KR20190006253A - Composition for Suppressing Cutaneous Pigmentation Containing Rab5A Inhibitors - Google Patents

Abstract

The present invention relates to a composition for suppressing skin pigmentation containing a Rab5a inhibitor as an active ingredient, wherein the composition can inhibit the expression of tyrosinase, reduce migration and predation of melanosome, and thus can be usefully used for suppressing pigment formation.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for suppressing skin pigment formation containing Rab5A inhibitor as an active ingredient,

The present invention relates to a composition for inhibiting skin pigment formation containing Rab5A inhibitor as an active ingredient.

Melasma is one of the most common skin diseases in which pigments are excessively deposited. Skin pigmentation is caused by the process of melanosome-generated melanocytes migrating to keratinocytes around melanocytes and degradation of keratinocytes . This movement of melanomas is known to require mechanisms such as the formation of filophodia filled with melanosomes in the cell body and dendrite of melanocytes.

Rab protein is a protein that forms a family of guanosine triphosphatase (GTP) -binding proteins and is known to be located in a specific compartment within the cell. This protein is known to be involved in the regulation of membrane transport in cells. For example, Rab27A / Melanophilin / Myosin5a protein complexes transfer melanosomes to the peripheral actin network of melanocytes (Westbroek et al., 2008). Humans have about 60 Rab genes (Seabra et al., 2002), and some Rab proteins such as Rab7 and Rab27a are known to be involved in skin pigmentation (Bahadoran et al., 2001, etc.).

The present inventors completed the present invention by confirming that Rab5A (RAS-associated protein Rab5A) increases the expression of proteins involved in pigment formation such as tyrosinase and increases melanosome migration and predation, thereby contributing to skin pigment deposition.

1. US registered patent US 9,561,167 2. US Published Patent US 2006/0252036

It is an object of the present invention to provide a composition for inhibiting pigment formation comprising Rab5A (RAS-associated protein Rab5A) inhibitor as an active ingredient.

Another object of the present invention is to provide a method for producing melanocytes which comprises contacting melanocytes with a test substance; Culturing the melanocytes in contact with the test substance; And tyrosinase, kinesin, Rab7, Rab27A, early endosome antigen (EEA1), Trp-1 (tyrosinase-related protein-1) and RAC1 (ras-related C3 botulinum toxin substrate 1), Rho (Ras homolog gene family member A), CDC42 (Cell division control protein 42 homolog), and Dynein, to determine the validity of the test substance And a method for screening an inhibitor of pigment formation.

One aspect of the present invention provides a composition for inhibiting pigment formation comprising a Rab5A inhibitor as an active ingredient.

As used herein, the term " Rab5A (RAS-associated protein Rab5A) " is one of the guanosine triphosphatase (GTP) -binding proteins and refers to the cytoplasmic surface of the cell membrane, (early endosome) and is known to be distributed mainly.

As used herein, the term " Rab5A inhibitor " refers to a substance that inhibits mRNA production by inhibiting the transcription of Rab5A gene or inhibits translation of mRNA, a substance that promotes mRNA degradation, a substance that promotes activity of Rab5A protein And substances capable of promoting degradation of the protein.

According to one embodiment of the present invention, the expression of tyrosinase involved in melanin synthesis increases when the expression of Rab5A increases, so that the Rab5A inhibitor can be usefully used for inhibiting pigment formation.

As used herein, the term 'composition for inhibiting pigment formation' means a composition capable of inhibiting the expression of a gene involved in pigment formation or translation of mRNA, or controlling the activity and decomposition level of proteins involved in pigment formation .

According to one embodiment of the present invention, the pigment may be melanin, but is not limited thereto.

As used herein, the term " melanosome " is a membrane-like granule containing melanin and serves to transfer melanin from melanocytes to keratinocytes. Melanomas migrate from the melanocytes to the keratinocytes through the dendrites. When the keratinocyte is degraded, melanomas are degraded and the melanin migrates to the stratum corneum of the skin, causing pigmentation symptoms such as spots.

