KR100912355B1 - Composition for controlling melanogenesis containing Rho GDI-?, and method for screening melanogenesis modulator using Rho GDI-? - Google Patents

Abstract

Disclosed are a composition for inhibiting melanogenesis using a Rho GDI-α protein or a gene encoding the protein, and a method for screening a substance for inhibiting melanogenesis using a Rho GDI-α protein. Also disclosed are compositions that promote melanin production using siRNA genes that inhibit Rho GDI-α expression, and methods for screening substances that promote melanin production using siRNA genes. Using the present invention, it is possible to effectively promote or inhibit melanin production and to effectively screen for substances that promote or inhibit melanin production.

Rho GDI-α, MITF, Tyrosinase, Promotes Melanin Production, Inhibits Melanin Production, Melanin

Description

Composition for controlling melanogenesis containing Rho GDI-α, and method for screening melanogenesis modulator using Rho GDI-α}

The present invention provides a melanin production amount using a Rho GDP dissociation inhibitor-alpha (Rho GDI-α, ARHGDIA) protein, a gene encoding the protein, or a gene having a sequence complementary to the gene or a fragment thereof. It relates to a composition to be adjusted. The present invention also relates to a method for screening a substance that promotes or inhibits melanogenesis using a Rho GDI-α protein, or a gene encoding the protein, or a gene having a sequence complementary to the gene or a fragment thereof. .

There are two types of melanin, eumelanin, which is black in purple, and pheomelanin, which is yellow in red. Melanin is produced by melanocytes in the basal layer of the epidermis and metastasizes to the surrounding keratinocytes to give a skin color. The change in skin color is determined by various factors such as the change in the number of melanocytes, abnormal production or structure of melanocytes, melaninization of melanocytes, migration of keratinocytes, and loss of melanin. These are highly dependent on heredity, metabolism, endocrine, nutrition, inflammation, infections, tumors, physical and chemical factors. Inhibiting the production of such melanin has been the mainstream of whitening cosmetic research.

Conventional whitening cosmetic research has been largely made in three directions: inhibiting melanin production in melanocytes, controlling melanocyte stimulants, and promoting melanin excretion.

Inhibiting melanogenesis controls copper (Cu), an essential component of the active site of tyrosinase, an enzyme involved in melanogenesis, or tyrosinase related protein I (TRP-1). And the development of substances that inactivate enzyme activity or inhibit the production of melanin intermediates by controlling iron ions (Fe), an essential component of tyrosinase related protein II (TRP-2). A week has come. Kojic acid, arbutin, vitamin C derivatives, rucinol, and the like are known as representative whitening agents. In addition to these whitening ingredients, whitening cosmetics often contain a sunscreen as an auxiliary ingredient, and research on technology development on stability and transdermal absorption for maximizing the effects of functional ingredients contained in cosmetics is being conducted. In particular, retinol, vitamin C, arbutin, sphingolipid, etc., which are key ingredients, are very unstable in light, temperature, air, etc., and thus, research on raw material stabilization technology is actively conducted to overcome them.

Whitening cosmetics are products developed for the purpose of relieving blemishes and freckles caused by ultraviolet rays and suppressing the production of melanin pigment. Representative new materials have been developed such as mulberrin extract, kazinol F extract, and halves extract that inhibit melanin production by inhibiting the activity of tyrosinase, an enzyme catalyzing the oxidation of tyrosine. It is used in products as raw materials. The raw materials of the whitening cosmetics currently announced by the KFDA include mulberry extract, arbutin, ethyl ascorbyl ether, oil-soluble licorice extract.

There are more than a dozen factors that activate melanocytes, including endothelin-1, melanosite stimulating hormone (α-MSH), nitric oxide, histamine, pyridine dimers (pTpT), and the like. Currently, a number of ingredients that inhibit the activity of information transfer substances that induce melanocyte activation are found in plant extracts. In addition, placenta extract is widely used as a whitening cosmetic material by promoting tissue metabolism or promoting delamination of the stratum corneum. It has been reported that retinoic acid, which promotes the division of epidermal cells, is effective for senile plaques.

In the future, it is expected that materials that restore the proliferation and differentiation of keratinocytes or promote the exfoliation of the old stratum corneum and consequently promote metabolism will have the function of promoting melanin excretion.

