The Essential Role of p53 in Hyperpigmentation of the Skin via Regulation of Paracrine Melanogenic Cytokine Receptor Signaling
2008; Elsevier BV; Volume: 284; Issue: 7 Linguagem: Inglês
10.1074/jbc.m805570200
ISSN1083-351X
AutoresDaiki Murase, Akira Hachiya, Yasuko Amano, Atsushi Ohuchi, Takashi Kitahara, Yoshinori Takema,
Tópico(s)Dermatologic Treatments and Research
ResumoHyperpigmentation of the skin is characterized by increases in melanin synthesis and deposition. Although considered a significant psychosocial distress, little is known about the detailed mechanisms of hyperpigmentation. Recently, the tumor suppressor protein p53 has been demonstrated to promote ultraviolet B-induced skin pigmentation by stimulating the transcription of a melanogenic cytokine, POMC (pro-opiomelanocortin), in keratinocytes. Given that p53 can be activated by various kinds of diverse stresses, including sun exposure, inflammation, and aging, this finding led us to examine the involvement of p53 in cytokine receptor signaling, which might result in skin hyperpigmentation. Immunohistochemical and reverse transcription-PCR analyses revealed the increased expression and phosphorylation of p53 in the epidermis of hyperpigmented spots, accompanied by the higher expression of melanogenic cytokines, including stem cell factor, endothelin-1, and POMC. The involvement of p53 in hyperpigmentation was also indicated by the significantly higher expression of p53 transcriptional targets in the epidermis of hyperpigmented spots. Treatment of human keratinocytes and melanocytes with known p53 activators or inhibitors, including pifithrin-α (PFT), demonstrated significant increases or decreases, respectively, in the expression of melanogenic factors, including cytokines and their receptors. Additionally, PFT administration abolished stem cell factor-induced phosphorylation of mitogen-activated protein kinase in human melanocytes. Furthermore, when organ-cultured hyperpigmented spots, in vitro human skin substitutes, and mouse skin were treated with PFT or p53 small interfering RNA, the expression of melanogenic cytokines and their receptors was significantly decreased, as were levels of tyrosinase and melanogenesis. Taken together, these data reveal the essential role of p53 in hyperpigmentation of the skin via the regulation of paracrine-cytokine signaling, both in keratinocytes and in melanocytes. Hyperpigmentation of the skin is characterized by increases in melanin synthesis and deposition. Although considered a significant psychosocial distress, little is known about the detailed mechanisms of hyperpigmentation. Recently, the tumor suppressor protein p53 has been demonstrated to promote ultraviolet B-induced skin pigmentation by stimulating the transcription of a melanogenic cytokine, POMC (pro-opiomelanocortin), in keratinocytes. Given that p53 can be activated by various kinds of diverse stresses, including sun exposure, inflammation, and aging, this finding led us to examine the involvement of p53 in cytokine receptor signaling, which might result in skin hyperpigmentation. Immunohistochemical and reverse transcription-PCR analyses revealed the increased expression and phosphorylation of p53 in the epidermis of hyperpigmented spots, accompanied by the higher expression of melanogenic cytokines, including stem cell factor, endothelin-1, and POMC. The involvement of p53 in hyperpigmentation was also indicated by the significantly higher expression of p53 transcriptional targets in the epidermis of hyperpigmented spots. Treatment of human keratinocytes and melanocytes with known p53 activators or inhibitors, including pifithrin-α (PFT), demonstrated significant increases or decreases, respectively, in the expression of melanogenic factors, including cytokines and their receptors. Additionally, PFT administration abolished stem cell factor-induced phosphorylation of mitogen-activated protein kinase in human melanocytes. Furthermore, when organ-cultured hyperpigmented spots, in vitro human skin substitutes, and mouse skin were treated with PFT or p53 small interfering RNA, the expression of melanogenic cytokines and their receptors was significantly