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Master's Thesis
in the Department of Biomedical Science
GDF15 derived from
Senescent Fibroblasts Stimulates
Melanogenesis in Human Melanocytes
Graduate School of Ajou University
Major in Molecular Medicine
GDF15 derived from
Senescent Fibroblasts Stimulates
Melanogenesis in Human Melanocytes
Hee Young Kang, Advisor
I submit this thesis as the Master's thesis
in the Department of Biomedical Science.
August, 2019
Graduate School of Ajou University
Major in Molecular Medicine
The Master's thesis of Yeongeun Kim in Department
of Biomedical Science is hereby approved.
Thesis Defense Committee President Hee Young Kang
Tae Jun Park Jang Hee Kim
Graduate School of Ajou University
-ABSTRACT-GDF15 derived from Senescent Fibroblasts Stimulates Melanogenesis in Human Melanocytes
Background
Senescent fibroblasts play a role in aging pigmentation. In our previous RNA sequencing data in senescent fibroblasts, the Growth differentiation factor 15 (GDF-15) gene was found to be significantly upregulated.
Objective
We investigated the role of senescent fibroblast-derived GDF15 in the regulation of human pigmentation.
Methods
Normal human melanocytes were co-cultured with fibroblasts infected with GDF15 lenti-virus or sh-GDF15 and melanogenesis was analyzed. The pigmentation were also assessed in the ex vivo skin culture.
Results
The melanin contents and tyrosinase activity were significantly increased in melanocytes co-cultured with fibroblasts infected with GDF15 lenti-virus. The mRNA and protein expression levels of melanogenesis-associated proteins, microphthalmia-associated transcription factor (MITF) and tyrosinase were significantly up-regulated. The GDF15 stimulated b-catenin signaling in melanocytes. Consistently, the pigmentation were significantly reduced in melanocytes co-cultured with fibroblasts infected with sh-GDF15. The stimulatory action of GDF15 in pigmentation was further confirmed in ex vivo cultured skin.
Conclusion
Senescent fibroblast-derived GDF15 stimulates skin pigmentation and it may play a role in the aging pigmentation.
TABLE OF CONTENTS
ABSTRACT
‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ ⅰTABLE OF CONTENTS
‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ ⅱLIST OF FIGURES
‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ ⅳINTRODUCTION
‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 1Ⅱ. MATERIALS AND METHODS
‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 41. Cell culture ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 4 2. Enzyme-linked immunosorbent assay (ELISA) ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 5 3. Senescence Associated b-Galactosidase (SA-b-Gal) Staining ‧‧‧‧‧‧‧‧ 5
4. In vitro model of senescent fibroblasts ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 5 5. Ex vivo skin organ culture and pigmentation assay in cultured skin ‧ 6 6. Melanin content and tyrosinase activity assay ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 6 7. Lentivirus and adenovirus production ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 7 8. Semiquantitative real-Time PCR Analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 7 9. Western blot analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 8 10. Immunocytochemistry and immunohistochemical analysis ‧‧‧‧‧‧‧‧‧‧‧ 8 11. Statistical analysis ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 9
Ⅲ. RESULTS
‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 10 1. UV induced senescent fibroblasts stimulate skin pigmentation.‧‧‧‧‧‧ 10 2. GDF15 expression is increased in senescent fibroblasts and photo-agedhyperpigmented skin. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧16 3. GDF15 derived from senescent fibroblasts stimulates melanogenesis in
human melanocytes. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 23 4. GDF15 stimulates b-catenin signaling. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 35
Ⅳ. DISCUSSION
‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 40Ⅴ. REFERENCES
‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 45LIST OF FIGURE