As human genome research has recently reached completion, inhibitors or tyrosinase can control the synthesis of tyrosinase protein by controlling the promoter region of the tyrosinase gene, which plays the most important role in the skin whitening mechanism. The development of a whitening agent that utilizes the results of research at the gene level, such as an inhibitor that inactivates mRNA of the agent, is expected to proceed.

The purpose of one embodiment of the present invention is to regulate melanin production.

An object of another embodiment of the present invention is to inhibit melanin production.

Another object of one embodiment of the present invention, to promote the production of melanin.

It is an object of another embodiment of the present invention to screen for substances that modulate melanin production.

It is an object of another embodiment of the present invention to screen for substances that promote melanin production.

It is an object of another embodiment of the present invention to screen for substances that inhibit melanin production.

Melanin production control composition according to an embodiment of the present invention, Rho GDI-α protein, or a gene encoding the protein, or a gene having a sequence complementary to the gene or a fragment thereof as an active ingredient do.

Melanin production regulator screening method according to an embodiment of the present invention, a method for screening whether or not melanin production control for the test substance, to determine whether the test substance modulates the expression, activity or function of Rho GDI-α It is characterized by including the.

Using the present invention, it is possible to inhibit or promote melanin production and to screen for substances that inhibit or promote melanin production. Through this, a substance useful for inhibiting or promoting melanin production can be obtained.

Melanoblasts, which are derived from neural crests, migrate to the epidermis and differentiate into melanocytes, which are cells with dendrite, where melanin is formed. I will mention factors that are essential in this process. Factors that control the survival, reproduction, and differentiation of melanocytes include Wnt, fibroblast growth factor (FGF / FGF2), bone morphogenetic protein (BMP) group, hepatocyte growth factor / scatter factor (HGF / SF), obesity / stem cell factor (M / SCF), endoserine, melanotropin and the like. When the Wnt / beta catenin signaling pathway is activated first, KIT, ET3 / endothelin B and hepatocyte growth factor (HGF) / MET are activated. Wnt is a signaling protein involved in a variety of developmental processes, such as stem cell regulation, in the embryogenic stage.It activates the Frizzled receptor group and activates beta-catenin through the glycogen synthase kinase 3β (GSK3β) enzyme. -catenin). Phosphorylated glycogen synthase kinase 3beta enzyme will induce ubiquitionation and degradation of MITF. As beta-catenin accumulates and migrates into the nucleus, it binds to the LEF / TCF transcription factor, which in turn activates MITF gene expression. Melanin stimulating hormone (α-MSH) binds to the melanocortin-1 receptor (MC1-R) to activate adenylyl cyclase, and cAMP-dependent protienkinase PKA) phosphorylates CAMP response element binding protein (CREB), which in turn activates melanocyte-specific MITF promoters.

The three major enzymes of melanin synthesis, tyrosinase, tyrosinase-related protein I, and tyrosinase-related protein II, all have MITF DNA binding sites, and these promoter regions are combined with MITF DNA to synthesize these melanin. Genes of related enzymes are activated. Steel factor / C-kit receptors play an essential role in the development of early melanocyte precursors. C-kit is a tyrosine kinase belonging to platelet-derived growth factor (PDGF), which affects the growth and differentiation of embryonic melanoblasts. In other words, the steel factor on the surface of the cell wall binds to the KIT receptor to replace the phosphate group in tyrosine residues and converts tyrosine from inactive to active form, thereby inducing embryonic melanoblast Regulates growth and differentiation

During melanosite development, survival and reproduction are manifested by the binding of activated MET to hepatocyte growth factor (HGF) ligands. Loss of the MET gene in melanoblasts leads to problems in early neuronal migration. Overexpression of HGF or increased melanocyte distribution in the skin epidermis. Endothelin (endothelins) is a growth factor (growth factor) endoserine protein is divided into three ET1, ET2, ET3, there is an endothelin receptor (endothelin receptor) of EDNRA, EDNRB. ET3 binding to EDNRB is essential for the survival and migration of melanoblast, resulting in the loss of melanocytes in the event of mutation.

The melanocyte activators mentioned above activate the melanocyte-specific final target genes and eventually form melanin synthesis-related enzymes. The melanin-related enzyme genes produced in the nucleus are endoplasmic reticulum and golgi. Glycosylation takes place as it moves through, which takes the form of a completed protein and acts as an important factor in melanin synthesis. It migrates to melanosomes, which are organelles associated with the cell wall, to form melanin. Eventually, melanin formed in melanocytes moves to keratinocytes through melanosomes to determine the color of the skin epidermis. Factors involved in the movement of melanosomes include Rab 27a and Myosin Va. Three hypotheses have been suggested for the transfer of melanosomes to keratinocytes. Absorption to keratinocytes after release from melanocytes to cells, plasma membrane fusion of melanocytes and keratinocytes, and exocytosis of the melanocyte dendritic region to keratinocytes. .