decreased, as were levels of tyrosinase and melanogenesis. Taken together, these data reveal the essential role of p53 in hyperpigmentation of the skin via the regulation of paracrine-cytokine signaling, both in keratinocytes and in melanocytes. There are various types of hyperpigmentations (pigmented spots) of the skin characterized by increases in melanin synthesis and deposition, such as freckles, postinflammatory hyperpigmentation, UV-induced pigmentation (UV-melanoses), senile lentigines (age spots), pigmentation petaloides actinica, and melasma. These hyperpigmentations are well known to cause significant psychosocial distress, but little is known about their detailed mechanisms. Hyperpigmentation generally results from three major steps in the epidermis: the proliferation of melanocytes, the synthesis and activation of tyrosinase to produce melanin, and the transfer of melanosomes to keratinocytes (1Rosdahl I.K. Szabo G. J. Invest. Dermatol. 1978; 70: 143-148Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 2Imokawa G. Mishima Y. Cancer Res. 1982; 42: 1994-2002PubMed Google Scholar, 3Mishima Y. Imokawa G. J. Invest. Dermatol. 1983; 81: 106-114Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 4Okazaki K. Uzuka M. Morikawa F. Toda K. Seiji M. J. Invest. Dermatol. 1976; 67: 541-547Abstract Full Text PDF PubMed Scopus (79) Google Scholar). 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Natl. Acad. Sci. U. S. A. 1995; 92: 1789-1793Crossref PubMed Scopus (347) Google Scholar, 15Chakraborty A.K. Funasaka Y. Slominski A. Ermak G. Hwang J. Pawelek J. Ichihashi M. Biochim. Biophys. Acta. 1996; 1313: 130-138Crossref PubMed Scopus (298) Google Scholar, 17Wintzen M. Gilchrest B.A. J. Invest. Dermatol. 1996; 106: 3-10Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 19Funasaka Y. Chakraborty A.K. Hayashi Y. Komoto M. Ohashi A. Nagahama M. Inoue Y. Pawelek J. Ichihashi M. Br. J. Dermatol. 1998; 139: 216-224Crossref PubMed Scopus (74) Google Scholar, 20Hedley S.J. Gawkrodger D.J. Weetman A.P. MacNeil S. Pigment Cell Res. 1998; 11: 45-56Crossref PubMed Scopus (43) Google Scholar), SCF (stem cell factor) (9Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (155) Google Scholar, 11Imokawa G. Kobayashi T. Miyagishi M. J. Biol. Chem. 2000; 275: 33321-33328Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 22Hachiya A. Kobayashi A. Ohuchi A. Takema Y. Imokawa G. J. Invest. Dermatol. 2001; 116: 578-586Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 23Hachiya A. Kobayashi A. Yoshida Y. Kitahara T. Takema Y. Imokawa G. Am. J. Pathol. 2004; 165: 2099-2109Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), nitric oxide (18Roméro-Gaillet C. Aberdam E. Clément M. Ortonne J.-P. Ballotti R. J. Clin. Invest. 1997; 99: 635-642Crossref PubMed Scopus (247) Google Scholar), and their specific receptors. During the process of melanosome transfer from melanocytes to keratinocytes, the expression of a melanosome phagocytic protein, PAR2 (protease-activated receptor 2), is induced by UV irradiation, and inhibition of PAR2 activation prevents UVB-induced pigmentation in Yucatan swine skin (24Seiberg M. Pigment Cell Res. 2001; 14: 236-242Crossref PubMed Scopus (199) Google Scholar, 25Scott G. Deng A. Rodriguez-Burford C. Seiberg M. Han R. Babiarz L. Grizzle W. Bell W. Pentland A. J. Invest. Dermatol. 2001; 117: 1412-1420Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In UVB-induced pigmentation, which disappears a few months after the initial exposure, we previously demonstrated that the expression of SCF and ET-1 is remarkably enhanced at the mRNA transcript and protein levels in the earlier and later phases of UVB-induced pigmentation, respectively (23Hachiya A. Kobayashi A. Yoshida Y. Kitahara T. Takema Y. Imokawa G. Am. J. Pathol. 