2025 Volume 48 Issue 10 Pages 1472-1484
Placental extract (PE) is employed as a skin-whitening agent in Japan, where this material is used in cosmetics and dietary supplements. However, the mechanism of PE’s anti-melanogenic activity remains poorly understood. The goal of the present study was to elucidate the mechanism of PE’s inhibitory effect on melanogenesis in B16 murine melanoma cells. Specifically, the activity of equine PE (EPE) against tyrosinase and melanogenic proteins was evaluated. The effects of EPE on tyrosinase activity and melanin content were assessed spectrophotometrically. This analysis showed that EPE inhibits melanogenesis in melanoma cells in a dose-dependent manner without affecting cell proliferation. EPE did not directly inhibit the enzymatic activity of tyrosinase. Western blot analysis demonstrated that EPE exposure also led to decreases in the protein levels of tyrosinase and tyrosinase-related protein 1 (TRP-1) in melanoma cells, without affecting the levels of the mRNAs encoding these proteins. This analysis further demonstrated that the EPE-induced depletion of tyrosinase and TRP-1 resulted from the induction, by EPE, of proteasome-mediated proteolytic degradation. Notably, the EPE-induced depletion of tyrosinase and TRP-1 was prevented by joint exposure to EPE and the proteasome inhibitor MG132. Similar experiments showed that the exposure of melanoma cells to MG132 abrogates the inhibition of melanogenesis by EPE. Immunoprecipitation analysis further revealed that EPE induces the ubiquitination of tyrosinase and TRP-1. Taken together, these results suggested that EPE induces proteasomal degradation of tyrosinase and TRP-1 by enhancing the ubiquitination of these targets, leading to the depletion of these proteins and the inhibition of melanogenesis.
The skin, the outermost layer of the surface of biological tissues, is constantly exposed to exogeneous stimuli such as UV radiation, harmful pollutants, and microorganisms, while also maintaining homeostasis as a first-line defense that separates the organism from the external environment. Melanin, a pigment that determines the color of skin and hair, is produced by pigment cells (melanocytes) located in the basal layer, the lowest layer of the epidermis in human skin. Melanin plays an important role in protecting the skin by absorbing harmful UV radiation; however, excessive melanogenesis in the skin is associated with hyperpigmentation disorders (e.g., melasma and solar lentigo), which are considered to be serious cosmetic problems. Recent research has focused on anti-melanogenic substances, especially on treatments derived from natural sources, reflecting the public’s desire for safe and effective ingredients.1)
Tyrosinase, a melanocyte-specific type I membrane glycoprotein, is the rate-limiting enzyme of melanogenesis; expression of the tyrosinase-encoding gene is modulated by the microphthalmia-associated transcription factor (MITF), as is the expression of the loci encoding other tyrosinase-related proteins (e.g., tyrosinase-related protein 1 (TRP-1), TRP-2).2–4) During translation, melanogenic proteins (tyrosinase, TRP-1, TRP-2) are produced as immature proteins that are localized to the endoplasmic reticulum (ER), where these polypeptides acquire the secondary (folded) structure and glycosyl modifications that facilitate the proteins’ functions. Following processing in the ER, these proteins translocate to the Golgi apparatus, where further glycosylation and processing render these polypeptides into a mature form.5) Notably, the glycosylation of tyrosinase is known to be an important modification that determines the enzyme’s activity and contributes to melanogenesis.6,7) Eventually, tyrosinase and TRPs are localized to specialized organelles (melanosomes) in melanocytes, where the mature proteins promote a series of reactions leading to the synthesis of melanin.8) Mature melanosomes accumulate in the dendrites of melanocytes, from which the organelles are transferred to keratinocytes, ultimately providing pigmentation in hair, eyes, and skin.9)
Melanogenic proteins that are non-functional or not properly transported to melanosomes are endogenously degraded via the cytosolic ubiquitin (Ub)-proteasome pathway. Ubiquitination is a post-translational modification in which Ub, a small (approx. 8.6 kDa) regulatory protein composed of 76 amino acids, is conjugated sequentially to the target protein, resulting in the formation of a polyubiquitin chain. Subsequently, ubiquitinated proteins are recognized by the proteasome, a multi-subunit protease complex that degrades the targets into peptide fragments.10,11) Mature melanogenic proteins are exported (from the melanosome) to the cytoplasm, where these proteins are (sequentially) deglycosylated, ubiquitinated, and digested by the proteasome.12–14) These mechanisms maintain an intracellular balance between the synthesis and degradation of melanogenic proteins, thereby contributing to the regulation of tissue pigmentation.15) Many substances that are well known as skin-whitening agents have been shown to inhibit melanogenesis in melanocytes by attenuating tyrosinase enzymatic activity (e.g., kojic acid (KA), arbutin, and ellagic acid) or by decreasing the level of the tyrosinase-encoding transcript (e.g., 2-amino-3H-phenoxazin-3-one).1,16–19) Furthermore, ubiquitination of tyrosinase and subsequent regulation of the level of tyrosinase protein by proteasomal degradation are newly recognized as mechanisms that regulate tyrosinase activity.20–24)
The placenta is an organ that forms in the maternal uterus during pregnancy in mammals; along with the fetus, this tissue is excreted from the body at delivery. Placental extract (PE), a hydrolysate of the placenta, contains several kinds of amino acids, peptides, and minerals,25,26) and has been reported to have beneficial effects, including anti-inflammatory, antioxidant, and anti-aging effects, on human health.27–30) For example, PE derived from humans has been approved in Japan as an injectable drug for the treatment of gynecological diseases such as menopause, and hepatic diseases.31,32) On the other hand, PEs from non-human vertebrates (including horse, pig, and sheep) are known as skin-whitening agents, reflecting the ability of these extracts to suppress melanogenesis in the skin; therefore, PEs are employed as components in cosmetics and dietary supplements. However, PEs have two opposing properties, both inhibiting and promoting melanogenesis; notably, although crude PE exhibits anti-melanogenic activity,33,34) specific components isolated from PE have been shown to promote melanogenesis.35,36) Therefore, it has been suggested that the anti-melanogenic effects of PE are due not to a single component, but rather to the synergistic effect of multiple components. Nevertheless, the detailed mechanism by which crude PE inhibits melanogenesis remains unknown. Notably, it is unclear whether the anti-melanogenic effect of PE reflects direct inhibition of tyrosinase and/or of melanogenic proteins, which also play an important role in melanogenesis. The purpose of the present study was to characterize crude equine PE (EPE), a material employed in dietary supplements, with respect to its anti-melanogenic activity, specifically by evaluating the effect of EPE on tyrosinase and melanogenic proteins in a cell line derived from a murine melanoma. The results showed that EPE, a traditional material, promotes the ubiquitination of tyrosinase and TRP-1, a modification that potentiates the degradation of these proteins by the proteasome, and subsequently decreases the intracellular levels of tyrosinase and TRP-1, ultimately suppressing melanogenesis in melanoma cells.