Aplysia Ras-related homologs (ARHs) are commonly called Rho genes and belong to the Ras Superfamily gene family, which encodes small guanine nucleotide exchange (GTP / GDP) factors. ARH proteins remain inactive at GDP-attached state through interaction with the Rho GDI-α protein. The gene of Rho GDI-α (ARHGDIA) consists of six axons and is known to be expressed in various tissues and cells. Eukaryotic proteins or lipids require twice as much protein and lipids as the cells differentiate, and in addition, damaged proteins must be replaced on an ongoing basis. Therefore, the synthesized proteins move to intracellular organelles according to their signals and are used as constituent proteins of intracellular organelles or participate in maintenance of cell life by regulating activity as a regulatory protein. The synthesized protein is then transported to another final destination, the organelle, which is transported by vehicle. It is involved in protein transport by cycling the vehicle-binding and GDP-binding forms.

Small GTPases, made of single polypeptides, are also known as small GTP-binding proteins (small G proteins) by hydrolyzing GTP at low molecular weights of 20-40 KDa. These small GTPases have a variety of functions and are involved in gene expression, proliferation, chemotaxis, and cytoskeletal rearrangement. During cell signaling, small GTPases act as molecular switches, moving back and forth between inactivated GDP binding (mostly in the cytoplasm) and activated GTP binding (mainly in the cell membrane). Most bovine GTPases are activated by binding and post-translational modification of isoprenoids at the C-terminus. There are two types of isoprenoids: farnesyl and geranyl, Ras is activated by farnesylation, and Rho is activated by geranylation. Activation requires a guanine nucleotide exchange factor (GEF), which converts inactive GTPases into an activated state. Inactivation requires GTPase activating protein (GAP), which promotes GTP hydrolysis to convert GTPase into inactivated GDP bound state. In addition, there are GDP dissociation inhibitors (GDI) as GTPase function regulators, and their function research is being actively conducted.

Looking at the types and functions of Rho GTPase, so far, 20 kinds of Rho are known in the human body. Rho has several functions, including actin cytoskeleton assembly, smooth muscle (SMC) contraction, cell-to-cell adhesion, cell movement and migration, gene transcription, and enzyme activation. Rho can be divided into three categories, Rac, Rho, and Cdc42, each of which affects each other's function. RhoA, B, C, Rac 1, 2, 3, Cdc42, RhoD, Rnd1, Rnd2, PhoE / Rnd3, RhoG, TC10 and the like are known. In actin cytoskeletal assembly, Rho forms stress fibers and focal adhesions, Rac forms lamellipodia around the cells, and Cdc42 forms microspikes or phyllophodia filopodia). Rho is caused by lysophatidic acid (LPA), Rac is PDGF, insulin, Cdc42 is Bradykinin, IL-1, etc. Rho in the body is regulated by 85 activators (GEFs) and 70 inactivators (GAPs). GDIs are currently known in three types, Rho GDI-α, b and g. These regulators are not yet known for their exact function.

We have found that an increase in Rho GDI-α inhibits melanin synthesis and a decrease in Rho GDI-α increases melanin synthesis. Reduction of Rho GDI-α promotes melanin production by promoting the expression and activity of MITF and tyrosinase, and increase of Rho GDI-α inhibits the increase in expression and activity of MITF and tyrosinase. It can inhibit melanin production.

Melanin production control composition according to an embodiment of the present invention is a composition for inhibiting melanin production, the active ingredient may be a Rho GDI-α protein or a gene encoding the protein. Here, the gene encoding the Rho GDI-α protein may be in a free state or in a form inserted into a vector.

Melanin production control composition according to another embodiment of the present invention is a composition for promoting melanin production, the active ingredient may be a gene having a sequence complementary to the gene sequence encoding the Rho GDI-α protein or fragments thereof. In the case of fragments, the preferred length is 19-40 bp. More preferably, it is 19-30bp, More preferably, it is 19-25bp. The active ingredient may be siRNA (small interfering RNA) that binds to the mRNA of Rho GDI-α.

