 A

If the Sema3A expression can be suppressed endogenously, the nerve regeneration promoting action can be induced, and if it can be enhanced, the nerve axon elongation inhibiting action can be induced. However, a method for simply searching for or examining a substance that controls the expression of Sema3A has not been sufficiently developed. In particular, it has been known that skin pruritus is associated with a decrease in Sema3A in the epidermis, which has been shown to lead to the development of itch, so it is necessary to search for substances that enhance endogenous Sema3A. If a simple Sema3A expression regulator search method can be provided, it will be very useful for the development of refractory pruritus therapeutics.

The present invention provides a novel means useful for screening a substance that regulates the expression of Sema3A, evaluation of the expression regulating action, and a novel means useful for treating pruritus and treating nerve damage. The purpose is to do.

The inventors of the present application isolated the human Sema3A gene promoter, determined its characteristics, and identified a transcription factor group in the promoter region. These transcription factors have been intensively studied and succeeded in identifying a transcription factor group that is related to Sema3A expression enhancement and a transcription factor group that is related to suppression of expression. Furthermore, the expression of the Sema3A gene is inhibited by inhibiting these transcription factors. After confirming that it can be adjusted, the present invention was completed.

That is, the present invention is NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, RORα, ER, AP-1, At least one transcription factor that regulates any transcription factor selected from Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2. A semaphorin 3A expression regulator comprising a substance as an active ingredient is provided. The present invention also includes NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, RORα, ER, AP-1, Compounds with the regulation of at least one transcription factor selected from Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2 A method for screening a compound that modulates the expression of semaphorin 3A is provided. Furthermore, the present invention provides a cell containing a nucleic acid construct containing a semaphorin 3A promoter region and a reporter gene, and a substance that regulates a transcription factor, and measuring the expression level of the reporter gene, and then measuring the expression level of the reporter gene. A method for evaluating the expression-regulating action of 3A is provided. Furthermore, the present invention relates to cultured cells, NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, RORα, ER, Contact with a substance that regulates a transcription factor selected from AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2. And a method for evaluating an action of regulating the expression of semaphorin 3A by the substance, comprising measuring the expression level of semaphorin 3A in the cell.

The present invention provides a Sema3A expression regulator using a transcription factor that regulates Sema3A gene expression. An agent that enhances the expression of Sema3A is useful for the treatment and prevention of pruritus in pruritic skin diseases such as atopic dermatitis. An agent that suppresses the expression of Sema3A is useful for promoting nerve regeneration at the site of nerve injury. The present invention also provides a method for screening a compound that regulates Sema3A expression and a method for evaluating the strength of Sema3A expression enhancing action or expression inhibiting action. According to the screening method and the evaluation method of the present invention, it is possible to provide a novel pharmaceutical that exhibits an excellent therapeutic effect by regulating the expression of Sema3A.

It is a schematic diagram which shows the 5'-flanking region of the gene encoding human Sema3A. Based on the nucleotide sequence of the Sema3A upstream gene newly cloned by the inventors of the present application, the putative transcription factor binding site was clarified by a search using a P-Match-1.0 public program (BIOBASE). It is the base sequence (SEQ ID NO: 1) of the 5'-flanking region of the gene encoding human Sema3A. A putative transcription factor binding sequence is shown below the sequence. It is a graph which shows the result of the luciferase assay of the Sema3A upstream gene using normal human epidermal keratinocytes (NHEK). In order to determine the promoter region more precisely, analysis with 5'-end deletion mutants was performed using NHEK in the growth phase and NHEK after differentiation induction. The nucleotide sequence near the minimal promoter (-240 / -1) of Sema3A is shown. The putative transcription factor binding sequence is underlined and the minimal promoter sequence (-134 / -25) is surrounded by a dotted line. This shows the nucleotide sequence of the transcriptional repression region (-1444 / -975) upstream of the Sema3A gene. Putative transcription factor binding sequences are underlined. The transcription factor binding to the putative transcription factor binding sequence of the Sema3A promoter is shown. Experiments were performed with an electrophoretic mobility shift assay (EMSA) using a nucleoprotein extract from cultured NHEK and a biotin-labeled oligonucleotide probe containing a putative transcription factor binding sequence. Lane 1 shows the binding profile of the nuclear extract and biotin-labeled probe, and lane 2 shows the binding profile of the biotin-labeled probe after competing with a 400-fold excess of unlabeled probe. It is the result of the luciferase assay using the construct which introduce | transduced the site-specific mutation into the putative transcription factor binding sequence site | part of Sema3A. *** is significantly different at p <0.001. Gene expression measurement results by real-time PCR of each transcription factor and Sema3A using small interfering RNA (siRNA) to confirm that transcription factors RORα, Jun, Fos, Sox4, and NF-κB1 are involved in Sema3A expression It is. NHEK was transfected with RORα, Jun, Fos, Sox4, NF-κB1 siRNA (40 nM), and total RNA was isolated after 48 hours of culture. A shows the expression suppression efficiency of each transcription factor. B and C are Sema3A expression analysis results after each transcription factor knockdown. Transcription factors GABPα, Sox9, Rel (c-Rel), CREB, Pbx1, ER, JunB, JunD, Fra1, and Fra2 were used to confirm that each transcription factor and Sema3A used siRNA to confirm Sema3A expression. It is a gene expression measurement result by real-time PCR. NHEK was transfected with siRNA (40 nM) of each transcription factor, and total RNA was isolated after 48 hours of culture. A shows the expression suppression efficiency of each transcription factor. B is the Sema3A expression analysis result after each transcription factor knockdown. * Is significantly different from control for p <0.05 and *** for p <0.001. It is the result of investigating the effect of RORα agonists and antagonists on Sema3A expression in NHEK. After treating NHEK with each drug for a certain period of time, total RNA was recovered and Sema3A expression level was analyzed by quantitative real-time PCR. A is the result of the RORα agonist SR1078, B is the result of the RORα agonist cholesterol sulfate, and C is the result of the RORα antagonist SR1001. ** indicates p <0.01 and *** indicates p <0.001 for the untreated groups, respectively. It is the result of the experiment which applies RORα agonist cholesterol sulfate to the lesioned part of atopic dermatitis (AD) model NC / Nga mice. After applying the base or cholesterol sulfate solution to the affected area for 9 consecutive days, the skin was removed and the expression level of Sema3A in the skin tissue was measured by quantitative real-time PCR. ** is significantly different at p <0.01 compared to the base alone control.

