
Seborrheic keratosis frequently occurs in sun-exposed areas on the skin of the elderly. Based on the predictable role of photoaging in seborrheic keratosis, it is found that upregulated guanine deaminase (GDA) is involved in keratinocyte senescence induced by ultraviolet (UV) radiation. Reactive oxygen species (ROS) generated by a metabolic end product such as uric acid have been found to play a role in keratinocyte senescence (Cheong and Lee, 2020). The role of skin aging in melasma has also been identified (Kim
Photoaging is often accompanied by skin hyperpigmentation. As a mechanism for UV-induced melanogenesis, paracrine factors derived from keratinocytes have been established (López
Adult skin specimens obtained from Cesarean sections and circumcisions were used for establishing cells for cell culture. The epidermis was separated from the dermis. Suspensions of individual epidermal cells were prepared. Keratinocytes were suspended in EpiLife Medium (Invitrogen, Carlsbad, CA, USA) supplemented with bovine pituitary extract (BPE), bovine insulin (BI), hydrocortisone, human epidermal growth factor, and bovine transferrin (BT) (Invitrogen). Melanocytes were suspended in Medium 254 (Invitrogen) supplemented with BPE, fetal bovine serum, BI, hydrocortisone, bFGF, BT, heparin, and phorbol 12-myristate 13-acetate (Invitrogen). In case of keratinocytes, passages from 3 to 5 were used for experiments. For coculture of keratinocytes and melanocytes, keratinocytes were seeded at 2×105 cells/well to six-well plates and incubated for 24 h. Keratinocytes were transfected with indicated genes. Four hours later, 1×105 melanocytes/well in 2 mL of EpiLife media without any supplement were added to transfected cells. After 24 h or 48 h, cells and the supernatants were harvested for next experiments. In case of supernatant culture as a conditioned media, 4 h after transfection of keratinocytes, 2 mL of EpiLife media without any supplement were added to transfected cells. At the same time, 1×105 cells/well melanocytes were seeded to another six-well plates. At 24 or 48 h later, media of melanocyte were changed to supernatants obtained from transfected keratinocytes. One day later, cells and supernatants were collected for the next step. For neutralization of bFGF or SCF, neutralizing antibodies to bFGF (Millipore, Billerica, MA, USA) or SCF (Abcam, Boston, MA, USA) were added to keratinocytes transfected with GDA. All collected cells and supernatants were used for Western blot analysis, immunohistochemistry, and ELISA.
To construct GDA, amplified PCR products were inserted to pCMV vector. GDA mutants with decreased guanine deaminase activity were constructed using a previously described method (Akum
One day following keratinocyte seeding (2×105 keratinocytes/well), cells were treated with appropriate concentrations (10 and 20 µM) of xanthine with or without allopurinol (50 µM) or uric acid (5 and 10 µM) (Sigma-Aldrich, St. Louis, MO, USA) for 24 to 48 h. Cells and supernatants were harvested for subsequent experiments.
At 24 h after GDA transfection, media of keratinocytes were changed without any supplement. N-acetyl-L-cysteine (NAC, 5 mM) (Sigma-Aldrich) or H2O2 (100 uM) were added to cells. At 24 and 48 h later, harvested cells were subjected to Western blot analysis for protein expression.
To measure cell viability, cells were incubated with MTT for 4 h. Precipitated formazan was dissolved in dimethyl sulfoxide (DMSO). The optical density was measured at 570 nm with background subtraction at 630 nm using a spectrophotometer.
Equal amounts of extracted proteins were resolved and transferred to nitrocellulose membranes. These membranes were incubated with antibodies to GDA, tyrosinase, SCF, xanthine oxidase (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA), MITF, bFGF, p-CREB, CREB (rabbit polyclonal; cell signaling technology, Beverly, MA, USA), and β-actin (mouse monoclonal; Sigma-Aldrich). After incubating with appropriate anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (Thermo Fisher Scientific, Waltham, MA, USA) or with anti-goat horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology), enhanced chemiluminescence solution (Thermo Fisher Scientific) was applied and signals were captured with an image reader (LAS-3000; Fuji Photo Film, Tokyo, Japan). Protein bands were then analyzed by densitometry. Concentrations of bFGF (R&D Systems, Minneapolis, MN, USA) and SCF (Abcam) in culture supernatants were measured using ELISA kits according to the manufacturer’s instructions.