Incorporation of siRNA into cells may include microinjection (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate co-precipitation (Graham, FL et al., Virology, 52: 456 (1973)), electroporation Method (Neumann, E. et al., EMBO J., 1: 841 (1982)) or using liposomes (Wong, TK et al., Gene, 10:87 (1980)), etc. It is not limited to this.

If the composition according to one embodiment of the invention is a cosmetic formulation, it contains a cosmetically acceptable medium or base. These are all formulations suitable for topical application, for example emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase, and / or It may be provided in the form of a nonionic vesicle dispersant or in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. These compositions can be prepared according to conventional methods in the art.

When the composition according to one embodiment of the present invention is a pharmaceutical formulation, a solid, by adding a commercially available inorganic or organic carrier using a gene or protein encoding Rho GDI-α according to an embodiment of the present invention as an active ingredient It may be formulated as a parenteral administration (injection, transdermal or external application) in semisolid or liquid form. Formulations for parenteral administration include topical injection preparations, ointments, lotions, sprays, suspensions and the like.

In order to formulate the active ingredient of the composition according to an embodiment of the present invention, it can be easily formulated according to a conventional method. Surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspensions, and other commonly used Adjuvants may be used as appropriate.

Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remingon's Pharmaceutical Sciences (19 ^th ed., 1995).

The dose of Rho GDI-α and its genes according to an embodiment of the present invention may be determined by the age, sex, and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration, and the judgment of the prescriber. Will depend on. Dose determination based on these factors is within the level of those skilled in the art, and can be applied at the level of the person skilled in the art by external application to the site where melanin inhibition occurs for the formulations except injections, and in the case of injections, melanin suppressed skin It can be injected locally. Generally dosages range from 0.001 ng / kg / day to approximately 2000 mg / kg / day. More preferred dosages are 1 μg / kg / day to 1 mg / kg / day for proteins and 50 ng / kg / day to 200 ng / kg / day for genes.

 Rho GDI-α protein, which is an active ingredient of the composition according to an embodiment of the present invention, may be substituted, deleted, and / or added with an amino acid residue of a natural amino acid sequence as long as it can inhibit melanin production or promote melanin production. It may be in various modified forms, and may also be in a form in which modifications such as phosphorylation, saccharification, methylation, and pansylation have occurred.

The gene of the present invention and fragments thereof may be composed of modified nucleotides. Nucleotides, including, for example, 2'-substituted ribose, such as 2'-0-alkyl (e.g. methyl, ethyl), 2'-0-allyl, 2'-0-allyl; have. It may also comprise partially or wholly nucleotides with substituted bases.

In one embodiment of the present invention, various methods may be used to administer a gene encoding Rho GDI-α or a similar protein or siRNA that inhibits the expression of Rho GDI-α. Viral vectors or non-viral vectors may be used to administer genes. Viral vectors include retroviruses, adenoviruses, adeno-associated viruses, and the like, and non-viral vectors may include micelles or liposomes. Structure and the like, and DNA may be administered by using electroporation or a gene gun without a vector. In particular, liposomes may be administered by carrying a gene encoding Rho GDI-α or a similar protein thereof.

In the melanin production regulator screening method according to an embodiment of the present invention, whether to regulate the expression of Rho GDI-α using RT-PCR or ELISA and Western blot (immuno blot) which is well known in the art Can be determined.

 In the melanin production regulator screening method according to an embodiment of the present invention, as a result of the confirmation, when the test substance promotes the expression, activity or function of Rho GDI-α may include determining as a melanin production inhibitor.

In the melanin production regulator screening method according to another embodiment of the present invention, when the test result inhibits the expression, activity or function of Rho GDI-α may include determining as a melanin production promoter. .

Melanin production regulator screening method according to another embodiment of the present invention, the method comprising the steps of transfecting a cell plasmid comprising a promoter and reporter gene of Rho GDI-α; Treating the cell with a test substance; And confirming whether the test substance promotes or inhibits expression of the reporter gene.

Screening of melanogenesis inhibitors or promoters using the expression level of Rho GDI-α can be confirmed by immunoassay (e.g., ELISA or Immunoblot) to determine whether the test substance promotes or inhibits the expression of Rho GDI-α protein. The method may be performed by transfecting a plasmid containing a reporter gene including a promoter into a cell and treating the cell with a test substance to determine whether the test substance promotes or inhibits expression of the reporter gene. Can be.