According to one embodiment of the present invention, the composition for inhibiting pigment formation inhibits the expression of kinesin, thereby inhibiting the movement of melanosomes and inhibiting the predation of melanosomes by keratinocytes.

The composition for inhibiting pigment formation of the present invention may be provided in various forms selected from a pharmaceutical composition or a functional cosmetic composition.

When the composition of the present invention is a pharmaceutical composition, suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, lubricants, flavors, antioxidants, buffers, , A diluent, a dispersant, a surfactant, a binder, and a lubricant.

When the composition of the present invention is a cosmetic composition, the composition of the cosmetic composition may be prepared in any form conventionally produced in the art, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, But are not limited to, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

Another aspect of the present invention is

(a) contacting the test substance with melanocytes;

(b) culturing the melanocytes in contact with the test substance; And

(c) tyrosinase, kinesin, Rab7, Rab27A, early endosome antigen 1 (EEA1), tyrosinase-related protein-1 (Trp-1), and ras- related C3 botulinum toxin substrate 1), Rho homolog gene family member A (RhoA), CDC42 (cell division control protein 42 homolog), and dynein to determine the effectiveness of the test substance A method for screening an inhibitor of pigment formation.

According to one embodiment of the present invention, the melanocyte of (a) may be a cell overexpressing Rab5A. The method of overexpressing Rab5A can be performed by a conventional method known in the art, for example, a method of transforming a vector containing a gene encoding Rab5A into a cell, but is not limited thereto.

According to an embodiment of the present invention, the step of judging whether the test substance of (c) is effective or not can be carried out by measuring the activity of tyrosinase, kinesin, Rab7, Rab27A, EEA1, Trp1, RAC1, RhoA and CDC42 in melanocytes If the level decreases or the level of dinine increases, the test substance may be judged to be effective as an inhibitor of pigment formation.

According to an embodiment of the present invention, measuring the 'level' means measuring the protein concentration, activity, and mRNA expression level. The concentration of the protein can be measured by a conventional method known in the art, for example, Western blot, ELISA (enzyme-linked immunospecific assay) or the like.

The composition according to one embodiment of the present invention can be usefully used for inhibiting pigment formation because it inhibits the expression of tyrosinase and reduces the migration and predation of melanosome.

FIG. 1 shows the results of measuring the activity of the miR-675, the pMIR-RAB5A vector, the pMIR-RAB5A mutant 1 vector, and the pMIR-RAB5A mutant 2 vector, or a combination thereof, on the melanocytes and measuring the activity of the mutant.
FIG. 2 is a graph showing the results of confirming the expression level of Rab5A protein after transfection of miR-675 analog or miR-675 inhibitor into keratinocytes and melanocytes.
Fig. 3 is a graph showing the results of measurement of mRNA levels of Rab5A, Rab7 and Rab27A in skin tissue samples of patients diagnosed with spots.
Fig. 4 shows results of double staining of normal pigmented skin tissue and hyperpigmented skin tissue from Rab5A and c-Kit, which were separated from patients diagnosed with spots.
FIG. 5 is a graph showing the results of confirming the expression of Rab5A and tyrosinase in keratinocytes and melanocytes overexpressing Rab5A.
FIG. 6 is a graph showing the results of confirming mRNA levels of Rab5A, Rab7 and Rab27A in exosomes isolated from cell culture of melanocyte-keratinocyte (Rab5A overexpression) cells.
FIG. 7 shows the result of confirming the level of Rab5A protein in exosome isolated from cell culture of melanocyte-keratinocyte (Rab5A overexpression).
FIG. 8 is a graph showing the results of confirming protein levels of Rab5A, tyrosinase and EEA1 (early endosome antigen 1) after treating normal melanocytes with exosomes isolated from cells overexpressing Rab5A.
FIG. 9 is a graph showing the results of confirming the expression of tyrosinase, tyrosinase-related protein-1, Trp-2, Rab7 and Rab27A after overexpression or knockdown of Rab5A in melanocytes.
FIG. 10 is a graph showing the result of immunoprecipitation using Rab5A antibody after knocking down Rab5A in melanocytes. FIG.
FIG. 11 shows the results of confirming the expression changes of tyrosinase, Trp-1, Trp-2, EEA1, Rab7 and Rab27A after transfection of melanocytes with WT Rab5A, Rab5A S34N and Rab5A Q79N, respectively.
FIG. 12 shows the result of confocal microscopy of the intracellular locations of Rab5A and Trp-1 after transfection of melanocytes with WT Rab5A, Rab5A S34N and Rab5A Q79N, respectively.
FIG. 13 is a graph showing the results of confirming the expression of kinesin and dinine after overexpression or knockdown of Rab5A in melanocytes.
FIG. 14 is a graph showing the results of confirming the expression of kinesin and tyrosinase by confocal microscopy after knocking down Rab5A in melanocytes. FIG.
FIG. 15 is a graph showing the results of confirming changes in expression of dinine and tyrosinase by confocal microscopy after knocking down Rab5A in melanocytes. FIG.
FIG. 16 is a graph showing the results of FACS analysis using TYRP-1 (Tyrosinase-related protein 1) and K14 antibody in keratinocytes and / or melanocytes knocked out of Rab5A.
FIG. 17 is a graph showing the results of Western blotting the protein levels of RAC1 (ras-related C3 botulinum toxin substrate 1), RhoA and CDC42 in melanocytes overexpressing Rab5A.
FIG. 18 is a graph showing the result of treating melanocytes with keratinocytes overexpressing Rab5A and then dyeing pigment globules. FIG.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Experimental Method