Figure 1. Establishment of senescent fibroblasts by UV irradiation. ‧‧‧‧ 11 Figure 2. Senescent marker expression in UV induced senescent fibroblasts. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 12 Figure 3. Senescent fibroblasts by UV irradiation increased melanogenesis in human normal melanocytes. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 13 Figure 4. UV-induced senescent fibroblasts increased MITF and tyrosinase expression level in melanocytes. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧14 Figure 5. Pigment level of ex vivo cultured skin in presence of senescent fibroblasts.‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 15 Figure 6. GDF15 expression level in skin cells. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 17 Figure 7. Immunocytochemical analysis of GDF15 in skin cells. ‧‧‧‧‧‧‧‧ 18 Figure 8. Immunohistochemical analysis of GDF15 in normal human skin. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 19 Figure 9. GDF15 is upregulated in UVA induced senescent fibroblasts. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 20 Figure 10. GDF15 expression in young and old skin. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 21 Figure 11. GDF15 expression in melasma and perilesional normal skin. ‧ 22 Figure 12. Fibroblasts were infected with a GDF15-overexpressing lentivirus. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 24 Figure 13. GDF15 overexpression did not affect fibroblast proliferation. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 25 Figure 14. Fibroblasts derived GDF15 stimulated pigmentation. ‧‧‧‧‧‧‧‧‧‧26 Figure 15. GDF15 overexpressing fibroblasts increased MITF and tyrosinase expression level in melanocytes. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 27 Figure 16. Pigment level of ex vivo cultured skin in presence of GDF15
overexpressing fibroblasts. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 28 Figure 17. GDF15 down-regulation in fibroblasts using a shGDF15 lentivirus. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 29 Figure 18. GDF15 knockdown did not affect fibroblast proliferation. ‧‧‧‧ 30 Figure 19. Fibroblasts infected with shGDF15 inhibited pigmentation. ‧‧‧ 31 Figure 20. Fibroblasts infected with shGDF15 down-regulated MITF and tyrosinase expression level in melanocytes. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 32 Figure 21. GDF15 knockdown decreased pigmentation in ex vivo. ‧‧‧‧‧‧ 33 Figure 22. GDF15 stimulated melanogenesis through β-catenin signaling. ‧‧ ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 36 Figure 23. GDF15 knockdown reduced the phosphorylation of the β-catenin. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 37 Figure 24. β-catenin nuclear translocation was observed in the melanocytes stimulated with GDF15. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 38 Figure 25. β-catenin nuclear translocation was observed in the melanocytes co-culture with fibroblasts infected shGDF15. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 39 Figure 26. GFRAL expression in skin cells. ‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧‧ 44
Ⅰ. INTRODUCTION
Melanogenesis is the process of formation of melanin. When skin is exposed UV, the melanin is produced in melanosome by melanocytes in a complex process called melanogenesis. Melanogenesis is a complex process with different stages. When disturbed, it may determine different type of pigmentation disorders, which are known as hyper-pigmentatary disorder (eg. melasma, lentigo) or hypo-pigmentatary disorder (eg. vitiligo).
Despite skin pigmentation is primarily depends on the melanocyte functionality, the surrounding keratinocytes and dermal fibroblasts also contribute to skin pigmentation (Bastonini et al., 2016). Several studies have highlighted the epidermal/dermal cross-talk. For example, fibroblast-derived pleiotrophin (PTN) inhibits melanogenesis in melanocytes by means of the degradation of MITF via Erk activation (Choi et al., 2015). Fibroblast derived clusterin (CLU) inhibits pigmentation via TGF-b signaling pathway (Lee et al., 2017).
Cellular senescence is an irreversible growth arrest. Senescent cells effect on development, wound healing, tumor suppression and tissue aging. Skin also become aging. Skin aging driven by various factor is complex process affects skin biology and function (Wang and Dressen, 2018). Skin aging results in a progressive decline of skin function, leading to fragility, and increased risk of cancer (Lewis et al., 2011; Maru et al., 2014). Aging skin also occur pigmentation. Aging pigmented skin conditions such as senile lentigo or melasma also show increased numbers of senescent fibroblasts, and phenotype switching in these cases is thought to contribute
to aging pigmentation (Kim et al., 2019). There is increasing evidence of a crucial role of senescent fibroblasts and senescence-associated secretor phenotype (SASP) in melanogenesis (Salducci et al., 2014; Kovacs et al., 2010). The crosstalk between melanocytes and senescent fibroblasts during the aging process plays an important role in the stimulation of melanogenesis and in subsequent aging-related pigmentation (Yoon et al., 2018).
Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine which belongs to the transforming growth factor beta super-family (Adela and Banerijee, 2015). GDF15 expression and serum levels rise in response to many stimuli that initiate cellular stress and as part of a wide variety of disease processes, including those associated with cardiovascular disease and cancer (Fujita et al., 2016). Studies have implicated GDF15 in aging and age-related disorders and have demonstrated the possibility of GDF15 as a biomarker for aging and age-related comorbidity (Fujita et al., 2016).
Involvement of GDF15 in skin biology has been suggested (Unal et al., 2015). GDF15 is overexpressed in melanoma cells and is associated with tumor invasion and metastasis (Boyle et al., 2009). GDF15 expression levels are increased in melanocytes in response to UVB irradiation or a histamine treatment (Lee et al., 2012; Yang et al., 2006). Dermal fibroblasts also express GDF15 when induced by ROS or visible light (Akiyama et al., 2009). Although a role of fibroblast-derived GDF15 in systemic sclerosis has been suggested, its roles in skin pigmentation and in melanocyte biology have not been extensively studied (Bruzzese et al., 2014).
UV-induced senescent fibroblasts. In our RNA sequencing data, the GDF15 expression level in senescent fibroblasts increased by 5.47 fold compared to that in young fibroblasts (GEO accession number GSE 109778). We therefore investigated the role of GDF15 in the regulation of skin pigmentation in line with the epidermal/dermal crosstalk between senescent fibroblasts and melanocytes.
Ⅱ. MATERIALS AND METHODS
1. Cell culture
Normal human melanocytes, keratinocytes and fibroblasts were isolated from skin biopsies. Melanocytes at passages 2~7 were incubated in a F12 medium (Gibco-BRL, Bethesda, MD) supplemented 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL), 1% penicillin/streptomycin (Gibco-BRL), 24 mg/ml 3-isobutyl-1-methylxanthine, 80 nM 12-O-tetradecanoylphorbor 13-acetate, 1.2 ng/ml basic fibroblast growth factor and 0.1 mg/ml cholera toxin (All from Sigma, St. Louis, MO). Keratinocytes at passages 2~3 were grown in the Epilife medium supplemented with human keratinocyte growth supplement (HKGS; Gibco-BRL, Bethesda, MD). Fibroblasts at passages 3~7 were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) supplemented with 10% FBS and 1% penicillin/streptomycin. In co-culture, melanocytes and fibroblasts were maintained in MCDB-153 (Welgene, Gyeongsan, Korea) containing 4% FBS, 0.6 ng/ml basic fibroblast growth factor, 5 mg/ml insulin, 1 mg/ml vitamin E, and 1 mg/ml transferrin. To investigate the functional role of GDF15. GDF15 overexpressed or knockdown fibroblasts were seeded into insert Transwell chambers (Corning, Tewksbury, MA), and melanocytes were seeded at the bottom of 6-well plates. After 24 hr, the insert chambers were translocated into melanocytes seeded in 6-well plates and then maintained with a melanocytes culture medium for five days. The inserts with fibroblasts were changed to fresh ones at three days.
2. Enzyme-linked immunosorbent assay (ELISA)
Cells were cultured for 48 hr, after which the media were harvested. GDF15 secretion levels in the cell culture media were measured using GDF15 ELISA kits (R&D Systems, Minneapolis, MN) used according to the manufacturer’s instructions.
3. Senescence Associated β-Galactosidase (SA-b-Gal) Staining The cells were fixed with 4% formalin for 15 min and then incubated with SA-β-Gal solution (X-gal, 1mg/ml; citric acid/sodium phosphate, pH 5.8, 40 mM; potassium ferrocyanide, 5 mM; potassium ferricyanide, 5 mM; NaCl, 150 mM; MgCl2, 2 mM) for 12 hr at 37°C. After PBS washing, SA-β-Gal –positive cells were analyzed under light microscopy.
4.