EPE powder (Lot No. SRK21AG01H) was provided by Sankyo Biochemicals Co., Ltd. (Tokyo, Japan); the characterization and composition of this reagent is detailed in Supplementary Tables S1 and S2. RPMI 1640 medium (ThermoFisher Scientific, Inc., Waltham, MA, U.S.A.), fetal bovine serum (FBS; Biowest, Nuailé, France), KA, PYR41 (Sigma-Aldrich, St. Louis, MO, U.S.A.), arbutin (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), bovine serum albumin (BSA; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), MG132 (FUJIFILM Wako), and the ISOGEN II RNA extraction reagent (NIPPON GENE Co., Ltd., Tokyo, Japan) were obtained from commercial vendors. The primary antibodies (Abs) used in the study were supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.), and included anti-tyrosinase (Clone T311), anti-MITF (Clone D-9), anti-TRP-1 (Clone G-9), anti-TRP-2 (Clone C-9), anti-Ub (Clone P4D1), and anti-β-actin (Clone C-4). The secondary Ab consisted of horseradish peroxidase (HRP)-conjugated goat anti-mouse Ab (Enzo Life Sciences, Inc., Farmingdale, NY, U.S.A.). Immunoprecipitation (IP) employed immunoglobulin G2b (IgG2b) anti-mouse monoclonal Ab (mAb) (Cell Signaling Technology, Inc., Danvers, MA, U.S.A.) as an isotype control Ab.
Cell CultureB16F1 murine melanoma cells were provided by the RIKEN BRC (Ibaraki, Japan) and were maintained in RPMI 1640 containing 10% (v/v) FBS, 100 units /mL penicillin (Gibco/Thermo Fisher), and 100 μg/ mL streptomycin (Gibco/ThermoFisher) at 37°C in a humidified 5% CO2 atmosphere. Cells were seeded at a density of 1 × 104 cells/well in 96-well plates, 8 × 104 cells/well in 12-well plates, 4 × 105 cells/dish in 60-mm dishes, and 5.5 × 105 cells/dish in 100-mm dishes. Following seeding, cells were allowed to attach to the plates for 48 h before the initiation of experiments. EPE was suspended in FBS-free RPMI 1640 medium and exposed to cells at 2.5–10 mg/mL.
Cell CytotoxicityCell cytotoxicity was quantified using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (NACALAI TESQUE, Inc., Kyoto, Japan). Briefly, cells were seeded into 96-well plates and exposed to the test reagents (EPE at 2.5–10 mg/mL, arbutin at 0.54 mg/mL, or KA at 0.028 mg/mL) for an additional 24, 48, and 72 h. After incubation, the number of viable cells was assessed by the MTT assay according to the manufacturer’s instructions, and the absorbance of the resulting reactions was measured at a wavelength of 570 nm with an SH-9000 microplate reader (CORONA ELECTRIC Co., Ltd., Ibaraki, Japan).
Measurement of Melanin ContentCells were seeded into 12-well plates and exposed to test chemicals (EPE at 5–10 mg/mL, arbutin at 0.54 mg/mL, or KA at 0.028 mg/mL) for an additional 72 h. After detachment from the dishes with trypsin/ethylenediaminetetraacetic acid (EDTA), the cells were dissolved in 1 n NaOH, and the melanin content was determined spectrophotometrically by measuring the absorbance at 405 nm. The amount of cellular melanin was corrected for the protein content of the samples, as quantified using the bicinchoninic acid (BCA) assay. The results were expressed as a percentage of the control, which was defined as 100%.
Cellular Tyrosinase Activity AssayCellular tyrosinase activities were determined spectrophotometrically by monitoring (at 450 nm) the oxidation of l-3,4-dihydroxyphenylalanine (l-DOPA) to dopachrome by the tyrosinase enzyme present in whole-cell lysates of melanoma cells. To obtain cellular tyrosinase, cells were seeded on 100-mm dishes in standard medium (without test reagents) and incubated for 72 h. Then, the cells were lysed with 0.1 M sodium phosphate buffer (pH 6.8) containing 5 mM EDTA, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The resulting cell lysate was aliquoted to the individual wells of a 96-well plate at a volume (80 μL/well) corresponding to 100 μg crude protein; each well had been pre-filled with 10 μL of test reagent (EPE at 5–10 mg/mL or KA at 28–56 μg/mL). Reactions were initiated by the addition of 10 mM l-DOPA in phosphate-buffered saline (PBS) at 10 μL/well. Following incubation of the mixture for 1 h at 37°C, the absorbance was measured at a wavelength of 450 nm.