1. Skin tissue isolation and human epidermal cell culture

A part of the skin tissue of hyperpigmentation site and normal pigmentation area was separated from biopsy from seven female patients (age 44 to 65 years, mean age 51.2 years) diagnosed with spots.

An epidermal cell was prepared by separating adult skin tissue isolated during dental surgery into dermis and epidermis.

In the epidermal cells, keratinocytes are composed of bovine pituitary extract (hereinafter referred to as BPE), bovine insulin (hereinafter referred to as BI), hydrocortisone, human epidermal growth factor (Invitrogen) containing human epidermal growth factor (hereinafter referred to as hEGF) and bovine transferrin (hereinafter referred to as BT; Invitrogen, Carlsbad, Calif.).

The melanocytes may be selected from the group consisting of BPE, BI, fetal bovine serum (hereinafter referred to as FBS), hydrocortisone, BT, basic fibroblast growth factor (hereinafter referred to as bFGF) And medium 254 (Invitrogen) containing phorbol 12-myristate 13-acetate. The keratinocytes and melanocytes were used for experiments while subculturing 3-5 and 7-15 times, respectively.

Fibroblasts were cultured in DMEM (Invitrogen) medium containing 10% FBS (Invitrogen) by dripping dermal cells and used for experiments while subculturing from 5 to 10 times.

2. Rab5A  Overexpression overexpression ) And knockdown

The pCMV plasmid (pCMV-CDH11) containing the Rab5A gene was transfected into cells according to the manufacturer's protocol using Lipofectamine 2000 (Invitrogen) for Rab5A overexpression.

In addition, human Rab5A siRNA (50 nM or 100 nM) or a negative control (ON-TARGETplus SMARTpool or Non-targeting siRNA; Dharmacon) was transfected into cells according to the manufacturer's protocol using TransIT-siQUEST transfection reagent for Rab5A knockdown Respectively. After 24 hours, the cells were used for further experiments.

3. Real-time RT- PCR (Real-time RT- PCR ) analysis

Mature miRNA expression levels were measured according to the manufacturer's protocol using TaqMan MicroRNA Assays (Applied Biosystems, Foster, Calif.) Specific for the human miR-675 stem loop. The housekeeping gene for the normalization of the experimental results was U18 (Roche, Mannheim, Germany).