2004; 165: 2099-2109Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). SCF and ET-1 are both potent mitogens and melanogens, and they synergistically activate the phosphorylation of mitogen-activated protein kinase (MAPK) 2The abbreviations used are: MAPK, mitogen-activated protein kinase; UVB, ultraviolet B; 5-FU, 5-fluorouracil; PFT, pifithrin-α; NHEK, normal human epidermal keratinocyte; NHEM, normal human epidermal melanocyte; HSS, human skin substitute; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA; RT, reverse transcription. in human melanocytes (9Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (155) Google Scholar, 11Imokawa G. Kobayashi T. Miyagishi M. J. Biol. Chem. 2000; 275: 33321-33328Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). SCF also stimulates the expression of ETBR (endothelin B receptor) so that its enhanced expression is harmonized with that of ET-1 in human epidermis (23Hachiya A. Kobayashi A. Yoshida Y. Kitahara T. Takema Y. Imokawa G. Am. J. Pathol. 2004; 165: 2099-2109Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Taken together, those findings suggest that the biphasic expression of SCF and ET-1 intrinsically plays a pivotal role in the proliferation and melanogenesis of human melanocytes in vivo during UVB-induced hyperpigmentation. In addition to UVB-induced pigmentation, the contribution of two receptor-mediated signaling cascades (ET-1/ETBR and SCF/KIT) has also been reported to play a role in the formation of one type of persistent hyperpigmentation, senile lentigines, in which both long term sun exposure and chronological aging are thought to be involved (26Kadono S. Manaka I. Kawashima M. Kobayashi T. Imokawa G. J. Invest. Dermatol. 2001; 116: 571-577Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 27Hattori H. Kawashima M. Ichikawa Y. Imokawa G. J. Invest. Dermatol. 2004; 122: 1256-1265Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The higher expression of both ET-1 and SCF has been confirmed in the lesional skin of senile lentigines compared with control perilesional skin. These cytokine receptor networks have also been reported to be involved in other human hyperpigmentation disorders, such as dermatofibromas, café-au-lait macules, and seborrehoic keratoses (28Teraki E. Tajima S. Manaka I. Kawashima M. Miyagishi M. Imokawa G. Br. J. Dermatol. 1996; 135: 918-923Crossref PubMed Scopus (37) Google Scholar, 29Shishido E. Kadono S. Manaka I. Kawashima M. Imokawa G. J. Invest. Dermatol. 2001; 117: 627-633Abstract Full Text Full Text PDF PubMed Google Scholar, 30Okazaki M. Yoshimura K. Suzuki Y. Uchida G. Kitano Y. Harii K. Imokawa G. Br. J. Dermatol. 2003; 148: 689-697Crossref PubMed Scopus (57) Google Scholar). Such evidence indicates that the mechanisms of several types of hyperpigmentation, such as UV-induced pigmentation (suntanning) and senile lentigos, commonly share increased SCF/KIT and ET-1/ETBR signaling and reveals their coordinated roles in regulating epidermal melanogenesis as a part of skin homeostasis consistent with a previous study (31Grichnik J.M. Burch J.A. Burchette J. Shea C.R. J. Invest. Dermatol. 1998; 111: 233-238Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Additionally, Motokawa et al. (32Motokawa T. Kato T. Katagiri T. Suzuki I. J. Dermatol. Sci. 2004; 37: 120-123Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) reported that expression of the POMC (pro-opiomelanocortin) gene, which encodes a precursor of α-melanocyte-stimulating hormone, is higher in the epidermis of senile lentigines than in peripheral epidermal controls. Recently, Cui et al. (33Cui R. Widlund H.R. Feige E. Lin J.Y. Wilensky D.L. Igras V.E. D'Orazio J. Fung C.Y. Schanbacher C.F. Granter S.R. Fisher D.E. Cell. 2007; 128: 853-864Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar) focused on the role of p53, a tumor suppressor protein, in the induction of UV-induced epidermal hyperpigmentation by its direct activation of POMC transcription in keratinocytes, which results in the enhanced melanogenesis of melanocytes in a paracrine manner. They demonstrated multiple instances of in vivo hyperpigmentation due to activation of the p53 cascade, which mimics the pathway of UV-induced pigmentation. Given that p53 can be activated by various diverse stresses, including sun exposure, inflammation, and aging, their findings led us to assess the potential involvement of p53 in melanogenic paracrine cytokine receptor signaling that causes hyperpigmentation disorders, such as senile lentigines, which are strongly related to photoaging and