I n vitro
model of senescent fibroblastsA primary culture of HDF was prepared and maintained in our laboratory in DMEM (Gibco-BRL) supplemented with 10% FBS (Gibco-BRL). For the UVA-induced senescent fibroblasts, HDF samples were washed once with phosphate-buffered saline (PBS) and placed in fresh PBS. The cells were irradiated with 5 J/cm2 UVA (wavelength 320-400 nm, maximum peak 350 nm) using a LZC-1 photoreactor system (Luzchem Research Inc. Ontario, Canada). Sham-irradiated HDF was rinsed and placed into an irradiator box without UV irradiation. After irradiation, the cells were maintained in DMEM for seven days.
5.
Ex vivo
skin organ culture and pigmentation assay in cultured skinNormal human skin samples obtained during surgery were placed on a sterilized stainless steel grid in 6-well plates containing DMEM supplemented with 5% FBS. The senescent fibroblasts were seeded in the bottom of 6-well plates. After three days of culturing in an incubator at 37°C with 5% CO2, the specimens were fixed in 10% formalin and embedded in paraffin sections. Melanin pigments were detected with Fontana-Masson staining. An image analysis was performed using Image Pro Plus Version4.5 (Media Cybernetics Co., Rockville, MD) and the pigmented area per epidermal area (PA/EA) was measured.(AJIRB-BMR-SMP-14-387, AJIRB-BMR-SMP-17-438)
6. Melanin content and tyrosinase activity assay
The cells were lysed with a 0.1 M phosphate buffer (pH 6.8) containing 1% Triton X-100 with a protease inhibitor cocktail (Roche, Basel, Switzerland). The supernatants were measured to determine the protein concentration using the Lowry assay system. Pellets were solubilized in 100 ml of 1 N NaOH for 3 hr at 60°C and the absorbance was measured at 490 nm to determine the melanin content relative to a standard curve using synthetic melanin (Sigma). For the assay of the tyrosinase activity, each sample was incubated with 2 mM L-DOPA (Sigma) in a 0.1 M phosphate buffer (pH 6.8) for 90 min at 37°C. After incubation, the tyrosinase activity was measured at 490 nm.
7. Lentivirus and adenovirus production
The human GDF15 cDNA was amplified by the polymerase chain reaction (PCR) from fibroblasts in our laboratory and then subcloned into pCDH-CMV-MCS-EF1-Puro lentivirus vector (System Biosciences, Mountain View, CA). For the knockdown of GDF15 expression, shRNA was prepared in a pLKO lentiviral vector (Sigma) and then amplified in 293TN cells. Fibroblasts were plated and grown in 6 cm culture dishes. After culturing overnight, they were infected with the lentivirus and the cells were then selected with 3.5 μM puromycin for one week. The shRNA sequences were as follows: sh-GDF15 #1: 5′ -CCGGTCTCAGATGCTCCTGGTGTTGCTCGAGCAACACCAGGAGCATCT
GAGATTTTTG-3′; sh-GDF15 #2: 5′
-CCGGCCGGATACTCACGCCAGAAGTCTCGAGACTTCTGGCGTGAGTAT CCGGTTTTTG-3′. To generate lentiviral particles, HEK-293TN cells were transfected with plasmid DNA (pGag-pol, pVSV-G, and pCDH-GDF15 or shGDF15) using Lipofectamine (Invitrogen, Carlsbad, CA). Viral supernatant was collected after 48 hr and transduced into normal human fibroblasts.