Relative expression levels of mRNA relative to GAPDH were measured by qPCR using the Light Cycler Real-Time PCR (Roche), and the primers used were as follows:

Rab5A-F: 5'-AGCTGGTCAAGAACGATACCA-3 '' (SEQ ID NO: 1)

Rab5A-R: 5'-TTTTGCTCTTGCAAAGGACTC-3 '(SEQ ID NO: 2)

Rab7-F: 5'-GATTGCACGGAATGCACTTA-3 '(SEQ ID NO: 3)

Rab7-R: 5'-TGATAGGTTCAGGAAATTCGTTG-3 '(SEQ ID NO: 4)

Rab27a-F: 5'-AGGCCAGAGAATCCACCTG-3 '(SEQ ID NO: 5)

Rab27a-R: 5'-CGCTGTCGTTAAGCTACGAA-3 '(SEQ ID NO: 6)

GAPDH-F: 5'-TCCACTGGCGTCTTCACC-3 '' (SEQ ID NO: 7)

GAPDH-R: 5'-GGCAGAGATGATGACCCTTT-3 '(SEQ ID NO: 8)

4. Western Blot (Western blot analysis)

20 ㎍ of the protein was separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), the protein was transferred to nitrocellulose membranes (hereinafter referred to as NC membrane), and the NC membrane was reacted with the primary antibody . The primary antibodies used were: Rab5a, Rab7, Rab27A, RAC1, RhoA, CDC42 and Dynein (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA); Trp-1, Trp-2 and Tyrosinase (goat; Santa Cruz Biotechnology); And Kinesin (rabbit polyclonal; Abcam, Cambridge, UK).

The primary antibody and the reacted NC membrane were further reacted with anti-rabbit or anti-goat secondary antibody (horseradish peroxidase-conjugated antibodies) and treated with ECL solution (Thermo, Rockford, Ill., USA) . The color signal was confirmed with Image Reader (LAS-3000; Fuji Photo Film, Tokyo, Japan) and the protein band was analyzed with densitometry.

5. Immunoprecipitation method ( Immunoprecipitation )

Cell lysates were reacted with Rab5A antibody and resin (Pierce ^TM Direct IP kit; Thermo Scientific, Waltham, MA, USA) at 4 ° C and proteins not bound to the resin were removed according to the manufacturer's protocol. Subsequently, the proteins bound to the resin were analyzed by Rab5A, Rab7, Trp-1 and tyrosinase antibodies.

6. Immunohistochemical staining ( immunohistochemistry )

The skin tissue isolated from the stool patient was embedded in paraffin to make a paraffin block, and a skin tissue slide was prepared from the paraffin block. After deparaffinization and rehydration on the slide, the slides were reacted with 3% bovine serum albumin. Then, the skin slides or cells were reacted with an anti-Rab5A antibody and 1: 200 Alexa Fluor 488-labeled goat anti-rabbit IgG (488; Molecular Probes, Eugene, OR, USA) The antibody and Alexa Fluor 594-labeled goat anti-mouse IgG (594; Molecular Probes) antibody were reacted. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich) and fluorescence images were analyzed with an image analysis system (Dp Manager 2.1; Olympus Optical Co., Tokyo, Japan).

7. Confocal  Confocal microscopy observation

Rab5A overexpressed or knocked down cells were reacted with anti-Rab5A or anti-Trp-1 antibody and then stained with Alexa Fluor-labeled goat anti-rabbit IgG or Alexa Fluor-labeled donkey anti-goat IgG diluted 1: Probes, Eugene, OR), respectively. The nucleus was cross-stained with Hoechst 33258. Dye images were acquired using EZ-C1 software (EZ-C1; Nikon, Tokyo, Japan) and fluorescence signals were measured using NIS-Elements AR 3.2 software (Nikon Instruments, Melville, NY).

8. Luciferase Assay ( luciferase  activity assay)

The pMIR-RAB5A vector was constructed by amplifying the 3'-UTR region (SEQ ID NO: 9) from the human Rab5A gene by PCR and inserting the amplified product after the luciferase gene of the pMIR-REPORT luciferase vector (Ambion, Austin, TX).