Tyrosinase ZymographyTyrosinase zymography was performed as described previously, with modifications.37–39) Briefly, cells were seeded into 60-mm dishes and exposed to test reagents (EPE at 5–10 mg/mL, arbutin at 0.54 mg/mL, or KA at 0.028 mg/mL) for an additional 72 h. Following detachment from the dishes using trypsin/EDTA, the cells were lysed with 0.1 M sodium phosphate buffer (pH 6.8) containing 5 mM EDTA, 1% Triton X-100, and 1 mM PMSF. Aliquots of the resulting whole-cell lysates (60 μg/lane) were subjected to 7.5% polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions (without 2-mercaptoethanol or prior heating of samples). Following electrophoresis, the gel was washed (30 min, room temperature, gentle shaking) by soaking first in rinse buffer (0.1 M sodium phosphate buffer, pH 6.8) and then in distilled water. The gel then was stained with l-DOPA staining solution (rinse buffer supplemented with 0.5 mM l-DOPA) by soaking in the dark for 3 h at 37°C. The intensity of the bands was quantified using a CS Analyzer 4 image analysis system (Version 2.2.1; ATTO Corporation, Tokyo, Japan). The tyrosinase activities in EPE-exposed cells were calculated as the sum of intensities of the two bands.
Western Blot (WB) AnalysisProtein levels were quantified by WB analysis. Cells exposed to the indicated reagents were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.1 mM PMSF). The resulting whole-cell lysates (50 μg/lane) were resolved by SDS-PAGE on a 7.5% gel and transferred to polyvinylidene difluoride (PVDF) membranes (ATTO Corporation). The resulting blots were blocked (1 h, room temperature) with 3% skim milk in TBS-T (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), then probed (overnight, 4°C) with primary Abs, including anti-MITF (1 : 200), anti-tyrosinase (1 : 200), anti-TRP-1 (1 : 5000), anti-TRP-2 (1 : 1000), and anti-β-actin (1 : 2000). Following hybridization with the primary Abs, the probed membranes were incubated (1.5 h, room temperature) with the secondary Ab (HRP-conjugated goat anti-mouse; 1 : 1000). The bands were visualized with ECL reagent (ThermoFisher Scientific, Inc.), and band intensities were quantified using the CS Analyzer 4. Values were normalized to those of the loading control (β-actin) in the respective lane, and then to those in the corresponding control sample.
RT-PCRTotal RNA was isolated from EPE-exposed cells using ISOGEN II according to the manufacturer’s instructions. cDNA was synthesized using 1 μg of total RNA with random primers and PrimeScript RTase (TaKaRa Bio, Inc., Shiga, Japan). The resulting cDNA was amplified using primers specific for the genes encoding tyrosinase (forward, 5′-CCA GAA GCC AAT GCA CCT AT-3′; reverse, 5′-ATA ACA GCT CCC ACC AGT GC-3′), MITF (forward, 5′-CCC GTC TCT GGA AAC TTG ATC G-3′; reverse, 5′-CTG TAC TCT GAG CAG CAG GTG-3′), TRP-1 (forward, 5′-GCT GCA GGA GCC TTC TTT CTC-3′; reverse, 5′-AAG ACG CTG CAG TGC TGG TCT-3′), TRP-2 (forward, 5′-GGA TGA CCG TGA GCA ATG GCC-3′; reverse, 5′-CGG TTG TGA CCA ATG GGT GCC-3′), and glyceraldehyde phosphate dehydrogenase (GAPDH, a housekeeping protein; forward, 5′-ACC ACA GTC CAT GCC ATC AC-3′; reverse, 5′-TCC ACC ACC CTG TTG CTG T-3′). Reactions were conducted using a TP350 thermal cycler (TaKaRa Bio). Aliquots of the resulting PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV illumination. Band intensities were measured with the CS Analyzer 4. Values were normalized to those of the internal control (GAPDH) in the respective samples, and then to those in the corresponding control sample.
Immunoprecipitation (IP)IP was performed using Protein G magnetic beads according to the manufacturer’s instructions (ThermoFisher Scientific), with slight modifications. Briefly, an aliquot of 5 μg of the anti-tyrosinase Ab, the anti-TRP-1 Ab, or the IgG2b isotype control mAb was diluted in 200 μL of PBS containing 0.02% Tween 20, then incubated with 25 μL of magnetic beads under continuous mixing for 20 min at room temperature to form the bead-Ab complexes. The tubes containing the resulting mixtures were placed in a magnetic separation rack for 15 s at room temperature, and the bead–Ab complexes were washed with PBS containing 0.02% Tween 20. Separately, whole-cell lysates were prepared using Lysis Buffer (Cell Signaling Technology) according to the manufacturer’s instructions. Subsequently, cell lysate containing a mass of 300 μg of total protein (in 300 μL of Lysis Buffer) was combined with the bead–Ab complexes, and the mixture was incubated for 20 min at room temperature with continuous mixing, to permit the formation of the bead–Ab–antigen (Ag) complexes. The tubes again were placed in a magnetic separation rack for 15 s at room temperature, and the bead–Ab–Ag complexes were washed four times with ice-cold PBS. The adsorbed proteins then were eluted by incubation (5 min, 95°C) in 30 μL of Tris-glycine-SDS sample buffer containing 2-mercaptoethanol. The eluted immunocomplexes were separated by SDS-PAGE, blotted to PVDF, and probed with the anti-Ub Ab (Clone P4D1) to detect ubiquitinated tyrosinase (as precipitated with anti-tyrosinase Ab) or ubiquitinated TRP-1 (as precipitated with anti-TRP-1 Ab). The levels of ubiquitinated tyrosinase were quantified following normalization first to that of tyrosinase (the loading control) in the respective sample, and second to that of the control (+MG132, no EPE) sample. The same evaluation was applied for the TRP-1 protein. Values were expressed as a fold of those in the control sample.
Statistical AnalysisResults are expressed as the mean ± standard deviation (S.D.). Differences between two groups were analyzed by Student’s t-test using Excel software (Office 2019, Microsoft Corporation, Redmond, WA, U.S.A.). Multiple comparisons of data were analyzed using one- or two-way ANOVA with post hoc Dunnett’s tests or Tukey’s tests (Excel and R software, ver. 4.1.0; R Foundation for Statistical Computing, Vienna, Austria). All comparisons were performed as two-tailed tests. Differences with p < 0.05 were considered statistically significant.