8. Semiquantitative real-Time PCR Analysis
Total cellular RNA was extracted using RNeasy Mini kit (Qiagen, Valencia, CA) and the cDNA was obtained using SuperScriptTMIII Reverse Transcriptase Kit (Invitrogen, Waltham, MA). Real-timePCR was carried out with iQTM SYBR® Green Supermix (Bio-Rad) using the following conditions: initial activation at 95°C for 5 min, followed by 40 cycles for 95°C for 15sec and 60°C for 1min. The primers used for real-time PCR as
follows: human 18S: 5’-CGGCTACCACATCCAAGGAA-3’, 5’-GCTGGAATTACCGCGGCT-3’, GDF15: 5′-CCCATGGTGCTCATTCA AAAG-3′, 5′-GCTCATATGCAGTGGCAGTCTT-3′, GFRAL: 5′-ACACGTTCCCATCATGGATTC-3′, 5′-GCTCATATGCAGTGGCAGTC TT-3′, MITF: 5’-AGAACAGCAACGCGCAAAAGAAC-3’, 5’-TGA TGATCCGATTCACCAAATCTG-3’, Tyrosinase: 5’-CACCACTTGGGCCT CAATTTC-3’, 5’-AAAGCCAAACTTGCAGTTTCCAC-3’, p16INK4: 5’-CCCAACGCACCGAA TAGTTA-3’, 5’-ACCAGCGTGTCCAGGAAG-3’
9. Western blot analysis
Cells were lysed in RIPA buffer (1% NP-40, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA) with a complete protease inhibitor (Sigma). The proteins were separated by SDS-polyacrylamide gel and transfer to a PVDF membrane (Millipore, Billerica, MA). The antibody against MITF was obtained from Abcam (Cambridge, UK), GDF15 and p-b-catenin (Ser675) were obtained from Novus Biologicals (Littleton, CO), p-GSK3b (Ser9) and GSK3b were obtained from Cell Signaling Technology (Danvers, MA), active b-catenin was obtained from Millipore, GFRAL was obtained from Invitrogen (Carlsbad, CA), and tyrosinase, p53, b-catenin, GAPDH, and actin were obtained from Santa Cruz Biotechnology (Dallas, TX), respectively.
10. Immunocytochemistry and immunohistochemical analysis
International, Rochester, NY) were fixed in 4% paraformaldehyde for 15 min at room temperature and permeated with 0.2% Triton X-100. Non-specific antibody binding was blocked by 1% BSA for 1 hr, and the cells were then incubated with anti-GDF15 (Novus Biologicals) overnight at 4°C. Immunohistochemical staining was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections. The histological sections (4 μm) were de-paraffinized and rehydrated in two changes of xylene and ethanol series. They were incubated in 0.05% trypsin in Tris-buffered saline for 20 min at 37°C for antigen retrieval. The primary antibodies used were as follows: anti-GDF15 (Novus Biologicals) and b-catenin (Millipore). All fluorescence photographs were taken using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and a Zeiss LSM 710 microscope and analyzed with Zeiss Axio Imager software (Carl Zeiss) at room temperature.
11. Statistical analysis
Data are presented as means +SD of independent determinations and were analyzed using Wilcoxon or paired Student's t-tests, with a p value < 0.05 considered as significant. IBM SPSS ver. 25 (IBM Corp., Armonk, NY) was used for all statistical analyses.
Ⅲ. RESULTS
1. UV induced senescent fibroblasts stimulate skin pigmentation.
To investigate the role of the senescent fibroblasts in skin pigmentation, I established in vitro models of UVA-induced senescent fibroblasts. Fibroblasts were irradiated with 5 J/cm2 UVA and incubated for seven days (Figure 1). Cellular senescence was analysed by senescence-associated β -galactosidase (SA-β-Gal) staining. In UV-induced senescent fibroblasts, 75.1% of fibroblasts were stained (Figure 2). The mRNA level of p16INK4 and protein level of p53 were increased in UV-induced senescent fibroblasts. which was detected by real-time PCR and western blotting (Figure 2). Normal human melanocytes were then co-cultured with fibroblasts (UVA, Normal or Sham) using Transwell system. In presence of UVA induced senescent fibroblasts, the melanin content and tyrosinase activity levels were significantly increased (Figure 3). The mRNA and protein expression levels of melanogenesis-associated proteins, microphthalmia-associated transcription factor (MITF) and tyrosinase were significantly up-regulated (Figure 4). The stimulatory action of UVA induced senescent fibroblasts in pigmentation was further confirmed in the ex vivo cultured skin. In presence of UVA induced senescent fibroblasts, increased basal pigmentation of the human skin was demonstrated by Fontana-Masson staining (Figure 5). The pigmented area/epidermal ared (PA/EA) ratio was measured by an image analysis. Taken together, these results indicate that senescent fibroblasts by UV irradiation stimulate skin pigmentation.