The primers used for PCR in the construction of pMIR-RAB5A vector are as follows:

Rab5A-3UTR-F: 5'-AAAGCTGCGCACTAGTGAATGCAGAGAGAGGAGAAG-3 '(SEQ ID NO: 10)

Rab5A-3UTR-R: 5'-ATCCTTTATTAAGCTT TTGGATTTGCAAATGACT-3 '(SEQ ID NO: 11)

In addition, according to the protocol of the manufacturer, some base sequences are deleted from the 3'-UTR region of the human Rab5A gene using a QuickChange site-directed mutagenesis kit (Stratagene, Lo Jolla, CA, USA) (SEQ ID NO: 12: 3'-UTR mutant 1; SEQ ID NO: 13: 3'-UTR mutant 2) was constructed by inserting pMIR-RAB5A mutant 1 and pMIR-RAB5A mutant 2 into pMIR-REPORT luciferase vector.

Melanocytes were plated into 6-well plates and the pMIR-Rab5A 3UTR vector or pMIR-Rab5A 3UTR (pRIV-Rab5A 3UTR) was added to each well with pRL-TK vector containing Renilla luciferase The mutant vector was transfected and 50 nM of miR-675, or negative control, was transfected into each well. Transfection was performed using Lipofectamine 2000 and luciferase activity was measured 24 hours later using Dual-Luciferase Reporter Assay (Promega).

9. Melano  move( melanosome  transfer) confirmation

The amount of melanosomes migrating from melanocytes to keratinocytes was analyzed using FACS (Lin et al., 2008). Cells were first fixed with 2% formaldehyde at room temperature and washed with PBS containing BD PhosFlow Perm / Wash Buffer I (BD Biosciences, San Jose, Calif.) For 10 min. The cells were then incubated with FITC-conjugated goat anti-TYRP-1 (Santa Cruz Biotechnology) and phycoerythrin-conjugated mouse anti-Cytokeratin 14 (BD Biosciences) for 30 min at room temperature. Cells were then washed with PBS and cells were analyzed using a Cytomics FC500 Flow Cytometer (Beckman Coulter, Hialeah, FL) and CXP software (Beckman Coulter). Melanocyte migration efficiency was calculated by dividing the number of double stained cells with cytokeratin 14 (Cytokeratin 14) and tyrosinase-related protein 1 (TYRP-1) by the number of cells with cytokeratin 14 (Cytokeratin 14) positive.

10. Statistical Analysis

Statistical analysis was performed using Student's t-test, and the results were expressed as means ± SD (means ± SD). A p- value less than 0.05 was considered statistically significant.

Experiment result

One. MiR By -675 Rab5A's  Identification of expression changes

As a result of the luciferase assay, it was confirmed that miR-675 significantly reduced the luciferase activity of Rab5A 3'-UTR compared to the non-targeting control gene ( p = 0.05). On the other hand, miR-675 did not decrease the luciferase activity of the modified Rab5A 3'-UTR in some sequences.

In addition, when the miR-675 mimic was transfected into keratinocyte and melanocyte cells, the level of Rab5A protein was decreased in a concentration-dependent manner, and when the miR-675 inhibitor was transfected, the level of Rab5A protein was increased . In addition, Rab5A was more expressed in keratinocytes and melanocytes than fibroblasts.

In addition, the relative expression levels of Rab5A mRNA in the skin tissue samples of patients with reduced miR-675 levels were measured. As a result, mRNA levels of Rab7 and Rab27A were increased in hyperpigmented skin tissues compared with normal pigmented skin tissues, Rab5A mRNA levels were also significantly increased ( P <0.05). In addition, as a result of staining the skin tissue, it was found that the hyperpigmented skin tissue was stained with Rab5A and the expression was increased.

1 is a graph showing the results of measuring luciferase activity after transfection of miR-675, pMIR-RAB5A vector, pMIR-RAB5A mutant 1 vector and pMIR-RAB5A mutant 2 vector or a combination thereof to melanocytes.