A “transcription factor” is a group of proteins that specifically bind to DNA, and binds to a region that regulates transcription 5 ′ upstream of the transcription region of the gene, such as a promoter or enhancer on the DNA, thereby inheriting the DNA. It is a protein that plays a role in promoting or repressing the process of transcription of information into messenger RNA. A transcription factor exerts a transcriptional regulation (promotion or repression) function when one protein molecule is used alone or forms a complex with a plurality of protein molecules. The term “transcription factor” in the present invention includes not only a single protein molecule but also a multimer or complex form in which a plurality of identical or different proteins are complexed. Also included are individual protein molecules that make up the body or complex. It is estimated that there are about 1,800 genes encoding transcription factors on the human genome, but there are various transcription factors for target proteins.

In the present invention, “regulation of transcription factor” refers to inhibition or promotion of transcription factor.

“Transcription factor inhibition” refers to any process that begins with transcription of mRNA from a gene that encodes a transcription factor and leads to regulation of Sema3A expression by binding to the promoter region of the Sema3A gene. Inhibiting this step. Use of antisense RNA, microRNA, siRNA and the like for transcription factors is an example of inhibition of transcription factors.

“Transcription factor promotion” refers to any process that begins with transcription of mRNA from a gene that encodes a transcription factor and leads to regulation of Sema3A expression when the produced transcription factor binds to the promoter region of the Sema3A gene. It means to promote the step. Applying the transcription factor protein itself to the affected area, or enhancing the expression of the transcription factor gene in the affected area is an example of promoting the transcription factor.

The putative transcription factor binding site shown in FIG. 2 is based on the base sequence (SEQ ID NO: 1) of the promoter region upstream of the Sema3A gene newly cloned by the present inventors, and the Match-1.0-Public program and P-Match-1.0 It was identified by performing a search using the Public program (BIOBASE). This program is a database that comprehensively contains information on transcription factors and experimentally proven transcription factor binding sequences, and is software that can predict new transcription factor binding sequences. By analyzing the database, it was possible to estimate an unknown transcription factor binding sequence upstream of the Sema3A gene and to predict transcription factors involved in Sema3A expression.

Based on the prediction results of transcription factor binding sequences, reporter assays of 5 'end deletion mutants in the promoter region, and the results of transcription factor knockdown experiments with siRNA, the following transcription factors are responsible for regulating Sema3A gene expression. Identified. Regulation of these transcription factors can regulate Sema3A expression.
NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2 (also referred to as Nrf2), Fra1 (also referred to as Fosl1), RORα, ER (also referred to as Esr1) , AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB (also called Atf2), JunB, JunD, Fra2

The above transcription factors can be classified into the following groups.
(A) Transcription factors that suppress Sema3A expression
NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, Fra1
(B) Transcription factors that promote Sema3A expression
RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB, JunB, JunD, Fra2

The transcription factor group (A) may preferably be NF-κB. The regulation of NF-κB may be performed by the regulation of its subunit NF-κB1.

The transcription factor group (B) is preferably a transcription factor group consisting of RORα, AP-1, Sox-4, Jun, Fos, STATx (especially STAT3), GABPα, Sox-9, CREB, JunB, JunD, and Fra2. More preferably a transcription factor group consisting of RORα, AP-1, Sox-4, Jun, Fos, CREB, and JunB, more preferably a transcription factor group consisting of RORα, AP-1, Jun, Fos, and CREB, or It may be a transcription factor group consisting of RORα, AP-1, Jun, and Fos. Among these, RORα is a particularly preferred transcription factor as a target for regulating expression of Sema3A. Jun and Fos are proteins constituting AP-1, and these are also included in the “transcription factor” in the present invention.

Inhibiting the transcription factor (A) enhances the expression of the Sema3A gene. Therefore, a substance that inhibits any of the transcription factors (A) can be used as a Sema3A expression enhancer. Such an agent is useful as a therapeutic or prophylactic agent for a disease or condition for which enhanced expression of Sema3A is desired. Specific examples of such diseases or conditions include pruritus, allergic rhinitis and osteoporosis in skin diseases such as atopic dermatitis and xeroderma. In pruritic skin diseases such as atopic dermatitis, it is known that the expression of Sema3A inherent in normal skin is decreased, and it is known that pruritus can be suppressed by supplementing the affected area with Sema3A protein itself (

Non-patent Documents

3 and 4 and Patent Document 2 etc.). In allergic rhinitis, the expression of Sema3A decreases in the nasal mucosal epithelium, peripheral nerve fibers in the epithelial layer and the lamina propria of the nasal turbinate increase, and symptoms of rhinitis caused by intranasal administration of Sema3A protein (Sawaki et al., J Pharmacol Sci 117, 34-44 (2011)). Sema3A is a factor that increases bone mass and is known to be useful for the treatment of osteoporosis (Hayashi et al., Experimental Medicine, Vol. 31, No. 4, 2013). Therefore, it is possible to treat and prevent pruritus, allergic rhinitis, osteoporosis and the like in skin diseases by enhancing Sema3A expression by inhibiting transcription factors that suppress Sema3A gene expression. That is, the substance that inhibits any of the transcription factors in (A) is used as a therapeutic or preventive agent for pruritus, allergic rhinitis, osteoporosis, etc. in skin diseases, and in particular, treatment or prevention of pruritus or allergic rhinitis in skin diseases. It can be preferably used as an agent, particularly as an agent for treating or preventing pruritus in skin diseases. However, the disease or condition for which enhanced expression of Sema3A is desired is not limited to the above specific examples.