2. GDF15 expression is increased in senescent fibroblasts and photo-aged hyperpigmented skin.
The endogenous expression level of GDF15 in cultured skin cells was examined first. GDF15 mRNA and proteins were expressed in fibroblasts and also in melanocytes and keratinocytes analyzed by real-time PCR, ELISA western blotting (Figure 6) and immunohistochemical staining (Figure 7). Cytoplasmic and perinuclear staining outcomes of GDF15 were noted. Vimentin (green) / GDF15 (red) double-positive cells were observed in the in vivo skin, confirming GDF15 expression in fibroblasts (Figure 8). The GDF15 expression levels were significantly increased when the cultured fibroblasts senesce with UVA irradiation analyzed by real-time PCR and ELISA. Senescent fibroblasts by irradiated UVA secreted much more GDF15 compared to sham-irradiated normal cells (Figure 9). Furthermore, the GDF15 expression level was examined in young-age skin, aged skin and melasma lesional skin. We found that GDF15 expression levels are increased in aged skin compared to young-age skin in all 8 cases (Figure 10, Young vs Old, epidermis, 0.015 ± 0.013 vs. 0.06 ± 0.05, p=0.03 ; dermis, 0.0013 ± 0.001 vs. 0.021 ± 0.02, p=0.02, n=8). The GDF15 expression levels are increased in Melasma, a common pigmentary disorder associated with photoaging, compared to perilesional normal human skin in all 7 patients (Figure 11, Normal vs Melasma, epidermis, 0.026 ± 0.04 vs. 0.055 ± 0.04, p=0.07 ; dermis, 0.002 ± 0.001 vs. 0.008 ± 0.003, p=0.01, n=7). These findings indicated that senescent fibroblasts have increased GDF15 levels and that GDF15 may play a role in the development of aging pigmentation.
3. GDF15 derived from senescent fibroblasts stimulates melanogenesis in human melanocytes.
To investigate the role of GDF15 in skin pigmentation, young fibroblasts were infected with a GDF15-expressing lentivirus (Figure 12). GDF15 overexpression did not affect fibroblast proliferation analyzed by cell counting (Figure 13). Then, GDF15-overexpressed fibroblasts co-cultured with human melanocytes. In the presence of GDF15-overexpressed fibroblasts, the melanin contents and tyrosinase activity levels were significantly increased (Figure 14). The mRNA and protein expression levels of melanogenesis-associated proteins, microphthalmia-associated transcription factor (MITF) and tyrosinase were significantly up-regulated (Figure 15). GDF15 down-regulation in young or senescent fibroblasts using a shGDF15 lentivirus (Figure 17) was associated with decreased melanogenesis. GDF15 knockdown did not affect fibroblast proliferation analyzed by cell counting (Figure 18). Then, GDF15-downregulated fibroblasts co-cultured with human melanocytes. In the presence of GDF15-downregulated fibroblasts, the melanin contents and tyrosinase activity levels were significantly decreased (Figure 19). The mRNA and protein expression levels of melanogenesis-associated proteins, microphthalmia-associated transcription factor (MITF) and tyrosinase were significantly down-regulated (Figure 20). A stimulatory role of GDF15 on pigmentation was further demonstrated in the ex vivo human skin (Figure 16). GDF15-overexpressed or downregulated fibroblasts co-cultured with human normal skin. In the presence of GDF15-overexpressed fibroblasts, increased basal pigmentation of the skin was demonstrated by Fontana-Masson staining. Consistently , GDF15 down-regulated UV induced senescent fibroblasts decreased basal
pigmentation of the skin was demonstrated by Fontana-Masson staining (Figure 21). Taken together, these results indicate that senescent fibroblast-derived GDF15 has a stimulatory effect on skin pigmentation.