FIG. 2 is a graph showing the results of confirming the expression level of Rab5A protein after transfection of miR-675 analog or miR-675 inhibitor into keratinocytes and melanocytes. In Figure 2, 3P is the miR-675 analog; 3PI refers to the miR-675 inhibitor.

FIG. 3 is a graph showing the results of measurement of mRNA levels of Rab5A, Rab7 and Rab27A in a skin tissue sample of a patient diagnosed with spots.

FIG. 4 shows results of double staining of normal pigmented skin tissue and hyperpigmented skin tissue from Rab5A and c-Kit, which were separated from patients diagnosed with spots.

2. Rab5A  Identification of protein expression changes by overexpression

Because Rab5A is highly expressed in keratinocytes, the effect of Rab5A on melanogenesis was confirmed by co-culturing melanocytes alone or melanocyte-keratinocytes with Rab5A overexpressing or knockdown. When co-cultured, experiments were carried out with rabb5A overexpressed or knocked down in keratinocytes or melanocytes, respectively.

As a result, tyrosinase expression was increased in all cells overexpressing Rab5A.

In addition, mRNA and protein levels of Rab5A were significantly increased by isolating exosome from the cell culture of melanocyte-keratinocyte (Rab5A overexpression), and Rab5A mRNA and protein levels were remarkably increased, while Rab7 and Rab27A mRNA levels were increased by about 2-fold.

In addition, when the exosomes isolated from cells overexpressing Rab5A were treated with normal melanocytes (melanocytes not overexpressing Rab5A), the protein levels of tyrosinase and EEA1 (early endosome antigen 1) also increased over time I could confirm.

FIG. 5 shows the results of confirming the expression of Rab5A and tyrosinase in keratinocytes and melanocytes overexpressing Rab5A. KC means keratinocyte, MC means melanocyte, and KC (Rab5A) and MC (Rab5A) means keratinocyte and melanocyte which overexpress Rab5A, respectively.

FIG. 6 shows the results of confirming mRNA levels of Rab5A, Rab7 and Rab27A in exosomes isolated from cell culture of melanocyte-keratinocyte (Rab5A overexpression) cells.

Fig. 7 shows the results of confirming Rab5A protein levels in exosomes isolated from cell culture of melanocyte-keratinocyte (Rab5A overexpression).

FIG. 8 shows the results of confirming the protein levels of Rab5A, tyrosinase and EEA1 (early endosome antigen 1) after treating normal melanocytes with exosomes isolated from cells overexpressing Rab5A.

3. Rab5A  Confirm the effect on melanin synthesis

Overexpression of Rab5A in melanocytes resulted in an increase in protein levels of tyrosinase, tyrosinase-related protein-1, Rab7 and Rab27A, and knockdown of Rab5A.

Immunoprecipitation experiments using the anti-Rab5A antibody showed that the binding of Rab5A to Rab7 or Rab5A and Trp-1 decreased by Rab5A knockdown.

In addition, the protein changes were confirmed by transfection of WT (wild) Rab5A, Rab5A S34N (GTPase activity removed, dominant negative mutant) and Rab5A Q79N (increased GTPase activity; active mutant) to melanocytes. As a result, when the WT Rab5A or Rab5A Q79N was transfected, the protein levels of tyrosinase, Rab7, Trp-1 and EEA1 were increased, but when the Rab5A S34N was transfected, the levels of the proteins were decreased there was. Confocal microscopy also revealed that WT Rab5A and Rab5A Q79N were located at the same position in Rab5A and Trp-1, but Rab5A S34N was in different positions.

FIG. 9 shows the results of confirming the expression of tyrosinase, Trp-1, Trp-2, Rab7 and Rab27A when Rab5A was overexpressed or knocked down in melanocytes.

FIG. 10 shows the result of immunoprecipitation using Rab5A antibody after knocking down Rab5A in melanocytes.

FIG. 11 shows the results of confirming the expression changes of tyrosinase, Trp-1, Trp-2, EEA1, Rab7 and Rab27A after transfection of melanocytes with WT Rab5A, Rab5A S34N and Rab5A Q79N, respectively.