chronological aging. Therefore, we investigated the involvement and the role of p53 in the regulation of melanogenic paracrine cytokine receptor networks in hyperpigmentation of the skin. We report for the first time that p53 plays a pivotal role in the formation of hyperpigmentation via the stimulation of paracrine cytokine networks in human epidermis. Materials—Normal human epidermal keratinocytes (NHEKs), melanocytes (NHEMs), and three-dimensional human skin substitutes (HSSs; MEL-300A) were purchased from Kurabo Co. (Osaka, Japan). C57BL/6J female mice 5–7 weeks old were supplied by CLEA Japan, Inc. (Tokyo, Japan). Human recombinant SCF, polyclonal antibodies specific for SCF and KIT, and the ET-1 ELISA kit were provided by Immuno-Biological Laboratories Co. (Gunma, Japan). Human recombinant ET-1 and anti-β-actin-specific antibody were supplied by Sigma. Antibodies specific for p53 (DO-7 and DO-1) and phospho-p53 were purchased from Dako Inc. (Carpinteria, CA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and Cell Signaling Technology, Inc. (Danvers, MA), respectively. Specific antibodies for p21, MDM2, and Gadd45a were supplied by Santa Cruz Biotechnology. The specific siRNA directed against human or mouse p53 and the control siRNA were provided by Invitrogen. Other chemicals were of reagent grade. Cell Culture—NHEKs were preliminarily incubated in Epi-Life medium (Kurabo) supplemented with 10 μg/ml insulin, 0.1 μg/ml human recombinant epidermal growth factor, 0.5 μg/ml hydrocortisone, 50 μg/ml gentamycin, 50 ng/ml amphotericin B, and 0.4% (v/v) bovine pituitary extract at 37 °C in an atmosphere of 5% (v/v) CO2. NHEMs were maintained in Medium 254 (Kurabo) supplemented with 5 μg/ml insulin, 5 μg/ml transferrin, 3 ng/ml human recombinant fibroblast growth factor, 0.18 μg/ml hydrocortisone, 3 μg/ml heparin, 10 ng/ml phorbol 12-myristate 13-acetate, 0.2% (v/v) bovine pituitary extract, and 0.5% (v/v) fetal bovine serum at 37 °C with 5% CO2, as previously described (6Yada Y. Higuchi K. Imokawa G. J. Biol. Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar). Cells were seeded in 6-well plates (BD Biosciences) at a density of 1.0 × 105 cells/well and then were incubated for 24 h, followed by incubation in the medium without bovine pituitary extract and human epidermal growth factor for NHEK and in the medium without phorbol 12-myristate 13-acetate for NHEM. Following another 24-h incubation, cells were treated with each reagent and incubated according to each experimental design. siRNA Transfection—NHEKs and NHEMs were transfected with 40 nm specific siRNA directed against p53 or a control siRNA, using Lipofectamine 2000 (Invitrogen) or TransIT-TKO Transfection Reagent (Mirus Bio Co., Madison, WI) according to the manufacturer's instructions. Suction Blister Method—Human epidermal sheets were obtained from the upper arms of five healthy 43–52-year-old Japanese subjects using the suction blister technique (34Furukawa F. Huff J.C. Weston W.L. Norris D.A. J. Invest. Dermatol. 1987; 89: 460-463Abstract Full Text PDF PubMed Google Scholar) with approval by the Ethical Committee of Kao Biological Science Laboratories. Following informed consent from the volunteers, samples were collected from each subject at three sites: a hyperpigmented spot in a sun-exposed area (site 1), a sun-irradiated control skin in the peripheral area of site 1 (site 2), and a nonexposed control skin from the ventral side of the upper arm (site 3). Blisters were induced with a 1- or 2.5-mm diameter syringe (Terumo, Tokyo, Japan) at each site for ∼1 to 2 h. The blister roof was removed with sterile scissors. Organ Culture of Human Pigmented Spots—Punch biopsy skins, with or without hyperpigmentation spots on the upper arm or shoulder from healthy 50–59-year-old Caucasian females with Type II skin (Stephens and Associates, Carrollton, TX) were used in this study. Collection of the skins was approved by the Institutional Review Board of IntegReview Ltd. (Austin, TX), and informed consent was obtained from the volunteers prior to the procedure. Each punch biopsy skin with hyperpigmentation was divided into two equal pieces, put onto collagen sponges (MedChem Products Inc., Woburn, MA), and incubated in Dulbecco's modified Eagle's medium or in a mixture of 50% Epilife, 50% Medium 254 without phorbol 12-myristate 13-acetate (Cascade Biologics, Portland, OR). One half was then treated with 10 μm PFT for 72 h, and the other half was incubated in medium without PFT. Media were changed every day, and finally the epidermal sheets were peeled off and used for gene expression analysis. Injection of siRNA into Mouse Ears Followed by UVB Irradiation—C57BL/6J female mice, 8 weeks old at the beginning of the study, were used. The animals had free access to food and chlorinated water and were housed in cages. Mouse ears were injected subepidermally with 5 μg of mouse p53-specific siRNA or control siRNA with a cationic polymer transfection reagent, in vivo-jetPEI™ (Polyplus-transfection SA, Illkirch, France). Immediately after the injection, they were irradiated with UVB irradiation with a dose of 50 mJ/cm2 of UVB light, at which dose most of the energy is emitted with a peak of emission near 306 nm, using UVB lamps (Sankyo Denki Co., Kanagawa, Japan). In total, they were administrated with siRNA injection and UVB irradiation three times at 24-h intervals. Measurement of Skin Color—The intensity of UVB-induced pigmentation was measured by a color difference meter (Nippon Denshoku Industries, Tokyo, Japan) 6 days after the first siRNA injection and UVB exposure and was expressed as the L* value. Immunohistochemistry—The specimens, obtained from hyperpigmented (spots) and from peripheral areas, were fixed in 10% buffered formalin and then embedded in paraffin. Following antigen retrieval by heating in a target retrieval solution (10 mm Tris-HCl buffer, including 1 mm EDTA (pH 9.0) or 10 mm sodium citrate buffer (pH 6.0)) at 95 °C for 45 min, the sections were treated with 0.3% H2O2 solution at room temperature for 30 min. Immunoreactivity was assessed using antibodies specific for p53 or p53 phosphorylated at serine 392 as primary antibodies and peroxidase-labeled anti-mouse or anti-rabbit IgG polyclonal antibodies (Dako Inc.) as secondary antibodies after blockage of nonspecific binding using 3% bovine serum albumin and 3% goat normal serum (Vector Laboratories, Burlingame, CA). Normal rabbit or mouse IgG (Vector Laboratories) in place of the primary antibody was used as a negative control. The sections were developed using the HistoMark® TrueBlue™ peroxidase system (Kirkegaard & Perry Laboratories, Gaithersburg, MD), rinsed in distilled water, and counterstained with NUCLEAR FAST RED (Vector Laboratories), followed by washing in running water. Quantitative Real Time RT-PCR—Total RNA extracted from human epidermal sheets and from cultured cells using an RNeasy microkit (Qiagen, Valencia, CA) and TRIzol reagent (Invitrogen), respectively, were used for the single-stranded cDNA synthesis. Real time quantitative RT-PCR was performed with the TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA), and its on-demand probes for genes of interest were normalized against RPLP0 (ribosomal protein large P0) using an ABI PRISM 7500 Sequence Detector System (Applied Biosystems). Western Blotting Analysis—Whole skin of mouse ears injected with p53-specific siRNA or control siRNA with or without UVB irradiation and cultured cells treated with 5 μm nutlin-3 (EMD Biosciences, Inc., San Diego, CA), 10 μg/ml 5-fluorouracil (5-FU; EMD Biosciences, Inc.), 10 μm pifithrin-α (PFT; EMD Biosciences, Inc.) or 40 nm siRNA specific for p53 together with Lipofectamine 2000 or TransIT-TKO, in the presence or absence of 10 nm SCF and/or 10 nm ET-1, were washed with phosphate-buffered saline, solubilized in 0.1 ml of radioimmune precipitation buffer (Sigma) supplemented with a protease inhibitor mixture (Roche Applied Science) and then homogenized using ultrasonication and/or a glass homogenizer. The resulting supernatants were collected and recovered as whole cell lysates, and their protein concentrations were determined using the BCA protein assay reagent (Pierce). The whole cell lysates were separated using 7.5, 10, or 15% SDS-polyacrylamide gel electrophoresis and transferred to Sequi-Blot® polyvinylidene