4. GDF15 stimulates b-catenin signaling.
Although the specific receptor for GDF15 remains to be identified in the peripheral tissue, several signaling pathways have been shown to be regulated by secreted GDF15. GDF15 activates the AKT signaling pathway through the activation of the ErbB2 receptor tyrosine kinase in several cancer cells (Kim et al., 2008). To elucidate the molecular mechanism by which GDF15 stimulates melanogenesis, the effects of GDF15 in the b-catenin signaling pathway were examined. GDF15 upregulated fibroblasts was associated with increased levels of b-catenin-Ser675 phosphorylation in human melanocytes, resulting in an increase in the total b-catenin level, phosphorylation of the GSK3b-Ser9 level, phosphorylation of the b-catenin-Ser675 level, which translocates into the nucleus, and active b-catenin level, which is a non-degraded b-catenin in human melanocytes (Figure 21). Consistently , GDF15 downregulated Young or UV induced senescent fibroblasts significantly reduced the phosphorylation of GSK3b-Ser9 and b-catenin-Ser675 in human melanocytes (Figure 22). Furthermore, an increased b-catenin nuclear translocation was observed in the human melanocytes cocultured with GDF15 overexpressed fibroblasts (Figure 23, 22.8% vs. 66.1%,). Consistently, a decreased b-catenin nuclear translocation was obseved in the melanocytes co-cultured with UV induced senescent fibroblasts infected shGDF15 (Figure 24, 69.6% vs. 26.8% vs.
26.4% ). These data strongly suggest that the GDF15 stimulates melanogenesis through b-catenin signaling.
Ⅳ. DISCUSSION
In skin pigmentation, interactions between melanocytes and neighboring skin cells (eg. keratinocytes, or fibroblasts) are very important. Ultraviolet (UV) is a major inducer of skin pigmentation. When exposed to UV, these neighboring skin cells secret many paracrine factors, such as α-MSH, endothelin-1, or secreted frizzled-related protein (sFRP2) (Kim et al., 2016; Rees, 2004). These paracrine factors up-regulate melanogenesis (Schallreuter et al., 2008; Yamaguchi and Hearing, 2009; Abdel-Malek et al.,2010).
In recently study, senescent fibroblasts and senescence-associated secretor phenotype (SASP) play an important role in the human skin pigmentation (Salducci et al., 2014; Kovacs et al., 2010). The interaction between melanocytes and senescent fibroblasts during the aging process contributes to the stimulation of melanogenesis. In senescent fibroblast, stromal cell-derived factor 1 (SDF1) deficiency drives skin pigmentation (Yoon et al., 2018).
In this study, senescent fibroblasts induced by UV irradiation stimulated skin pigmentation. I also found that GDF15 mRNA and protein were mainly expressed in fibroblasts in vivo and in vitro and much more increased in UV induced senescent fibroblasts and aged skin and melasma skin. In our previously RNA sequencing data in senescent fibroblast, GDF15 gene was found to be significantly up-regulated.
GDF15, also known as macrophage inhibitory cytokine-1 (MIC-1), nonsteroidal anti-inflammatory drug-activated gene (NAG-1), or placental
bone morphogenetic protein (PLAB), is a member of transforming growth factor beta (TGF-b) (Bootcov et al., 1997; Hromas et al., 1997; Lawton et al. 1997). In stressed condition, GDF15 is highly up-regulated and can be utilized as a biomarker for various disease (Emmerson et al., 2018). In skin, GDF15 is overexpressed in melanoma cell involved in histamine treatment ( Lee et al., 2012). In fibroblasts, GDF15 expression is seen in malignant skin pathologies and they have an effect on extracellular remodeling.(Unal et al., 2018).
Therefore, I hypothesized that GDF15 derived from UV induced senescent fibroblasts could stimulate melanogenesis in human melanocytes. I showed that GDF15 derived from UV induced senescent fibroblasts stimulates pigmentation in vitro. Moreover, the stimulatory action of GDF15 in pigmentation was further confirmed in human skin. In presence of GDF15 overexpressed fibroblasts, there was increased basal pigmentation of the human skin. Consistently, I showed that GDF15 down-regulated fibroblasts inhibits pigmentation in vitro. Moreover, the inhibitory action of shGDF15 in pigmentation was further confirmed in human skin. In presence of GDF15 downregulated fibroblasts, there was decreased basal pigmentation of the human skin.