First, the effect of EPE on cell viability was evaluated using the MTT assay. Cells were exposed for 72 h to a range of concentrations of EPE (2.5–10 mg/mL). As shown in Fig. 1A, EPE did not exhibit cytotoxicity at these concentrations when compared with growth in the unexposed cells. Similar results were obtained in time-course studies evaluating the cytotoxicity of EPE in cells exposed to EPE at a single concentration (10 mg/mL) for up to 72 h (Fig. 1B). A parallel analysis confirmed that two known skin-whitening compounds, arbutin (0.54 mg/mL) and KA (0.028 mg/mL), did not adversely affect cell survival at the tested concentrations.
(A) B16F1 cells were incubated with various concentrations of equine placental extract (EPE; 2.5–10 mg/mL) for 72 h, or with (B) arbutin (0.54 mg/mL), kojic acid (KA; 0.028 mg/mL), or EPE (10 mg/mL) for the indicated times. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Separately, cells were cultured in the presence of EPE (10 mg/mL) for the indicated times (in hours) (C), or incubated with various concentrations of EPE (2.5–10 mg/mL), arbutin (0.54 mg/mL), or KA (0.028 mg/mL) for 72 h (D); the cells were then harvested by centrifugation, and the resulting pellets were photographed. Next, melanin content was measured as described in “Materials and Methods.” (E) The activities of tyrosinase enzyme extracted from melanoma whole-cell lysates were measured following exposure to the indicated dose of EPE or KA. For panels (A) to (E), data represent the mean ± S.D. (n = 4, C; n = 5, A and E; n = 6, B and D) of values normalized to tyrosinase activity in the control. **p < 0.01, ***p < 0.001, and NS (not significant; p ≥ 0.05) by two-tailed one-way ANOVA with post hoc Dunnett’s tests (vs. control). (F) Zymographic analysis of tyrosinase activity. (Left) Total cellular protein was subjected to electrophoresis and tyrosinase activity was assessed by in-gel zymography following staining with 0.5 mM l-3,4-dihydroxyphenylalanine (l-DOPA). Lane 1, control (Ctrl); Lane 2, 5 mg/mL EPE; Lane 3, 10 mg/mL EPE; Lane 4, 0.028 mg/mL KA. The approximate molecular weights (kDa), based on comparison with the molecular weight marker ladder, are indicated on the left of the gel image. The gel image shown is representative of four independent experiments. (Right) Relative intensities of the bands were determined and expressed following normalization to that of the control; intensities for the EPE-exposed cells correspond to the sum of intensities for the pair of bands in each lane. Data represent the mean ± S.D. (n = 4). *p < 0.05, **p < 0.01, and NS by two-tailed one-way ANOVA with post hoc Dunnett’s tests (vs. control).
To evaluate the effect of EPE on melanogenesis, melanoma cells were cultured in the presence of EPE (10 mg/mL) for various time intervals. Following the addition of EPE, melanin content decreased in a nominally time-dependent manner and, after 72 h, decreased to approximately 40% of the control level (Fig. 1C). Next, melanoma cells were grown for 72 h in the presence of various concentrations of EPE, at which point the melanin content was quantified. As shown in Fig. 1D, exposure to EPE (at concentrations of 2.5 mg/mL or higher) led to a significant decrease in melanin content compared with cells grown in the absence of EPE (p < 0.001). Similar results were observed with the two known skin-whitening compounds. Given that previous work has shown that many skin-whitening agents control melanogenesis by directly inhibiting tyrosinase activity, the direct effect of EPE on tyrosinase activity was examined by assessing enzyme activity in crude lysates of melanoma cells. As expected, KA, a known tyrosinase inhibitor that chelates copper ions from the active site of the enzyme, inhibited tyrosinase activity in a dose-dependent manner (Fig. 1E). By contrast, EPE showed no inhibitory effect on cell-free tyrosinase activity when tested at concentrations (5–10 mg/mL) that inhibited melanogenesis in melanoma cells. Given that the inhibitory effect of EPE on melanogenesis in melanoma cells was not explained by direct inhibition of tyrosinase activity, the cellular tyrosinase activity of EPE-exposed cells was evaluated by zymography. Specifically, zymography was conducted as a two-step process: first, the protein content of a lysate of melanoma cells was subjected to electrophoresis, permitting separation of tyrosinase from other factors; second, tyrosinase activity was quantified by DOPA staining of the resulting electrophoresed gel, thereby localizing DOPA oxidative activity and assessing the size (glycosylation status) of tyrosinase.38,39) In this zymographic analysis, the band detected in the gel following staining with a solution containing l-DOPA indicated the location of the active tyrosinase enzyme.40) Cellular tyrosinase activity in cells treated with KA, which was used as the control, was significantly decreased compared with that in untreated cells (Fig. 1F, right panel, p < 0.01). Interestingly, zymographic detection of cellular tyrosinase activity in EPE-exposed cells revealed the presence of a lower-molecular-weight band than that seen in the unexposed control cells (Fig. 1F, left panel, open arrowheads), with the decrease in molecular weight indicating deglycosylation of the enzyme. Quantification of the tyrosinase activities (calculated as the sum of the intensities of the two bands) revealed that the level in EPE-exposed cells was significantly lower than that in the control (unexposed) cells (Fig. 1F, right panel, p < 0.05). These results suggested that the inhibitory effect of EPE on melanogenesis in melanoma cells is not explained by the direct blocking of tyrosinase activity, but may instead indicate regulation by some other mechanism.
EPE Exposure Depletes Tyrosinase and TRP-1 Protein Levels in B16F1 Melanoma CellsTo investigate whether EPE affects the levels of tyrosinase, the level of the protein in EPE-exposed melanoma cells (over time) was assessed via WB analysis. As shown in Fig. 2A, tyrosinase was detected as an approx. 80-kDa band, a size consistent with that expected for the mature protein.41) Notably, the level of tyrosinase protein decreased in an apparently time-dependent manner following exposure of the melanoma cells to EPE, with the protein level becoming significantly lower at 72 h when comparing EPE-exposed cells to control (unexposed) cells (Fig. 2B, p < 0.01).