Enhance expression of Sema3A is also possible by promoting the transcription factor (B). Therefore, a substance that promotes any of the transcription factors of (B) can be used as an Sema3A expression enhancer, as well as an inhibitor of the transcription factor of (A). It is useful as an agent for treating or preventing a condition. Preferred examples of diseases or conditions for which enhanced expression of Sema3A is desired are as described above. In particular, substances that promote RORα are particularly preferred examples of substances that can be used to enhance the expression of Sema3A.

The expression enhancer of Sema3A may contain one or more substances that inhibit the transcription factor (A), or one or more substances that promote the transcription factor (B). Alternatively, it may contain one or more substances that inhibit the transcription factor (A) and one or more substances that promote the transcription factor (B).

On the other hand, the expression of the Sema3A gene is suppressed by inhibiting the transcription factor (B). Therefore, a substance that inhibits any of the transcription factors (B) can be used as a Sema3A expression inhibitor. Such an agent is useful as a therapeutic or prophylactic agent for a disease or condition for which suppression of Sema3A expression is desired. Specific examples of such diseases or conditions include traumatic nerve injury, olfactory dysfunction, cerebral infarction neuropathy, facial paralysis, diabetic neuropathy, glaucoma, retinitis pigmentosa, Alzheimer's disease, Parkinson's disease, muscle Examples include spinal nerve injury or peripheral nerve damage in developmental lateral sclerosis, amyotrophic lateral sclerosis, Huntington's disease, cerebral infarction, traumatic neurodegenerative disease, and the like. It is known that nerve regeneration can be promoted by inhibiting Sema3A, and a nerve regeneration promoter using this is known (Patent Document 1). Therefore, by suppressing the expression of Sema3A by inhibiting a transcription factor that promotes the expression of the Sema3A gene, nerve regeneration in the nerve damage site can be promoted. That is, a substance that inhibits any of the transcription factors (B) can be preferably used as a nerve regeneration promoter. However, the disease or condition for which Sema3A expression suppression is desired is not limited to the above specific examples.

Sema3A expression suppression can also be achieved by promoting the transcription factor (A). Therefore, a substance that promotes any of the transcription factors of (A) can be used as an inhibitor of Sema3A expression, as well as an inhibitor of the transcription factor of (B), It is useful as an agent for treating or preventing a condition, such as a nerve regeneration promoter.

The expression inhibitor of Sema3A may contain one or more substances that promote the transcription factor (A), or one or more substances that inhibit the transcription factor (B). Alternatively, it may contain one or more substances that promote the transcription factor (A) and one or more substances that inhibit the transcription factor (B).

Drugs provided by the present invention (such as pruritus, allergic rhinitis or osteoporosis treatment or prevention agent for skin diseases, treatment or prevention agent for diseases or conditions in which expression enhancement of Sema3A is desired, and nerve regeneration promoting agent, etc. Therapeutic or preventive agent for diseases or conditions for which suppression of Sema3A expression is desired) is mainly used by local administration. For example, administration by injection or the like to the affected area where nerve regeneration is desired, administration by application to the affected area where itching should be suppressed, intranasal administration for suppressing nasal inflammation, and the like can be mentioned. As a method of administering an agent for treating or preventing osteoporosis, for example, a nucleic acid drug using a promoter that is specifically expressed in bone tissue (for example, siRNA for (A) transcription factor or (B) transcription factor itself is used as bone. Systemic administration in the form of a vector or the like that is expressed under the control of a promoter specific in the tissue), and thereby the expression of Sema3A can be locally enhanced in the bone tissue. Depending on the specific route of administration, it can be formulated into an appropriate dosage form such as an injection or ointment. The dose is appropriately selected according to the size of the affected area, the intensity of symptoms, etc., and is not particularly limited. The amount of active ingredient is about 0.01 μg to 1 g, for example, about 0.01 mg to 10 mg per administration. obtain.

As a substance that inhibits any of the transcription factors of (A) used as an expression enhancer of Sema3A, for example, as defined above, antisense RNA, microRNA, siRNA, etc. that inhibit the expression of each transcription factor are used. be able to. In this case, an expression vector capable of producing such an RNA molecule for the transcription factor of interest can be prepared by a known technique, and a Sema3A expression enhancer can be formulated using the vector as an active ingredient. The amino acid sequences of various transcription factors and the gene sequences encoding them are known, and vectors for producing siRNA and miRNA in living cells are known and commercially available. A Sema3A expression enhancer using RNA, microRNA, siRNA or the like can be prepared. Alternatively, it may be a substance already known as an inhibitor of each transcription factor. (A) Transcription factor inhibitors include IMD-0354 (Tanaka (et al, Blood, 105: 2324-31, 2005), DHMEQ (Ariga et al, J BiolChem, 277: 24625-30, 2002) Is also known to inhibit NF-κB). Alternatively, the compound group can be screened using the inhibition of any of the transcription factors of (A) as an index and provided as an expression enhancer of Sema3A.

The screening of a compound using as an index the inhibition of any of the transcription factors (A) can be performed, for example, as follows.

Step 1: A transcription factor and a compound are contacted, and the presence or absence of binding is examined. High throughput screening is possible by examining the presence or absence of binding in a microplate using an assay robot.