The reporter gene assay for screening the melanin inhibitor or promoter uses a reporter gene vector prepared by a general molecular biology technique. As the reporter gene, a firefly luciferase gene or a Gaussian luciferase gene (GLuc) may be used. Promoters of Rho GDI-α can be readily designed and obtained by those skilled in the art using gene IDs from the US NCBI database. After transfection of the prepared plasmid to primary cultured melanocytes or immortalized melanocytes or melanoma cells, the melanogenesis inhibitor or promoter candidate drug was treated and the luciferase activity was measured by a luminometer (Luminometer). ), The effect of the inhibitor or promoter candidate drug on the expression of Rho GDI-α can be confirmed.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited only to these examples.

Example 1 Inhibition and Production Confirmation of Melanin Production by Rho GDI-α Expression Change

In this experiment, RT-PCR was performed by culturing Melan-a melanocytes (DCBettett, St. George's hospital medical school, USA) derived from C57 / BL mice. Cells were plated in a RPMI-1640 medium containing 10% fetal bovine serum with 1 × 10 ⁵ cells per plate hole with 6 holes. The cells were allowed to adhere to the wall for 24 hours, and then experimental samples of each experimental group were administered.

We investigated whether Rho GDI-α could actually promote or reduce melanogensis. First, we examine whether the activity of this gene changes in relation to the microphthalmia-associated transcription factor (MITF) and tyrosinase (MITF), which are known to be important in the early stages of melanin synthesis. The amount of produced was also measured.

Concentrations of 0.5 and 1 μg / ml of the pCMV-SPORT6 vector (invitrogen, USA) in which the Rho GDI-α gene was inserted into the 6-hole cell culture plate were injected into cells using Fugene 6 HD (Roche, USA). The control group introduced a vector without the Rho GDI-α gene into pCMV-SPORT6. After injection, mRNAs of MITF and tyrosinase were identified by RT-PCR for 12, 24 and 48 hours, respectively. The annealing temperatures of MITF and tyrosinase were 58 degrees and 60 degrees, respectively, and the number of cycles was 27 and 24, respectively.

As a result, it can be seen that transiently expressing Rho GDI-α decreases the amount of mRNA of MITF and tyrosinase. This indirectly proves that melanin synthesis is inhibited (FIG. 1A).

In addition, the siRNA of Rho GDI-α (invirogen, USA) 20nmol / ml using Fugene 6 HD (Roche, USA) reduced the expression of Rho GDI-α mRNA. At this time, the control group introduced Rho GDI-α Scrambled siRNA (invitrogen, USA). As a result, the amount of mRNA of MITF and tyrosinase can be observed to increase. This indirectly proves that melanin synthesis is increased (FIG. 1 b).

On the other hand, the effect of Rho GDI-α on the expression and activity of tyrosinase, an important protein related to melanin synthesis, was also tested. Rho GDI-α was transiently expressed using 1 μg / ml of the pCMV-SPORT6 vector (invitrogen, USA). Using the cells thus prepared, changes in the expression and activity of tyrosinase and total melanin were measured. Tyrosinase expression was measured using Immuno Blotting. Activity inhibition was measured using Dopa staining. Total melanin was dissolved in 1N NaOH and absorbance was measured at 405 nm.

After washing once with cold PBS in melan-a cells transiently expressing the Rho GDI-α obtained in the above step and melan-a cells treated with the siRNA of Rho GDI-α, the cells were lysed and partially Immunoblot was performed to confirm the protein expression of tyrosinase, and some of them were loaded on Tris-Glycine gel and subjected to Dopa staining to confirm the activity of tyrosinase. Dopa staining was confirmed by incubation with 0.5M phosphate buffer and 0.1% Dopa / 80mM phosphate solution and drying the gel.

In addition, the cells were lysed and the remaining pellets were dissolved in 1N NaOH to measure absorbance at 405 nm to determine total melanin relative to total protein. The results are shown in FIG.

As shown in Figure 2, increasing the amount of Rho GDI-α, such as MITF can be observed to reduce both the expression of tyrosinase (a in Figure 2) and activity (b in Figure 2). This indirectly proves that melanin synthesis is inhibited.

In addition, by reducing the expression of Rho GDI-α mRNA using siRNA (invirogen, USA) 10 and 20 nmol / ml of Rho GDI-α, the protein expression of tyrosinase (FIG. 2A) and activity (FIG. 2B) were increased. Can be observed. This indirectly proves that melanin synthesis is increasing.