(A) Levels of tyrosinase protein in EPE-exposed cells, as assessed by Western blotting. Cells were cultured in the presence of EPE (10 mg/mL) for the indicated times (in hours); whole-cell lysates were then separated by SDS-PAGE, blotted to membranes, and probed with anti-tyrosinase antibody. β-Actin was used as a loading control. The blot image shown is representative of four independent experiments. (B) Quantification of tyrosinase band intensities in EPE-exposed cells, as assessed by densitometric analysis. Values were normalized first to those of β-actin (the loading control) in the respective sample, and second to that in the control (0 h). Data represent the mean ± S.D. (n = 4). Not significant (NS; p ≥ 0.05) and **p < 0.01 by two-tailed one-way ANOVA with post hoc Dunnett’s tests (vs. control).
The levels of tyrosinase and melanogenic proteins (including TRP-1, TRP-2, and MITF) then were assessed in melanoma cells grown (for 72 h) in the presence of a range of EPE concentrations (0, 5, and 10 mg/mL). As shown in Fig. 3A, tyrosinase protein levels were significantly decreased, in a dose-dependent manner, by EPE exposure; by 72 h, the enzyme levels were decreased (compared with control cells) by 20% in the presence of 5 mg/mL EPE (p < 0.05) and by 40% in the presence of 10 mg/mL EPE (p < 0.01) (Fig. 3B). Unexpectedly, exposure to 5 and 10 mg/mL EPE also resulted in a significant (70%) decrease in TRP-1 protein levels (p < 0.001) (Fig. 3C). By contrast, the levels of other melanogenic proteins (including TRP-2 and MITF) were not significantly altered by 72 h of growth in the presence of EPE at these concentrations (Fig. 3C).
(A) Effect of EPE on the levels of melanogenic proteins. The expression of tyrosinase and other melanogenic proteins (MITF, TRP-1, and TRP-2) was determined using Western blot analysis; the levels of the proteins were normalized to that of β-actin (the loading control) in the respective sample. The levels of (B) tyrosinase protein or (C) melanogenic proteins in EPE-exposed cells were then quantified by densitometric analysis. Values were normalized first to the level of β-actin protein (the loading control) in the respective sample, and second to that of the target protein in the control (0 mg/mL). Data represent the mean ± S.D. (n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001 by two-tailed one-way ANOVA with post hoc Dunnett’s tests (vs. level of the respective protein in the control culture). (D) Effect of EPE on the levels of transcripts encoding the melanogenic proteins. Cells were cultured with EPE (10 mg/mL) for the indicated times and processed to total RNA. The levels of the respective transcripts were determined by RT-PCR with gene-specific primers; the amplification products were then subjected to agarose gel electrophoresis and visualized following ethidium bromide staining. The mRNA encoding the housekeeping protein glyceraldehyde phosphate dehydrogenase (GAPDH) was assessed as an internal control. (E) Band intensities were quantified by densitometric analysis. Values were normalized first to the level of GAPDH mRNA (the internal control) in the respective sample, and second to that of the respective transcript in the control (0 h). Data represent the mean ± S.D. (n = 5). The levels of the transcripts encoding tyrosinase, TRP-1, TRP-2, or MITF were assessed by two-tailed one-way ANOVA with post hoc Dunnett’s tests (vs. level of the respective transcript in the control culture). The images shown are representative of four (A) or five (D) independent experiments. TRP-1, tyrosinase-related protein-1; TRP-2, tyrosinase-related protein-2; MITF, microphthalmia-associated transcription factor.
In extension of this analysis, RT-PCR was used to determine whether the observed depletion of tyrosinase protein was mediated by changes in transcript levels. Notably, the levels of the transcripts encoding tyrosinase, TRP-1, TRP-2, and MITF (all normalized to the levels of GAPDH mRNA, which served as an internal control) did not differ significantly when comparing the levels of the respective transcripts between EPE-exposed cells and control cells (Figs. 3D, 3E).
EPE Induces Proteasomal Degradation of Tyrosinase and TRP-1 ProteinsMelanogenic proteins are known to be degraded endogenously by proteasomes, which selectively degrade intracellular ubiquitinated proteins. To determine whether the EPE-mediated decrease in tyrosinase and TRP-1 protein levels results from proteasomal degradation, the effects of EPE on melanin content and on the levels of these proteins were evaluated in melanoma cells grown in the presence or absence of MG132, a proteasome inhibitor. The resulting cellular melanin content values were analyzed by two-way ANOVA to assess the effect on cellular melanin content of MG132 and EPE, both alone and together. A significant difference (compared with the control) was detected for cells grown in the presence of MG132 (p < 0.001), of EPE (p < 0.05), or of both (p < 0.05). Subsequent Tukey’s multiple comparisons showed that co-incubation with MG132 and EPE significantly counteracted the decrease in melanin levels otherwise seen with EPE alone (p < 0.05, Fig. 4A). Next, tyrosinase and TRP-1 protein levels were examined in melanoma cells grown in the presence of MG132 and/or EPE. Analysis of the densitometric data by two-way ANOVA revealed a significant difference in band intensity among the various cultures (p < 0.05). Subsequent post hoc analysis showed that growth in the presence of both MG132 and EPE significantly counteracted the decreases in tyrosinase and TRP-1 protein levels seen in cultures grown in the presence of EPE alone (Figs. 4B, 4C, p < 0.05). These results suggested that EPE inhibits melanogenesis by inducing proteasomal degradation of the tyrosinase and TRP-1 proteins in melanoma cells.