Step 2: It is examined whether or not a compound that has been confirmed to have a binding action has an effect of increasing the expression level of the Sema3A gene. This step can be performed, for example, by a reporter assay using cells transfected with a nucleic acid construct containing a Sema3A promoter region and a reporter gene. The expression level of the reporter gene may be examined by bringing the cell containing the nucleic acid construct into contact with the compound confirmed to be bound in step 1 in a well of a microplate. The reporter assay itself is a well-known conventional method and can be easily performed using a commercially available kit or the like. As the sequence of the Sema3A promoter region, the sequence shown in SEQ ID NO: 1 can be preferably used. The region to which the transcription factor of (A) binds is from position 1 to position 471 in SEQ ID NO: 1. Such screening makes it possible to newly identify a compound that enhances Sema3A expression, which is useful as a therapeutic or prophylactic agent for a disease or condition in which Sema3A expression enhancement is desired (preferred specific examples are as described above).

The substance that promotes any of the transcription factors of (B) used as an expression enhancer of Sema3A may be each transcription factor protein itself as defined above, for example. Specifically, for example, when used as a medicine, the transcription factor protein itself or a vector capable of expressing the transcription factor protein can be applied to the affected area where Sema3A expression is to be enhanced. Alternatively, it may be a substance already known as a promoter for each transcription factor. (B) as a transcription factor promoter, SR1078 (Wang et al, ACS Chem Biol. 5: 1029-34, 2010), cholesterol sulfate (Bitsch et al, Anal Biochem. 323: 139-49, 2003) ( All of them promote RORα). RORα agonists having RORα activation action are particularly promising as promoters of the transcription factor (B). Alternatively, the compound group can be screened and provided as an Sema3A expression enhancer using as an index the promotion of any of the transcription factors of (B). As described above, the Sema3A expression enhancer is useful as a therapeutic or prophylactic agent for pruritus, allergic rhinitis, osteoporosis, etc., particularly as a therapeutic or prophylactic agent for pruritus. For example, WO2011 / 115892 describes the use of SR1078 for the treatment of central nervous diseases, metabolic diseases, cancers, etc. as a medicinal use of RORα agonists of particular interest (however, WO2011 / 115892 describes the use of ROR It also describes the use of inverse agonists for the treatment of central nervous disease, metabolic diseases, cancer, etc., and it is not specifically disclosed which of ROR inhibition or promotion is useful for the treatment of these diseases ), There is no report on the therapeutic use of pruritus, which is a skin disease, and this is a new use proposed for the first time in the present invention.

(B) Screening of a compound using any of the transcription factors as an index is performed by, for example, reporter gene expression by reporter assay using a cell transfected with a nucleic acid construct containing a Sema3A promoter region and a reporter gene. This can be done by screening for compounds that increase the amount. The expression level of the reporter gene may be examined by bringing the compound into contact with a cell containing a nucleic acid construct containing the Sema3A promoter region and the reporter gene in a well of the microplate. In this case, as the Sema3A promoter region, the nucleotide sequence shown in SEQ ID NO: 1 can be used, or the region to which the transcription factor (B) binds exists, -134 to -24 positions of the Sema3A gene It is also possible to use only the region (positions 1311 to 1421 in SEQ ID NO: 1, the sequence of this region is taken out and shown in SEQ ID NO: 2). Such screening makes it possible to newly identify a compound that enhances the expression of Sema3A, which is useful as a therapeutic or prophylactic agent for a disease or condition in which Sema3A expression enhancement is desired (preferred specific examples are as described above).

The substance that promotes any of the transcription factors (A) used as an Sema3A expression inhibitor can be the same as the substance that promotes any of the transcription factors (B) described above. Each transcription factor protein itself may be a substance that is already known as a promoter of each transcription factor, or a compound that uses any of the transcription factors of (A) as an index. The group can also be screened and provided as an Sema3A expression inhibitor. (A) Transcription factor promoters include isohumulones (WO 2006/043671) and sulforaphane (Thimmulappa et al, Cancer Res, 62: 5196-203,2002) (all activate NF-E2) Are known. Compound screening can be carried out, for example, by screening a compound that increases the expression level of a reporter gene by a reporter assay using a cell transfected with a nucleic acid construct containing a Sema3A promoter region and a reporter gene. The region to which the transcription factor of (A) binds is from position 1 to position 471 in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 1 can be used as the Sema3A promoter region. By such screening, it is possible to newly identify a compound that suppresses the expression of Sema3A, which is useful as a therapeutic or preventive agent for a disease or condition for which suppression of Sema3A expression is desired, preferably as a nerve regeneration promoter.

The substance that inhibits any of the transcription factors (B) used as an Sema3A expression inhibitor can be the same as the substance that inhibits any of the transcription factors (A) described above. Antisense RNA, microRNA, siRNA, etc. for each transcription factor can be used, and it can be a substance already known as an inhibitor of each transcription factor, or any of the transcription factors of (B) It can also be obtained by screening a compound using as an index the inhibition. (B) transcription factor inhibitors include T-5224 (Uchihashihet al, Drug Metab Dispos.39: 803-13, 2011.AP-1 inhibition), SR1001 (Crumbley et al, PLoS One. 7: e33804, 2012. RORα inverse agonist), SR3335 (Solt and Burris, risTrends in Endocrinology and Metabolism. 23: 619-627, 2012. and Kumar et al., ACS Chem Biol. 18: 218-22, 2011. RORα Inverse agonists) are known. The compound screening may be performed in accordance with the above-described

steps

1 and 2, similarly to the screening of the substance that inhibits the transcription factor (A). As the Sema3A promoter region contained in the nucleic acid construct, the base sequence shown in SEQ ID NO: 1 can be used, or the region to which the transcription factor (B) binds exists, from position 1311 in SEQ ID NO: 1. Only the region at position 1421 (the sequence of this region is extracted and shown in SEQ ID NO: 2) can be used. By such screening, it is possible to newly identify a compound that suppresses the expression of Sema3A, which is useful as a therapeutic or preventive agent for a disease or condition for which suppression of Sema3A expression is desired, preferably as a nerve regeneration promoter.