difluoride membranes (Bio-Rad). The membranes were then incubated with the primary antibodies specific for p53 (DO-7 (1:1000 dilution) or DO-1 (1:400 dilution)), for p53 phosphorylated at serine 15 (1:1000 dilution), for SCF (1:100 dilution), for p21 (1:200 dilution), for MDM2 (1:200 dilution), for Gadd45a (1:200 dilution) for ERK1 and -2 (MAPK) (Thr(P)185/Tyr(P)187) (1:1000 dilution; Biomol, Plymouth Meeting, PA), for ERK1 and -2 (MAPK) (1:1000; Biomol dilution), for KIT (1:1000 dilution), or for tyrosinase (1.0 μg/ml; Upstate Biotechnology, Inc., Lake Placid, NY). The blots were then washed and incubated with appropriate secondary antibodies (anti-mouse IgG peroxidase-linked F(ab′)2 fragment (1:2500 dilution; GE Healthcare) or anti-rabbit IgG peroxidase-linked F(ab′)2 fragment (1:5000 dilution; GE Healthcare)). Immunoreactive protein bands were visualized with ECL Western blotting detection reagents (GE Healthcare). The amounts of protein loaded were normalized against a control protein, β-actin, using a monoclonal antibody specific for β-actin (1:5000 dilution; Sigma) as an internal standard. ELISA—NHEKs were seeded in 6-well plates at a density of 1 × 105 to 2 × 105 cells/ml and were treated with 5 μm nutlin-3, 10 μg/ml 5-fluorouracil, or siRNA specific for p53 together with Lipofectamine 2000. The conditioned media were then collected and quantified at 100 μl/well for the levels of ET-1 using an ET-1 ELISA kit (Immuno-Biological Laboratories Co.) according to the manufacturer's instructions. The standard curve was linear from 78.1 to 5000 pg/ml. Measurement of Melanin Contents in Three-dimensional HSSs and NHEMs—Three-dimensional HSSs were maintained in EPI-100NMM-113 medium (Kurabo Co.) at 37 °C with 5% CO2, according to the manufacturer's instructions. To investigate the effects of p53 on paracrine cytokine receptor signaling and melanin synthesis, HSSs were incubated with 10 μm PFT in the presence of 10 nm SCF and ET-1 for 14 days. Media were changed every other day. Photographs of HSSs were taken when cells were harvested. Cells were washed three times with phosphate-buffered saline, 5% (v/v) trichloroacetic acid, and diethyl ether mixed with three volumes of ethanol. Cells were then washed one time with diethyl ether and incubated at 50 °C for 2 h until dry. Cells were solubilized in 200 μl of 2 m NaOH, and melanin contents were measured using an absorbance meter (Microplate Reader model 550; Bio-Rad) at 405 nm and melanin standard (Sigma). To measure the amount of melanin content in human melanocytes, 5 × 104 NHEMs were cultured in the conditioned medium in 6-well plates and transfected with specific siRNA directed against p53 or a control siRNA. After the transfection, the wells were washed three times with phosphate-buffered saline, and cells were solubilized in 200 μl of 2 m NaOH on day 10. Melanin content in NHEMs was measured as described above. Statistics—The level of significance of differences was calculated by Student's t test, paired t test, ANOVA, or Fisher's exact test. A p value of <0.05 is considered statistically significant. The Expression and Phosphorylation of p53 Is Stimulated in Hyperpigmented Skin Accompanied by Significant Increases in the Expression of Paracrine Melanogenic Cytokines—In order to investigate the potential involvement of p53 in the increased expression of paracrine cytokines from keratinocytes, which might result in the formation of hyperpigmentation, mRNA transcripts or protein levels (and phosphorylations) were examined in pigmented spots, which are frequently observed as benign brownish patches on sun-exposed skin (Fig. 1A). Histologically, those tissues were clearly found to contain melanin granules in the basal and spinous layers of the epidermis accompanied by acanthosis (Fig. 1B). Quantitative real time RT-PCR analysis demonstrated significant increases in mRNA transcript levels of well known melanogenic paracrine cytokines (i.e. SCF, ET-1, and POMC, in the pigmented spots compared with peripheral control areas (Fig. 1C), which is consistent with previous studies (26Kadono S. Manaka I. Kawashima M. Kobayashi T. Imokawa
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