(A) MG132 abrogates the EPE-induced inhibition of melanogenesis in melanoma cells. Cells were cultured for 72 h with (+) or without (−) MG132 (100 nM) and with or without EPE (10 mg/mL); the cells then were harvested by centrifugation, and the resulting pellets were photographed. Next, cellular melanin content was determined spectrophotometrically (see section “Materials and Methods”). Values were normalized to that in the control culture (no MG132, no EPE) and expressed as percentages. Data represent the mean ± S.D. (n = 5). Statistically significant differences between groups were determined by two-way ANOVA with post hoc Tukey’s tests. Different lower-case letters on the plot indicate significant differences among the groups (p < 0.05). (B) Tyrosinase and TRP-1 protein levels in melanoma cells grown in the presence of MG132 and/or EPE (as above) for 48 h were analyzed by Western blotting and staining with anti-tyrosinase and anti-TRP-1 antibodies. The image shown is representative of four independent experiments. (C) Quantification of tyrosinase (upper panel) and TRP-1 (lower panel) protein levels by densitometric analysis of immunoblots. Relative intensities of the bands were determined and expressed following normalization first to that of β-actin (the loading control) in the respective sample, and second to that of the control (no MG132, no EPE). Data represent the mean ± S.D. (n = 5). Statistically significant differences between groups were determined by two-way ANOVA with post hoc Tukey’s tests. Different lower-case letters on the plot indicate significant differences among the groups (p < 0.05).
Given that ubiquitination is a prerequisite for the proteasomal degradation of many cytosolic proteins, the effect of EPE exposure on the induction of ubiquitination of tyrosinase and TRP-1 was assessed. Given that ubiquitinated proteins normally would be recognized and immediately degraded by proteasomes, this experiment was performed using cells exposed to MG132 for 3 h (for assessment of TRP-1) or 6 h (for assessment of tyrosinase).12,41,42) First, the status of ubiquitination in general cellular proteins was compared between cells exposed to MG132 for 3 h or 6 h, in the presence or absence of EPE. As shown in Fig. 5A, 3-h exposure of cells to EPE in the presence of MG132 resulted in increased (1.5 ± 0.32-fold; mean ± S.D., n = 3, p < 0.05) band intensities of ubiquitinated proteins (Lanes 2 and 3), whereas a 6-h exposure of cells resulted in decreased (0.7 ± 0.11-fold; mean ± S.D. n = 3, p < 0.05) band intensities of ubiquitinated proteins (Lanes 5 and 6) compared with the MG132-treated control. This experiment suggested that EPE regulates the ubiquitination of cellular proteins, which are subsequently degraded, via a proteasome-dependent pathway, within 3–6 h. IP assays then were conducted to directly assess tyrosinase and TRP-1 ubiquitination. Whole-cell lysates were first subjected to IP with an anti-tyrosinase (Fig. 5B) or anti-TRP-1 (Fig. 5C) Ab, and ubiquitinated forms of tyrosinase or TRP-1 were then detected by probing a WB of the IPed proteins using an anti-Ub Ab. As shown in Fig. 5B, ubiquitinated tyrosinase stained with anti-Ub and anti-tyrosinase Abs was detected as two bands with distinct mobilities, migrating as proteins between 70 and 100 kDa in weight (Lanes 3 and 4 of the top and bottom panels). I postulate that the upper (larger) band corresponds to a polyubiquitinated protein, attached to multiple, linked ubiquitin molecules. The lower (smaller) band, representing a smear of lower intensity, might then correspond to proteins decorated with smaller numbers of Ub molecules, ranging down to the size of a protein with a single attached Ub molecule. The level of ubiquitinated tyrosinase was calculated as the sum of the intensities of the two bands. As a result, the level of ubiquitinated tyrosinase in cells exposed to both MG132 and EPE was significantly elevated compared with that seen in cells exposed to MG132 alone (Lane 4 vs. 3, respectively), with band intensities differing by 1.46 ± 0.23-fold (mean ± S.D., n = 3; p < 0.05, Fig. 5D). In the control experiment, the same samples were subjected to IP with a mouse isotype control Ab; no bands corresponding to the ubiquitinated forms of tyrosinase were detected in the resulting blot (Fig. 5B, Lanes 1 and 2). When this membrane was stripped and reprobed with an anti-tyrosinase Ab to quantify the amount of tyrosinase protein IPed in each sample, no significant difference was observed between the two samples in the levels of the target protein (Fig. 5B, lower panel, Lanes 3 and 4). Thus, the level of tyrosinase protein was not decreased in cells grown in the presence of MG132 and EPE for 6 h, and the same band intensity was observed in the lysate from cells exposed to MG132 alone, confirming that the degradation of tyrosinase was prevented by MG132. In the case of TRP-1, even in cells grown in the presence of both MG132 and EPE, TRP-1 degradation was not prevented in cells exposed for 6 h, resulting in decreased levels of IPed TRP-1 protein (Supplementary Fig. S1, Lane 4, marked with an asterisk). TRP-1 is known to be the most abundant glycoprotein expressed in melanocytes.7) As shown in Fig. 5A, the band intensity of ubiquitinated proteins decreased at 6 h; this may reflect the proteasome-mediated degradation of TRP-1. I infer that TRP-1 is degraded more rapidly by proteasomes than is tyrosinase. Therefore, whole-cell lysates were prepared from cells treated with MG132, with or without EPE, for a shorter interval (3 h); these lysates were then subjected to IP with anti-TRP-1 Ab. While TRP-1 protein was detected as three bands by WB analysis, as shown in Figs. 3A and 4B, ubiquitinated TRP-1 was detected as a single band between 70 and 100 kDa (Fig. 5C, Lanes 3 and 4 of the upper and lower panels). The ubiquitination of membrane proteins such as tyrosinase and TRP-1 typically occurs at lysine residues located in the cytoplasmic domain.43) The C-terminal cytoplasmic tail of mouse tyrosinase contains seven lysine residues, whereas that of mouse TRP-1 has only one. This difference also is thought to be the reason why ubiquitinated tyrosinase was detected as two bands, while ubiquitinated TRP-1 was detected as a single band. As with tyrosinase, the level of ubiquitinated TRP-1 was significantly increased in cells exposed to both MG132 and EPE (compared with cells exposed to MG132 alone) (Fig. 5C, Lane 4 vs. 3, respectively), with a 1.35 ± 0.15-fold difference in band intensities (mean ± S.D., n = 3; p < 0.05, Fig. 5E). Together, these results demonstrated that EPE alters the protein levels of tyrosinase and TRP-1 by increasing the proteasomal degradation of these enzymes, an effect mediated by enhancing the ubiquitination of these proteins.