The base sequence shown in SEQ ID NO: 1 is the sequence of the promoter region of the Sema3A gene identified by the inventors of the present application, but is a small number of bases in SEQ ID NO: 1, particularly a cis element of Sema3A gene regulation -1444. If it is a base other than position -974 (positions 1 to 471 in SEQ ID NO: 1) and positions -134 to -24 (positions 1311 to 1421 in SEQ ID NO: 1), Even if there is some difference from the base sequence shown, it can be used as the Sema3A promoter region in the screening method. Therefore, in screening for compounds that enhance or suppress the expression of Sema3A, in addition to the nucleotide sequence shown in SEQ ID NO: 1 or the nucleotide sequence shown in SEQ ID NO: 2, it is highly identical to SEQ ID NO: 1 or SEQ ID NO: 2. A base sequence having (identity of 90% or more, preferably 95% or more, more preferably 98% or more) can also be used as the Sema3A promoter region.

Here, the “identity” of the base sequences is a percentage obtained by aligning both sequences so that the bases of the two base sequences to be compared match as much as possible, and dividing the number of matched bases by the total number of bases. It is represented by. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of bases is the number of bases obtained by counting one gap as one base. When the total number of bases counted in this way differs between the two sequences to be compared, the identity (%) is calculated by dividing the total number of bases of the longer sequence and dividing the number of matched bases. The However, if the sequence to be compared is in a state linked to any other sequence (for example, a state incorporated in an expression vector, a state linked to a desired gene to be expressed, etc.), it corresponds to a promoter. Only the region to be extracted is taken out, the sequences are compared, and the identity is calculated.

Also, as described above, inhibitors and promoters are known for various transcription factors. Among these known inhibitors and promoters, the Sema3A promoter region-reporter described in the above description of the screening method for the purpose of finding a compound having particularly strong activity of regulating (suppressing or enhancing) the expression of Sema3A. Reporter assays using cells containing the gene construct can be performed. That is, the present invention includes a cell comprising a nucleic acid construct comprising a semaphorin 3A promoter region and a reporter gene, NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP -1, HLF, NF-E2, RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and There is also provided a method for evaluating the action of regulating the expression of semaphorin 3A by the substance, which comprises contacting a substance that regulates any transcription factor selected from Fra2 and measuring the expression level of the reporter gene.

The reporter assay and the Sema3A promoter region are the same as described above for the screening method. As the Sema3A promoter region, one containing any one of the following base sequences (1) to (3) can be used.
(1) Base sequence shown in SEQ ID NO: 1
(2) Base sequence shown in SEQ ID NO: 2
(3) Base sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with (1) or (2)

When the above evaluation method is applied to the inhibitor of any of the transcription factors of (A) and the promoter of any of the transcription factors of (B), Sema3A by the inhibitor or the promoter is used. The expression enhancing action of is evaluated. On the other hand, when the above evaluation method is applied to any promoter of the transcription factor (A) and any inhibitor of the transcription factor (B), the promoter or the inhibitor Sema3A expression-suppressing action is evaluated. A promoter region using SEQ ID NO: 2 or a nucleotide sequence having 90% or more identity to this can be used when evaluating an inhibitor or promoter of the transcription factor (B).

Alternatively, it is also possible to evaluate the expression regulating action of Sema3A by a substance that regulates (inhibits or promotes) the above transcription factor using the expression level of Sema3A in cultured cells as an index, regardless of the reporter assay. In this case, the cultured cells, the transcription factor (A) (NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, Fra1) and the above (B) transcription factors (RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB, JunB, JunD , Fra2) is contacted with a substance that regulates (inhibits or promotes) a transcription factor selected from, and the expression level of Sema3A in the cell is measured.

When the evaluation method is applied to any one of the inhibitors of the transcription factor of (A) and the promoter of any of the transcription factors of (B), the expression of Sema3A by the inhibitor or the promoter is enhanced. The effect is evaluated. On the other hand, when the evaluation method is applied to any promoter of the transcription factor (A) and any inhibitor of the transcription factor (B), the promoter or the Sema3A by the inhibitor Expression inhibitory action is evaluated.

(A) The evaluation of the transcription factor inhibitor in (A) and the evaluation of the transcription factor promoter in (B) can be performed in combination to identify a combination of drugs that have a very high effect of enhancing the expression of Sema3A. Such a combination of agents can be particularly preferably used as a therapeutic or prophylactic agent for diseases or conditions in which enhanced expression of Sema3A is desired, such as a therapeutic or prophylactic agent for pruritus.