After deparaffinization and rehydration, sections were preincubated with 3% bovine serum albumin. These sections were reacted sequentially with anti-GDA antibody, 1:200 Alexa Fluor-labeled goat anti-mouse IgG (488; Molecular Probes, Eugene, OR, USA), anti-bFGF (BD biosciences, San Jose, CA, USA), or anti-SCF (Santa Cruz Biotechnology) and Alexa Fluor-labeled goat anti-mouse IgG (594; Molecular Probes). Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich). Fluorescence images were evaluated using an image analysis system (Dp Manager 2.1; Olympus Optical Co., Tokyo, Japan) and Wright Cell Imaging Facility (WCIF) ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Statistical analyses of experimental data were performed using Student’s t-test. Results are expressed as means ± SD. A
To examine a potential role of paracrine melanogenic growth factors from GDA-overexpressing keratinocytes in melanogenesis, expression levels of growth factors derived from keratinocytes with or without GDA overexpression were checked. Effect of culture supernatant on melanogenesis was also examined. ELISA results showed that concentrations of bFGF and SCF were increased in culture supernatants obtained from viable GDA-overexpressing keratinocytes (Fig. 1A). Western blot analysis showed that relative expression levels of microphthalmia-associated transcription factor (MITF), the most critical transcription factor required for melanogenesis (Lee and Noh, 2013), and tyrosinase proteins were increased in melanocytes cultured with supernatants obtained from GDA-overexpressing keratinocytes (Fig. 1B). These increases of MITF and tyrosinase proteins were restored by anti-bFGF and SCF antibodies (Fig. 1B).
GDAΔ(76-84) and GDAΔ(76-84 and 350-402) were loss-of-function mutants of GDA (Cheong and Lee, 2020). Overexpression of these mutants of GDA did not stimulate relative expression levels of MITF or tyrosinase proteins (Fig. 2A). Immunofluorescence staining intensities against anti-bFGF antibody were weaker in keratinocytes with overexpression of GDAΔ(76-84) or GDAΔ(76-84 and 350-402) compared to those with wild type GDA overexpression (Fig. 2B). Staining intensities against anti-SCF antibody did not increase in keratinocytes with either GDAΔ(76-84) or GDAΔ(76-84 and 350-402) overexpression either (Fig. 2C). Concentrations of bFGF and SCF proteins were not increased in culture supernatants from keratinocytes with GDAΔ(75-84) overexpression (Fig. 2D).
Overexpression of loss-of-function mutants of GDA significantly reduced uric acid, a metabolic end product of guanine, compared with wild type overexpression (Cheong and Lee, 2020). Allopurinol inhibits xanthine oxidoreductase (XO), an enzyme involved in metabolizing xanthine into uric acid. Thus, expression levels of bFGF and SCF were examined in GDA-overexpressing keratinocytes and normal keratinocytes after treatment with xanthine in the presence or absence of allopurinol. Increased expression levels of bFGF and SCF in GDA-overexpressing keratinocytes were reduced by allopurinol (Fig. 3A). ELISA results also showed increased bFGF and SCF concentrations in culture supernatants of GDA-overexpressing keratinocytes. These increases were restored by allopurinol (Fig. 3B). Allopurinol also decreased expression levels (Fig. 3C) and released concentrations (Fig. 3D) of bFGF and SCF from normal keratinocytes treated with exogenous xanthine.
Exogenous xanthine can increase uric acid generation and release, similar to GDA overexpression (Cheong and Lee, 2020), suggesting that uric acid could be involved in upregulation of bFGF and SCF. However, allopurinol is an inhibitor of XO, which generates ROS (Furuhashi, 2020; Liu
GDA overexpression upregulated keratinocyte-derived melanogenic growth factors such as bFGF and SCF (Fig. 1A). Expression levels of MITF and tyrosinase proteins upregulated by GDA-overexpressing keratinocytes were restored by neutralizing antibodies against bFGF and SCF (Fig. 1B). These results suggest a role of GDA-overexpressing keratinocytes in melanogenesis via bFGF and SCF upregulation. In addition, overexpression of loss-of-function GDA mutants did not increase expression levels of MITF or tyrosinase proteins (Fig. 2A). They did not increase the expression levels of bFGF or SCF proteins as well (Fig. 2B-2D), supporting the idea that upregulation of bFGF and SCF may be associated with melanogenesis in GDA-overexpressing keratinocytes. Previously, keratinocyte-derived paracrine factors have been reported in connection with seborrheic keratosis or GDA upregulation; increased levels of ET-1 and endothelin B receptor have been considered as potential mechanisms of hyperpigmentation in seborrheic keratosis (Manaka
Loss-of-function mutants of GDA, which reduced uric acid production (Cheong and Lee, 2020), did not increase intracellular or extracellular bFGF or SCF proteins (Fig. 2B-2D). Allopurinol treatment may inhibit the production of uric acid as a metabolic end product (Vickneson and George, 2020; Orhan and Deniz, 2021). Further, allopurinol did not increase the production or release of bFGF or SCF in GDA-overexpressing or normal keratinocytes to which exogenous xanthine was applied (Fig. 3A-3D). These results suggest that uric acid could be involved in melanogenesis via generation of bFGF and SCF. In fact, increased expression levels of bFGF and SCF by exogenous uric acid (Fig. 4A) supported the role of uric acid in upregulating of bFGF and SCF.
In addition to inhibition of uric acid production, allopurinol as an XO inhibitor could obstruct ROS generation by XO (Furuhashi, 2020; Liu
In summary, uric acid produced by GDA upregulation in keratinocytes could stimulate melanogenesis via bFGF and SCF production not via ROS generation.
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HP20C0131).
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