To determine the melanin synthesis inhibition and increase directly, the total amount of melanin was measured (FIG. 2 c). Compared with the control group, the treatment of Rho GDI-α clone reduced the melanin production by about 30%, and the treatment of Rho GDI-α siRNA (20nmol / ml) increased the melanin production by about 90%. Can be observed.

Example 2 Effect of Rho GDI-α on MITF Promoter Activity

In this experiment, the MITF activity against Rho GDI-α was measured using GLuc analysis using a melan-a cell system containing a MITF promoter. The amount of melanin was also quantified using the cells used at this time.

Specifically, a small amount of medium was removed from the cell culture dish and transferred to the measurement plate, and 1 × GLuc assay working solution (NEB) was added to the medium in a ratio of 4: 1, and the amount of light generated at 470 nM using a luminometer. Was measured. After ⁵ × 10 ⁴ cells were plated using a 24-hole plate, siRNA of Rho GDI-α was treated with 20 nmol / ml using Fugene 6 HD (Roche, USA). The pCMV-SPORT6 vector with Rho GDI-α (invitrogen, USA) was treated with Fugene 6 HD (Roche, USA) with 0.5 mg / ml with IBMX 100 μM. IBMX 100 μM was used as a positive control. After material treatment, GLuc analysis was performed at 12 hours, 24 hours, and 48 hours of culture. The analysis results are shown in a of FIG. 3.

After adding 1N NaOH to dissolve the cells, the absorbance at OD 405 nm was measured to determine the amount of melanin. The melanin amount measurement result is shown in b of FIG.

As can be seen from the results of Figures 3a and 3b, mRNA and protein reduction of Rho GDI-α by siRNA increases the MITF promoter activity by about 100-200% and increases melanin production by about 50% or more. Thus, it is shown that the reduction of Rho GDI-α has the effect of increasing melanin synthesis.

In addition, the increase in the mRNA and protein of Rho GDI-α decreases the MITF promoter activity by IBMX by about 80% and melanin production by about 40% or more, thereby increasing the Rho GDI-α reduces melanin synthesis. Shows.

Example 3 Screening of Melanin Inhibitors Induced by Reduction of Rho GDI-α Expression

In this experiment, Melan-a cells were screened using RT-PCR to reduce the expression of increased MITF and tyrosinase under conditions in which Rho GDI-α expression was reduced. In other words, it screens for substances that inhibit melanin production.

Rho GDI-α siRNA (invirogen, USA) 20nmol / ml reduced the expression of Rho GDI-α mRNA using Fugene 6 HD (Roche, USA) and increased the expression, activity and total tyrosinase expression Substances that reduce the amount of melanin were screened. Tyrosinase expression was measured by Immuno Blotting, activity inhibition was measured by Dopa staining, total melanin was dissolved in 1N NaOH, and absorbance was measured at 405 nm.

In detail, 20 nmol / ml of siRNA of Rho GDI-α gene (invitrogen, USA) was introduced into cells using Fugene 6 HD (Roche, USA) in a 6-hole cell culture plate. The control group introduced the same amount of Rho GDI-α Scrambled siRNA (invitrogen, USA). After injection, mRNAs of MITF and tyrosinase were identified by RT-PCR for 12, 24 and 48 hours, respectively. The annealing temperatures of MITF and tyrosinase are 58 degrees and 60 degrees, respectively, and the number of cycles is 27 and 24, respectively.

After washing with cold PBS in the melan-a cells treated with the siRNA obtained in the above step, the cells were lysed and the remaining pellets were dissolved in 1N NaOH to measure the absorbance at 405 nm to determine the total melanin amount relative to the total protein. The results are shown in FIG.

As a result, the expression of MITF and tyrosinase increased due to the reduction of Rho GDI-α was increased by ISAO-03 (IUPAC name: 3- (3,4-dihydroxyphenyl) -N-adamantyl having the structure of Formula 1 below. -propanamide, INCFA name registered in CTFA: adamntyl dihydrocaffeamide) was confirmed to be inhibited. This is shown in a of FIG. 4. This indicates that increased melanin synthesis is inhibited by ISAO-03 with a decrease in Rho GDI-α.

On the other hand, the results of measuring the total melanin amount is shown in b of FIG. Also through b of Figure 4 it was confirmed that the melanin synthesis amount was also reduced by ISAO-03.