In addition, the evaluation of the transcription factor promoter in (A) and the evaluation of the transcription factor inhibitor in (B) can be conducted in combination to identify combinations of drugs that have a very high effect of suppressing Sema3A expression. it can. Such a combination of drugs can be particularly preferably used as a therapeutic or prophylactic agent for a disease or condition for which suppression of Sema3A expression is desired, such as a nerve regeneration promoter.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Isolation of the human Sema3A gene promoter Based on the known nucleotide sequence of the coding region of the human Sema3A gene, the 5'-flanking region was replaced with gene-specific primer 1 (GSP1: 5'-TCTGATAGTTTGCTCTTGCTGTAAG-3 ', SEQ ID NO: 3). And gene-specific primer 2 (GSP2: 5'-AAGACAGACAATCCTAGTTAACCAG-3 ', SEQ ID NO: 4) according to the manufacturer's instructions and amplified using the Genome Walker Kit (Clontech, Mountain View, CA) . Briefly, the manufacturer recommended two-step PCR protocol using Ex Taqpolymelase: 94 ° C for 25 seconds, 72 ° C for 4 minutes for 7 cycles, followed by 94 ° C for 25 seconds, and Primary PCR using GSP1 and adapter primer (AP) 1 was performed using 32 cycles of 4 minutes at 67 ° C. and 4 minutes at 67 ° C. for the final extension reaction. The primary PCR mixture was then diluted and used as a template for secondary PCR amplification with GSP2 and AP2. The secondary PCR was identical to the primary PCR except that 5 cycles were used instead of 7 cycles followed by 22 cycles instead of 32 cycles. Sequential 5′-deletion mutants of the 5′-flanking region of Sema3A were generated by PCR using the primers listed in Table 1. After amplification, all PCR products were cloned into pGEM-T-Easy vector (Promega Co., Madison, Wis., USA) and sequenced using ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, Calif.) Were determined.

To construct the reporter plasmid pGL3-1444 / -1, PCR was first denatured at 94 ° C for 5 minutes, 94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 1 minute under the following conditions: A set of specific Sema3A primers containing a restriction enzyme site for the template pGEM-T-Sema3A-1444 / -1 and KpnI or HindIII for 30 minutes in minutes and finally 5 minutes at extension 72 ° C. Sema3A-1444 KpnI and Sema3A + 1 HindIII) were used. The resulting PCR product was digested with KpnI and HindIII and cloned into the pGL3-Basic vector (Promega). The pGL3-Basic vector contains a firefly luciferase gene. All of the constructs were prepared using QIAGEN Plasmid Maxi Ki (QIAGEN, Duesseldolf, Germany).

Furthermore, using Quikchange site-directed mutagenesis kit, site-directed mutagenesis was introduced into the putative transcription factor binding sequence site of Sema3A, and the RORα binding region, Sox-5 / 9 binding region and GABP binding region were deleted. A construct was made. Table 2 shows the primers used for site-directed mutagenesis.

2. Characterization of human Sema3A gene promoter (1) Sema3A promoter luciferase assay using NHEK Normal human epidermal keratinocytes (NHEK) were cultured according to the protocol of Lonza. NHEKs were cultured in 12-well tissue culture plates and transfected with 1 μg of each construct using X-treme GENE HD DNA transfection reagent (Roche Diagnostics, Basel, Switzerland). To correct for transfection efficiency, all cells were co-transfected with the pRK-TK vector (Promega) containing the Renilla luciferase gene under the control of the HSV-TK promoter. After transfection, add 250 μl of Passive Lysis Buffer to NHEK in the growth phase (80% confluence) or after differentiation (adding 1.4 mM calcium after confluence and continuing the culture for 4 days) to lyse the cells. It was. A dual-luciferase reporter assay system (Promega Co., Madison, WI, USA) was used for the measurement. 100 μl of Luciferase Assay Reagent II was added to a predetermined 96-well plate, 20 μl of sample was added thereto, and measurement was performed using a Wallac 1420 ARVOsxMultilabel counter (Perkin Elmer). Thereafter, 100 μl of Stop & Glo reagent was further added and measurement was performed in the same manner.

(2) Inhibition of transcription factor expression by siRNA Transcription factor RORα, Jun, Fos, Sox4, STAT3, NF-κB1, GABPα, Sox9, Rel (c-Rel), CREB, 40 nM of siRNA (Thermo Scientific) against Pbx1, ER, JunB, JunD, Fra1, and Fra2 was transfected using LipofectamineRNAi Max (Invitrogen, Carisbad, CA), respectively. The cells were cultured in antibiotic-free medium for 48 hours, after which total RNA was extracted and analyzed by real-time PCR. The sequences of each siRNA used (each using a mixture of four siRNAs) are shown in Table 3 below.

The transcription level of transcription factors was analyzed by quantitative real-time PCR. Total RNA was extracted from cultured cells using RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions and reverse transcribed into cDNA using PrimeScript RT reagent kit (Takara, Shiga, Japan). Quantitative real-time PCR was performed by Applied Biosystems 7900HT Fast Real-time PCR System (Applied Biosystems) using SYBR Premix Ex Taq (Takara) according to the manufacturer's instructions. Information on the primers used is shown in Table 4. Ribosomal protein S18 (RPS18) was used as a reference gene. The amount of mRNA was normalized to the amount of RPS18 mRNA and was finally expressed as a ratio to the untreated control mRNA.

(3) Electrophoretic mobility shift analysis (EMSA)
A double-stranded oligonucleotide probe was prepared by annealing a single-stranded biotinylated oligonucleotide and a complementary single-stranded unlabeled oligonucleotide. Table 5 shows the sequences (sense strand sequences) used for the probes. Nuclear protein extraction and EMSA were performed using NE-PER Nuclear Cytoplasmic Extraction Reagents and LightShift Chemiluminescent EMSA kit (Thermo Scientific). Nuclear protein extract (5 μg) was added to 1 × binding buffer and 50 ng / μl poly (dI · dC), 50 mM KCl, 5 mM EDTA, 2.5% glycerol, 0.05% NP-40, and −122 / of the Sema3A promoter region Incubated for 20 minutes at room temperature with biotinylated probes (50 pmol / μl) corresponding to the -93, -77 / -46, -47 / -18 regions. For competition assays, a 400-fold excess of unlabeled probe was added to the binding reaction prior to the addition of biotinylated probe. These incubation mixtures were then electrophoresed in a 6% polyacrylamide gel with 0.5 × TBE buffer and transferred to a Biodyne B nylon membrane (Pall, Port Washington, NY). Bands were visualized using the chemiluminescence detection reagent in the EMSA kit.