(A) Effects of EPE on general cellular protein ubiquitination, as assessed by Western blotting (WB). Cells were cultured for 3 h or 6 h with (+) and without (−) MG132 (100 nM), and in the presence or absence of EPE (10 mg/mL); whole-cell lysates (50 μg total protein/lane) were then separated by SDS-PAGE and transferred to membranes. (Upper panel) The membrane was probed with an anti-ubiquitin (Ub) antibody. (Lower panel) The same membrane was stripped and reprobed with anti-β-actin antibody. The position of the β-actin protein band is indicated by the filled arrowhead to the left of the WB. For each lane, the intensity of upper-blot staining was normalized to that of the lower-blot staining. The resulting mean values (from three independent experiments) of -fold increase or decrease compared with MG132-treated controls (+MG132, no EPE) are shown at the bottom of each lane in Fig. 5A. (B) Exposure of melanoma cells to EPE induces the ubiquitination of tyrosinase and TRP-1(C), as assessed by immunoprecipitation (IP) and WB. Cells were cultured for 3 h (TRP-1) or 6 h (tyrosinase) with MG132 (100 nM) in the presence or absence of EPE (10 mg/mL). Whole-cell lysates (300 μg total protein/reaction) were subjected to IP with anti-tyrosinase antibody, anti-TRP1 antibody, or with isotype anti-mouse immunoglobulin G (mIgG) antibody as the control. The IPed proteins were then separated by SDS-PAGE and transferred to membranes. (Upper panel) The membrane was probed with an anti-Ub antibody. The positions of the IgG heavy and light chains are indicated to the left of the WB. (Lower panel) The same membrane was stripped and reprobed with anti-tyrosinase (B) or anti-TRP-1 antibody (C). The positions of the tyrosinase and TRP-1 protein bands are indicated by the filled arrowheads to the right of the WB. For the samples IPed with anti-tyrosinase or anti-TRP-1 antibodies following growth in the absence and presence of EPE (Lanes 3 and 4, respectively), densitometric analysis was used to quantify the intensities of the paired anti-Ub-stained bands in the 70- to 100-kDa interval of the upper WB and in the bands labeled “tyrosinase” or “TRP-1” in the lower WB. For each lane, the intensity of upper-blot staining was normalized to that of lower-blot staining. The resulting values then were normalized to those in Lane 3 (-EPE) to determine the relative levels (fold) of ubiquitination of the tyrosinase or TRP-1 protein, as shown in (D) and (E). The images shown in panels A, B, and C are representative of the results obtained from each of the three independent experiments. Data represent the mean ± S.D. (n = 3). *p < 0.05 by two-tailed unpaired Student’s t-test.
The above results suggest that EPE promotes the ubiquitination of tyrosinase and TRP-1. This modification potentiates the degradation of these proteins by the proteasome and subsequently decreases the intracellular levels of these two proteins in melanoma cells. To further support these findings, the effects of EPE on melanin content and protein levels were assessed in melanoma cells cultured with or without PYR41, an inhibitor of ubiquitination and Ub-dependent protein degradation. As for Fig. 4A, the resulting cellular melanin content values were analyzed by two-way ANOVA with post hoc Tukey’s multiple comparisons. The results showed that co-incubation with the combination of PYR41 and EPE significantly counteracted the decrease in melanin levels otherwise seen in cultures incubated with EPE alone (p < 0.05, Fig. 6A). Tyrosinase and TRP-1 protein levels were then examined in melanoma cells grown in the presence of PYR41 and/or EPE. Analysis of the densitometric data by two-way ANOVA with post hoc Tukey’s tests showed that co-incubation with the combination of PYR41 and EPE prevented the decrease in tyrosinase and TRP-1 protein levels seen in cultures incubated with EPE alone (Figs. 6B, 6C, p < 0.05). These results strongly suggest that ubiquitination of the tyrosinase and TRP-1 proteins is a prerequisite for the EPE-induced depletion of their levels, leading to the inhibition of melanogenesis.
(A) Cells were cultured for 72 h with (+) or without (−) PYR41 (10 μM) and with or without EPE (10 mg/mL); the cells were then harvested by centrifugation, and the resulting pellets were photographed. Next, cellular melanin content was determined spectrophotometrically (see “Materials and Methods”). Values were normalized to that in the control culture (no PYR41, no EPE) and expressed as percentages. Data represent the mean ± S.D. (n = 5). Statistically significant differences between groups were determined by two-way ANOVA with post hoc Tukey’s tests. Different lower-case letters on the plot indicate significant differences among the groups (p < 0.05). (B) Tyrosinase and TRP-1 protein levels in melanoma cells grown in the presence of PYR41 and/or EPE (as above) for 48 h were analyzed by Western blotting using anti-tyrosinase and anti-TRP-1 antibodies. The image shown is representative of four independent experiments. (C) Quantification of tyrosinase (upper panel) and TRP-1 (lower panel) protein levels by densitometric analysis of immunoblots. Relative intensities of the bands were determined and expressed following normalization first to that of β-actin (the loading control) in the respective sample, and second to that of the control (no PYR41, no EPE). Data represent the mean ± S.D. (n = 4). Statistically significant differences between groups were determined by two-way ANOVA with post hoc Tukey’s tests. Different lower-case letters on the plot indicate significant differences among the groups (p < 0.05).
The present study sought to elucidate the mechanism of EPE’s anti-melanogenic effects, specifically by examining the effect of this extract on the activity and accumulation of tyrosinase and related melanogenic proteins in melanoma cells. This study revealed a new function for EPE, which appears to act as a regulator of the ubiquitin-proteasome system in mammalian cells, providing quantitative attenuation of the intracellular levels of the tyrosinase and TRP-1 proteins in melanoma cells (Fig. 7). To the best of my knowledge, this work represents the first report indicating that a PE regulates protein levels via the ubiquitin–proteasome axis.