Several studies have suggested that GDF 15 might mediate certain cellular responses through the Smad signaling pathway via the TGF-b receptor (Li et al., 2016). However, TGFb-Smad signaling pathway which downregulates PAX3 and MITF expression, resulted in decreased melanogenesis (Yang G et al., 2008). There were no change of Smad signaling pathway in human melanocytes cocultured with GDF15 overexpressing fibroblasts. GDNF family receptor alpha like (GFRAL) is a
receptor for GDF15 and ligand promotes weight loss (Breit et al., 2017; Mullican et al., 2017). GFRAL was not detected in human melanocytes and fibroblasts which was analyzed by western blotting (Figure. 26). GDF15 activates the AKT signaling pathway through the activation of the ErbB2 receptor tyrosine kinase in several cancer cells (Kim et al., 2008; Li S et al., 2018). GDF15 also activates AKT/ GSK-3b/ b-catenin pathway(Xu et al., 2017). Then, I identified b-catenin signaling in melanocyte cocultured GDF15 overexpressed or downreulated fibroblasts. The stimulatory effect of GDF15 was associated with b-catenin signaling. GDF15 increased the level of the active b-catenin, which is a non degraded form of b-catenin in melanocytes, and b-catenin-Ser675 phospholyation, which translocates into the nucleus. This result suggest that GDF15 derived from UV induced senescent fibroblasts increase melanogenesis in human melanocytes via b-catenin signaling.
The present study showed senescent fibroblasts have much more GDF15 and the GDF15 induces skin pigmentation. The stimulatory role of GDF15 in melanogenesis was thought to play a role in photo-aging pigmentation as the GDF15 expression was increased in the aged skin and melasma.
How the aged fibroblasts have upregulation of GDF15 is unclear, however, it has been recognized that GDF15 concentrations increase with aging and upregulated by p53 in response to various cellular stress (Fujita et al., 2016). The transcriptional activation of p53 in the skin has been known as an important factor in UV induced hyper-pigmentation (Murase et al., 2009). Collectively, it is likely that the elevation of GDF15 levels in aged fibroblasts of the skin may be due in part to the activation of p53 and AKT signaling caused by UV oxidative stress. It is speculated that aged
fibroblast-derived GDF15 induces skin pigmentation through crosstalk with melanocytes. This may provide valuable information regarding the development of target proteins for treatment strategies as well as a better understanding of the pathophysiology of aging pigmentation.
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이 증가하였으며, MITF와 티로시나아제 의 mRNA 양과 단백질 발현량이 증가 하였다. 반대로 GDF15 발현을 억제시킨 섬유아세포와 공동 배양한 멜라닌 세 포의 경우 멜라닌 양과 티로시나아제 활성이 감소하였으며, MITF와 티로시나 아제 의 mRNA 양과 단백질 발현량이 감소하였다. 더불어 GDF15 과발현 시킨 섬유아세포와 피부조직을 공동 배양한 경우에는 피부의 색소가 증가하는 것을 확인하였으며, GDF15 발현을 억제시킨 섬유아세포와 피부조직을 공동배양 후, 피부의 색소가 감소하는 것을 확인 하였다. GDF15는 멜라닌 세포에서 b-catenin 신호 전달 기전을 통해 MITF와 티로시나아제의 발현을 증가시켜 색 소형성과정에 영향을 미침을 확인하였다. 결론: 노화 섬유아 세포 유래 GDF15는 멜라닌 세포내에서 b-catenin 신호전달 기전을 통하여 멜라닌 형성을 자극하며, 노화 색소 침착에 GDF15가 중요한 역 할을 할 것으로 생각된다. 핵심어: 피부 색소, 노화 섬유아세포, 멜라닌 세포, GDF15, b-catenin