(4) Prediction of transcription factor binding sites A search using the P-Match1.0 Public program revealed a number of putative transcription factor binding sites within the 5'-flanking region of human Sema3A (Figs. 1 and 2). ). In particular, in the -134 / -1 region close to the position of the transcription start site, there was a sequence that closely matched the consensus sequence recognized by RORα / ER / AP-1, Sox-4 / 5/9, GABP. Therefore, it was suggested that these transcription factors are involved in the regulation of Sema3A promoter activity. In order to more precisely determine the promoter region of Sema3A, analysis with deletions was performed (FIG. 3).

The highest level of luciferase activity was detected in differentiated NHEK transfected with pGL3-134 (including -134 / -1 region within Sema3A promoter region). However, the relative luciferase activity of the deletion plasmid remained until the deletion proceeded to pGL3-24. Among the constructs, plasmids containing the pGL3-1444 / -1, -1244 / -1, -1044 / -1 fragment showed significantly lower activity in NHEK, and showed upstream suppressor activity in the -1444 / -844 region. Existence was suggested. These results demonstrated that the -134 / -24 region contains a minimal promoter for Sema3A gene transcription, so its nucleotide sequence is shown in Figure 4-1. Several transcription factor binding sites such as RORα / ER / AP-1, Sox-4 / 5/9, and GABP were present in this region. In addition, the sequence of the transcription repressing region (-1444 / -975) and the transcription factor binding site that suggested the presence of upstream suppressor activity are shown in FIG.

(5) Identification of cis element involved in Sema3A gene regulation Next, it was analyzed whether or not these transcription factors can actually bind to each of the putative binding sites. For this purpose, electrophoretic mobility shift assay (EMSA) was performed on the nucleoprotein extract of cultured NHEK and biotin corresponding to the -122 / -93, -77 / -46, and -47 / -18 regions of the Sema3A promoter region. This was carried out using a double-stranded oligonucleotide probe. As shown in FIG. 5, -122 / -93 containing the RORα / ER / AP-1 binding site, -77 / -46 containing the Sox 5/9 binding site, -47 /-containing the GABP binding site. A transcription factor was bound to the target sequence of each of the 18 probes, and a shifted band was detected. These results indicate that these binding sites in the -122 / -18 bp region are essential as cis elements for Sema3A transcription.

In order to identify transcription factor binding sequences important for Sema3A expression, each transcription factor binding sequence was deleted in a site-specific manner, and then a promoter assay was performed using NHEK in the growth phase. FIG. 6 shows the results of luciferase assay of each mutant in normal human epidermal keratinocytes (NHEK). Mutants lacking the -108 / -98 region where the RORα binding sequence was present showed marked suppression of luciferase activity, and it was revealed that transcription factors that upregulate Sema3A expression bind to this region.

Furthermore, RORα, Jun, Fos, Sox4, STAT3, NF-κB, GABPα, Sox9, Rel (c-Rel), CREB, Pbx1, ER, JunB, JunD, Fra1, and Fra2 are involved in Sema3A expression. To confirm, transcription factor expression was suppressed using small interfering RNA (siRNA), and gene expression of each transcription factor and Sema3A was analyzed using real-time PCR.

Results of knockdown of RORα, Jun, Fos, Sox4, STAT3, and NF-κB1 are shown in FIG. In NHEK transfected with siRNA against RORα, Jun, Fos, Sox4 and STAT3 transcription factors, Sema3A expression was significantly suppressed. On the other hand, in NHEK transfected with NF-κB1 siRNA, Sema3A expression was significantly enhanced (about 8 times). These results strongly suggest that RORα, Jun, Fos, Sox4, and STAT3 are indispensable for Sema3A expression, and that NF-κB1 is a suppressor of Sema3A expression.

Figure 8 shows the results of knockdown of GABPα, Sox9, Rel, CREB, Pbx1, ER, JunB, JunD, Fra1, and Fra2. Sema3A expression was significantly suppressed (*, p <0.05; ***, p <0.001) by suppressing the expression of GABPα, SSox9, CREB, JunB, JunD, and Fra2. In particular, Sema3A expression was significantly suppressed when CREB and JunB expression was suppressed.

(6) Effects of RORα agonists and antagonists on Sema3A expression in NHEK Based on the results of promoter assay of siRNA and site-directed mutagenesis construct, retinoid-related orphan receptor α (RORα) is important for up-regulation of Sema3A expression Was found to be one of the key transcription factors. Therefore, after adding RORα agonist or antagonist to NHEK, total RNA was collected after a certain period of time (48 hours for SR1078 and SR1001, 96 hours for cholesterol sulfate), and Sema3A expression was analyzed by quantitative real-time PCR did.

The results are shown in FIG. A is the result of the RORα agonist SR1078, B is the result of the RORα agonist cholesterol sulfate, and C is the result of the RORα antagonist SR1001. Sema3A expression was significantly enhanced in NHEK supplemented with RORα agonist SR1078 and cholesterol sulfate in a concentration-dependent manner. On the other hand, when RORα antagonist SR1001 was added, Sema3A expression was significantly suppressed in a concentration-dependent manner. Since any compound has a molecular weight of 500 Da or less that easily penetrates into the epidermal stratum corneum, it may be applicable as a topical drug.