This figure provides a schematic representation of the proposed model of the inhibition, by equine placental extract (EPE), of melanogenesis in melanoma cells. This inhibition model includes the following steps: 1. EPE enhances TRP-1 and tyrosinase ubiquitination, thereby 2. inducing proteasomal degradation of ubiquitinated tyrosinase and TRP-1, and resulting in 3. depletion of intracellular tyrosinase and TRP-1 protein levels and 4. suppression of melanogenesis. Tyr-ase, mature tyrosinase (white ovals); TRP-1, mature tyrosinase-related protein-1 (white triangles); Ub, ubiquitin (black circles); thin down arrows, decrease/depletion.
This study began by evaluating the effect of EPE on cell survival, while also confirming that EPE exhibits anti-melanogenic activity similar to that seen with well-known skin-whitening compounds in melanoma cells. However, the anti-melanogenic activity of EPE was observed at concentrations much higher than those required for arbutin or KA. Nonetheless, EPE did not exhibit cytotoxicity against melanoma cells, even at the highest tested concentrations. These results indicated that the inhibition of melanogenesis by EPE is not attributable to a cytotoxic effect on melanoma cells. EPE, a crude drug, is a mixture of multiple components derived from equine placenta; there may be combinations of the effects of different compounds in the mixture, such that the potencies of the active ingredients may be combinatorially increased or competitively decreased. Based on existing reports, the anti-melanogenic effects of EPE are speculated to be due to the synergistic effect of multiple components.33–36) This point may explain the apparently lower potency of EPE compared with those of purified compounds such as KA and arbutin. Furthermore, comparison with porcine liver extract (PLE) indicated that the anti-melanogenic effect is unique to the placental tissue (Supplementary Fig. S2B). In addition, the present study demonstrated that the EPE-associated decrease in tyrosinase and TRP-1 protein levels, and of tyrosinase activity levels, is mediated not by direct inhibition of enzymatic activity, but by protein degradation. Zymography of EPE-exposed cells revealed a banding pattern distinct from that observed in unexposed cells. The mouse homolog of this protein (NP_035791) is a type I membrane glycoprotein that is 533 amino acids long, an interval that includes an 18-amino-acid N-terminal signal sequence. The mature protein consists of three domains, including: (1) an N-terminal 477-amino-acid lumenal ectodomain that is essential for enzymatic activity and that contains two copper-binding sites and six glycosylation sites, (2) a transmembrane domain of 26 amino acids, and (3) a C-terminal cytoplasmic tail. Based on this domain organization, the 55-kDa band seen in most samples is presumed to be the native tyrosinase protein. On the other hand, the lower-molecular-weight band (approximately 50 kDa) seen only in EPE-exposed cells is thought to correspond to the deglycosylated form, lacking sugar modifications at the consensus glycosylation sites in mouse tyrosinase, suggesting that EPE exposure leads to changes in the glycosylation of this protein. In addition, WB analysis revealed that tyrosinase protein levels were decreased in a time-dependent manner during 72 h of EPE exposure, an observation consistent with the results of a zymographic assessment of cellular tyrosinase activity. Previous reports have suggested that biomolecules such as linoleic acid (a major component of cell membranes) and amino acids (components of the proteins that make up all living organisms) regulate the proteasomal degradation of proteins.20,21,44) Given that PE is a crude material that contains various biomolecules (e.g., amino acids) derived from the vertebrate placenta,25,45) the present study focused on the possible relationship between melanogenic proteins and proteasomal degradation in response to EPE treatment. As expected, studies using a proteasome inhibitor (MG132) and a ubiquitin E1 inhibitor (PYR41) conclusively demonstrated that the anti-melanogenic effect of EPE reflects proteasome-dependent degradation of tyrosinase and TRP-1, resulting from the ubiquitination of these proteins. At the same time, EPE was shown to not affect proteasomal activity in B16F1 cells (Supplementary Fig. S3). The results of the present study also showed that TRP-1 is ubiquitinated more rapidly than is tyrosinase in EPE-exposed cells. These distinctions may reflect varying maturation rates between tyrosinase and TRP-1.7) More importantly, EPE may control the trafficking (to melanosomes) of TRP-1, together with that of tyrosinase, by ubiquitinating these proteins. TRP-1 not only interacts with tyrosinase to stabilize its catalytic activities,8) but also controls efficient melanogenesis by correctly delivering tyrosinase to melanosomes, along with several important regulators of tyrosinase transport (e.g., the VPS9-ankyrin-repeat protein (VARP, aka ANKRD27)).46) Therefore, I hypothesize that the initial induction of TRP-1 ubiquitination by EPE suppresses the stabilizing influence of TRP-1 on tyrosinase and is closely associated with the subsequent induction of the ubiquitination and proteasomal degradation of tyrosinase.8,46) Specifically, I propose that TRP-1 is initially ubiquitinated in response to EPE exposure, resulting in the depletion of TRP-1, a decrease in the direct interaction between TRP-1 and tyrosinase, and a decrease in the correct delivery of tyrosinase to melanosomes; the mistargeted tyrosinase may be ubiquitinated and degraded by proteasomes. To confirm this possibility, further analysis will attempt to investigate the effect of EPE on the subcellular localization of these proteins.
Ubiquitination of proteins is known to involve the sequential catalytic action of three types of ubiquitinating enzymes: ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Notably, the E3 ligase is known to play an important role in identifying target proteins and determining the selectivity of the degradation of these targets.43,47,48) Based on the results of the present study, I postulate that EPE has some effect on E3 ligase activity; the E3 ligase is the most promising candidate for the mediator of the induction of the ubiquitination of tyrosinase and TRP-1, although the details of this effect were not apparent in the present study. Further studies will be needed to clarify the mechanism by which EPE specifically induces the ubiquitination of tyrosinase and TRP-1.
Thus, the present study elucidated that EPE, a material traditionally used as a skin-whitening agent, controls protein turnover and accumulation in cells by activating the selective proteolysis of the tyrosinase and TRP-1 proteins. These findings are expected to facilitate the development of drugs to treat hyperpigmentation diseases and to lead to novel applications of EPE in cosmetics and dietary supplements.
The author declares no conflict of interest.
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