In conclusion, we could screen ISAO-03 as a substance that inhibits melanin production.

Example 4

The experiment was carried out in the same manner as in Example 3 except that the concentration of ISAO-03 was changed to 1, 2, 4 μM and the treatment time was changed to 24, 48, 72 hours.

The results are shown in FIG. FIG. 5A shows the results of ISAO-03 reducing the amount of melanin by about 40% concentration, and FIG. 5B shows the time-dependent results from 24 hours to 72 hours with two ISAO-03 concentrations (2,4μM). Could see. These two results indicate that ISAO-03 obtained by the screening of Example 3 (Fig. 4A) effectively inhibits melanin production, and as a result, ISAO-03 effectively melanin concentration and time-dependently It can be seen that the production inhibitory effect.

Example 5 Inhibition of Melanin Production by ISAO-03 in Guinea Pigs

Brown guinea pigs weighing around 500 g were removed from the back hairs, anesthetized with pentobarbital (30 mg / kg), and burned with 500 mJ ultraviolet light. The burned area was circled about 1 cm in diameter. In the same way, the same site was irradiated with ultraviolet rays three times a week. After the last UV irradiation, each of the samples was treated with the artificial pigment plate from the next day to morning and evening.

Specifically, it was divided into a vehicle-treated control group, ISAO-03 0.1%, 0.5% treated experimental group, nothing treated untreated group and hydroquinone (Hydroquinone) 2% compared to the treated group. Each sample was applied for 5 weeks at 5 μl. 3 weeks after the application of the sample, the color of the artificial pigment plate of the control group treated with the vehicle only, the experimental group treated with ISAO-03, and the comparative group treated with hydroquinone was visually confirmed based on the untreated group and the relative color difference was observed. Was quantified. Six animals were tested simultaneously and each value was averaged and tabulated.

The results are shown in Figure 6a. In Figure 6a, 0.5 is a whitening effect that only an expert can know, 1.0 is a whitening effect that anyone can know, 3.0 is a whitening effect as white as the original skin color, 2.0 is a whitening effect between 1 and 3, 4.0 is the original skin color Whitening effect is shown.

As shown in FIG. 6A, the whitening effect of ISAO-03 0.5% was 2.1, and the pigmentation of the artificial pigmented plate by UV was reliably improved after 3 weeks. Even when 0.1% was applied, the whitening effect was superior to hydroquinone.

In addition, the color of the untreated group, the artificial pigment plate (control group) treated with the vehicle only, the artificial pigment plate treated with ISAO-03, and the artificial pigment plate treated with the hydroquinone were contrasted with a color difference meter (chromator). The average value of the numerical value (L value) obtained by quantifying the contrast of the artificial color plate was measured.

The results are shown in Figure 6b. In FIG. 6B, the larger the numerical value (L value), the brighter the color. ISAO-03 0.1% and 0.5% showed higher values than hydroquinone, indicating an excellent whitening effect.

Example 6 Effect on Normal Human Melanosite

To investigate the effects of Rho GDI-α on human melanocytes, we examined the effects of melanin synthesis on normal human melanocytes. To assess this, we measured tyrosinase activity, which plays an important role in the final stage of melanin synthesis. This was done by examining the tyrosinase activity on in situ using DOPA staining. In a 6-hole medium, normal human melanocytes (MTCC, South Korea), 1X10 ⁵ pieces were laid, and the siRNA of Rho GDI-α (invirogen, USA) was treated with 20 nmol / ml using Fugene 6 HD (Roche, USA), The pCMV-SPORT6 vector incorporating Rho GDI-α (invitrogen, USA) was treated with Fugene 6 HD (Roche, USA) with 0.5 mg / ml with IBMX 100 μM. IBMX 100 μM was used as a positive control. On day 3, the cells were washed with PBS and sonicated using 0.05M sodium phosphate buffer (pH6.8) containing PMSF and 1.0% Triton X-100. And 30 minutes at 4 ℃ to separate the tyrosinase from the melanosome membrane. Then, after centrifugation at 40000g for 20 minutes, only the supernatant was collected to obtain protein. 10 ug of protein is mixed with SDS sample buffer and subjected to electrophoresis using Tris-Glycine gel. After the end of electrophoresis the gels were incubated for 15 minutes in 0.5 M sodium phosphate buffer (pH 6.5). Thereafter, the gel was added to 0.1 M sodium phosphate buffer (pH6.8) containing 0.2% L-DOPA and reacted at 37 ° C. When the band appeared the reaction stopped. This was quantified and shown in FIG. 7 graphically. As shown in FIG. 7, the tyrosinase activity-promoting effect was shown upon reduction of Rho GDI-α, and the tyrosinase activity-promoting inhibition was confirmed when the increase was increased.