(7) Effects of RORα agonists on lesions of atopic dermatitis model NC / Nga mice An experiment was conducted in which cholesterol sulfate sulfate, a RORα agonist, was externally applied to lesions of atopic dermatitis (AD) model NC / Nga mice. . Biosta AD ointment containing mite antigen on the shaved back of NC / Nga mice with AD predisposition was applied twice a week for 3 weeks to develop AD. Mice that developed dermatitis were extracted and classified into 2 groups (4 animals each) of (i) base dimethyl sulfoxide only, (ii) 1 mM cholesterol sulfate, and then the base or cholesterol sulfate solution was applied to the affected area of the mice. It was applied for 9 consecutive days. After the external application was completed, the skin was removed, immersed in a 0.02% trypsin / EDTA solution overnight, the epidermis was peeled off, and RNA was extracted. The expression level of Sema3A in the epidermis by quantitative real-time PCR is shown in FIG. Sema3A expression was significantly (**, p <0.01) promoted in the group externally applied with 1 mM cholesterol sulfate compared with the group externally applied with the base.

Claims

NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, RORα, ER, AP-1, Sox-4, Sox- 5. Contains at least one substance that regulates any transcription factor selected from Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2 as an active ingredient A semaphorin 3A expression regulator.
Semaphorin 3A expression regulation is enhanced expression of semaphorin 3A, the active ingredient is NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2 And a substance that inhibits any transcription factor selected from Fra1, and RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, The agent according to claim 1, which is at least one substance selected from the group consisting of a substance that promotes any transcription factor selected from GABPα, c-Rel, CREB, JunB, JunD, and Fra2.
The agent according to claim 2, which is a therapeutic or prophylactic agent for a disease or condition for which enhanced expression of semaphorin 3A is desired.
The agent according to claim 3, wherein the disease or condition is pruritus in skin diseases, allergic rhinitis, or osteoporosis.
The agent according to claim 4, wherein the disease or condition is pruritus in skin diseases.
6. The agent according to claim 5, which is an agent for treating or preventing pruritus in skin diseases, comprising as an active ingredient at least one substance that promotes RORα.
The agent according to claim 6, wherein the at least one substance that promotes RORα is at least one selected from SR1078 and cholesterol sulfate.
Semaphorin 3A expression regulation is semaphorin 3A expression suppression, the active ingredient is NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2 And a substance that promotes any transcription factor selected from Fra1, and RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, The agent according to claim 1, which is at least one substance selected from the group consisting of a substance that suppresses any transcription factor selected from GABPα, c-Rel, CREB, JunB, JunD, and Fra2.
The agent according to claim 8, which is a therapeutic or prophylactic agent for a disease or condition for which suppression of semaphorin 3A expression is desired.
The agent according to claim 9, which is a nerve regeneration promoter.
NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, RORα, ER, AP-1, Sox-4, Sox- 5. screening a compound using as an index the regulation of at least one transcription factor selected from Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2. And a method for screening a compound that modulates the expression of semaphorin 3A.
Semaphorin 3A expression regulation is enhanced semaphorin 3A expression, selected from NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, and Fra1 Inhibition of at least one transcription factor, or RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, 12. The method according to claim 11, wherein the index is promotion of at least one transcription factor selected from CREB, JunB, JunD, and Fra2.
13. The method according to claim 12, which is a screening method for a therapeutic or prophylactic agent for a disease or condition for which enhanced expression of semaphorin 3A is desired.
14. The method of claim 13, wherein the disease or condition is pruritus in a skin disease.
Semaphorin 3A expression regulation is suppression of semaphorin 3A expression, selected from NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, and Fra1 At least one transcription factor promoted, or RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, 12. The method according to claim 11, wherein inhibition of at least one transcription factor selected from CREB, JunB, JunD, and Fra2 is used as an index.
The method according to claim 15, which is a screening method for a therapeutic or prophylactic agent for a disease or condition for which suppression of the expression of semaphorin 3A is desired.
The method according to claim 16, which is a screening method for a nerve regeneration promoter.
A cell comprising a nucleic acid construct comprising a semaphorin 3A promoter region and a reporter gene, and NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF -E2, RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2 A method for evaluating an action of regulating the expression of semaphorin 3A by the substance, which comprises contacting a substance that regulates the transcription factor and measuring the expression level of the reporter gene.
The method according to claim 18, wherein the semaphorin 3A promoter region comprises any one of the following base sequences (1) to (3):
(1) The base sequence shown in SEQ ID NO: 1.
(2) The base sequence shown in SEQ ID NO: 2.
(3) A nucleotide sequence having 90% or more identity with (1) or (2).
A substance that inhibits any transcription factor selected from NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, and Fra1, Or selected from RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB, JunB, JunD, and Fra2. The method according to claim 18 or 19, wherein any one of the transcription factors that promotes the transcription factor is evaluated, and the action of enhancing the expression of semaphorin 3A by the substance is evaluated.
A substance that promotes any transcription factor selected from NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, and Fra1, Or selected from RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB, JunB, JunD, and Fra2. 20. The method according to claim 18 or 19, wherein the substance inhibits any of the transcription factors, and the action of suppressing the expression of semaphorin 3A by the substance is evaluated.
Cultured cells and NF-κB, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, RORα, ER, AP-1, Sox- 4. Contact with a substance that regulates a transcription factor selected from Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, JunB, JunD, Fra1, and Fra2, and semaphores in the cells A method for evaluating an action of regulating the expression of semaphorin 3A by the substance, comprising measuring the expression level of phosphorus 3A.
A substance that inhibits any transcription factor selected from NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, and Fra1, Or selected from RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB, JunB, JunD, and Fra2. 23. The method according to claim 22, wherein the substance enhances the expression of semaphorin 3A by the substance.
A substance that promotes any transcription factor selected from NF-κB, Nkx2.5, Brn-2, Elk-1, HNF-4α1, XBP-1, HLF, NF-E2, and Fra1, Or selected from RORα, ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABPα, c-Rel, CREB, JunB, JunD, and Fra2. 23. The method according to claim 22, wherein the substance inhibits any of the transcription factors, and the action of suppressing the expression of semaphorin 3A by the substance is evaluated.