Figure 12 shows the result of confocal microscopy of the intracellular location of Rab5A and Trp-1 after transfection of melanocytes with WT Rab5A, Rab5A S34N and Rab5A Q79N, respectively.

4. In melanocytes Rab5A Melano  Identify the impact on movement

Melanomas migrate from dendrites of melanocytes to keratinocytes, move along microtubule tracks to antegrade by kinesin superfamily proteins, and then to dynein ) And dynein-related proteins. &Lt; / RTI &gt; Therefore, in order to confirm the effect of Rab5A on melanoma migration, expression of kinesin and dinine by overexpression or knockdown of Rab5A was confirmed in melanocytes.

As a result, kinesin levels were increased when Rab5A was overexpressed, and reversed when Rab5A was knocked down. On the other hand, the levels of Dinein decreased when Rab5A overexpressed, and increased when Rab5A knocked down. Confocal microscopy also revealed that when the Rab5A was knocked down, the fluorescence signal of kinesin was weakened, while the fluorescence signal of Dinein was stronger, and the expression of tyrosinase was also decreased.

FIG. 13 shows the results of confirming the expression changes of kinesin and dinine when overexpressing or knocking down Rab5A in melanocytes.

FIG. 14 shows the result of confirming the expression of kinesin and tyrosinase by confocal microscopy after knocking down Rab5A in melanocytes.

FIG. 15 shows the result of confocal microscopy of changes in expression of Dinein and tyrosinase after knocking down Rab5A in melanocytes.

5. Confirmation of reduction of melanoma mobility by Rab5A knockdown

Rab5A-knockdown cells were analyzed by FACS for keratinocytes or melanocytes and keratinocytes, in which melanocytes and keratinocytes were co-cultured, and Rab5A alone was knocked down. As a result, TYRP-1 (Tyrosinase-related protein 1 ) And K14 double - positive cells were decreased and the migration of melanomas decreased.

Furthermore, it was confirmed that the protein levels of RAC-1 (ras-related C3 botulinum toxin substrate 1), RhoA and CDC42 were increased by western blotting in the melanin cells overexpressing Rab5A, and when Rab5A was overexpressed, Of melanosomes were engulfed by keratinocytes.

FIG. 16 shows the results of FACS analysis using TYRP-1 and K14 antibodies in keratinocytes and / or melanocytes knocked down with Rab5A.

FIG. 17 shows Western blotting results of protein levels of RAC-1, RhoA and CDC42 in melanocytes overexpressing Rab5A.

FIG. 18 shows the result of treating melanocytes with keratinocytes overexpressing Rab5A and then dyeing pigment globules.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

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Claims (8)

A composition for inhibiting pigment formation comprising Rab5A (RAS-associated protein Rab5A) inhibitor as an active ingredient.
The composition of claim 1, wherein said Rab5A inhibitor inhibits the expression, transcription or activity of Rab5A.
The composition of claim 1, wherein the dye is melanin.
2. The composition of claim 1, wherein the composition reduces melanosome migration.
2. The composition of claim 1, wherein the composition inhibits melanosomal predation by keratinocytes.
(a) contacting the test substance with melanocytes;
(b) culturing the melanocytes in contact with the test substance; And
(c) tyrosinase, kinesin, Rab7, Rab27A, early endosome antigen 1 (EEA1), tyrosinase-related protein-1 (Trp-1), and ras- related C3 botulinum toxin substrate 1), Rho homolog gene family member A (RhoA), CDC42 (cell division control protein 42 homolog), and dynein to determine the effectiveness of the test substance &Lt; / RTI &gt;
7. The method of claim 6, wherein the melanocyte of (a) is Rab5A overexpressed.
7. The method according to claim 6, wherein the step (c) comprises determining the level of tyrosinase, kinesin, Rab7, Rab27A, EEA1, Trp1, RAC1, RhoA and CDC42 in melanocytes Or when the level of dinine is increased, the test substance is judged to be effective as an inhibitor of pigment formation.

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