The present invention can obtain a whitening effect by inhibiting the production of melanin and can be usefully used to prevent or treat blotch, blemishes, freckles and the like. In addition, by promoting the production of melanin may be useful for preventing or treating vitiligo and vitiligo.

1 is a RT-PCR result showing the change in mRNA of genes involved in melanin synthesis according to the change in the expression level of Rho GDI-α (increase: a, decrease: b).

Figure 2 is a result showing the change in melanin synthesis (c) according to the expression of Rho GDI-α (a) and the change in activity (a) and tyrosinase protein expression of the gene associated with it (b).

Figure 3 is a graph showing the MITF activity (a) and the amount of melanin (b) quantified from the cells for Rho GDI-α measured by GLuc analysis method using a melan-a cell system containing a MITF promoter.

Figure 4 is an example of screening a substance that inhibits melanin production, ISAO-03 result of reducing the expression level of genes related to increased melanin synthesis due to inhibition of Rho GDI-α expression (a) and ISAO-03 This increased result of melanin was again reduced (b).

5 is a result of reducing melanin synthesis in melanocytes produced by melanin synthesis inhibitor ISAO-03 obtained by the above screening concentration (Fig. 5a), hourly (Fig. 5b).

Figure 6 shows the results of animal experiments on the whitening effect of ISAO-03, a candidate melanin synthesis inhibitor obtained through the above screening. In the guinea pig animal model, whitening was observed visually and quantified (FIG. 6A) and the graph showing L value of the color system (FIG. 6B).

7 is a result of measuring tyrosinase activity in situ (insitu) to confirm the melanin synthesis or inhibitory effect of Rho GDI-α in human melanocytes.

Claims (12)

Rho GDI-α (Rho Guanine nucleotide Dissociation Inhibitor-α) protein, or melanin containing a gene encoding the protein, or a gene having a sequence complementary to the gene or a fragment thereof as an active ingredient Cosmetic composition for production control. The method of claim 1, The composition for controlling melanin production is a composition for inhibiting melanin production, The active ingredient is a Rho GDI-α protein or a cosmetic composition for controlling melanin production, characterized in that the gene encoding the protein. The method of claim 2, wherein the composition for controlling melanin production is a cosmetic composition for controlling melanin production, characterized in that to suppress melanin production by inhibiting MITF (Microphthalmia-associated transcription factor) and tyrosinase expression. The method of claim 2, The gene encoding the Rho GDI-α protein is a cosmetic composition for controlling melanin production, characterized in that the form is inserted into the vector. The method of claim 1, The composition for controlling melanogenesis is a composition for promoting melanin production, The active ingredient is a cosmetic composition for controlling melanin production, characterized in that the gene having a sequence complementary to the gene sequence encoding Rho GDI-α protein or fragments thereof. According to claim 5, The melanin production control composition, Melanin production control cosmetic composition, characterized in that to promote melanin production by promoting MITF and tyrosinase expression. The method of claim 5, wherein the active ingredient melanin production control cosmetic composition, characterized in that siRNA that binds to the mRNA of Rho GDI-α. As a method for screening whether or not melanin production is controlled for a test substance, Method of screening melanin production regulator comprising determining whether the test substance modulates the expression, activity or function of Rho Guanine nucleotide Dissociation Inhibitor-α (Rho GDP dissociation inhibitor-alpha). The method of claim 8, wherein whether to regulate the expression of Rho GDI-α is determined using RT-PCR or ELISA and Western blot (immuno blot). The method according to claim 8, wherein the identification result, when the test substance promotes the expression, activity or function of Rho GDI-α melanin production inhibitor screening method comprising the step of determining as a melanogenesis inhibitor. The method of claim 8, wherein the test substance comprises a melanin production promoter when the test substance inhibits the expression, activity, or function of Rho GDI-α. The method of claim 8, The melanin production regulator screening method, Transfecting the cells with a plasmid comprising a promoter and a reporter gene of Rho GDI-α; Treating the cell with a test substance; And Melanin production regulator screening method comprising the step of identifying whether the test substance promotes or inhibits the expression of the reporter gene.

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