Bone Morphogenetic Protein-4, a Novel Modulator of Melanogenesis
2006; Elsevier BV; Volume: 281; Issue: 35 Linguagem: Inglês
10.1074/jbc.m600580200
ISSN1083-351X
AutoresMina Yaar, Christina Wu, Heeyoung Park, Izabela Panova, Günther Schütz, Barbara A. Gilchrest,
Tópico(s)Cell Adhesion Molecules Research
ResumoBone morphogenetic proteins (BMPs), members of the transforming growth factor-β family, signal in many cells including neural precursors. Two receptors, types 1 and 2, coordinately mediate BMP signaling, and type 1 receptor has two forms: A and B. Using RT-PCR we found that neural crest-derived human melanocytes express BMP receptor-1A, -1B, and -2. Furthermore, melanocytes and the surrounding keratinocytes express BMP-4, suggesting both autocrine and paracrine effects of this molecule. Moreover, BMP-4 supplementation of cultured human melanocytes decreases melanin synthesis, tyrosinase mRNA, and protein. The mechanism of this BMP-4 effect on tyrosinase and ultimately on melanogenesis involves modest decreases of tyrosinase transcription rate and mRNA stability. Moreover, ultraviolet irradiation, the best recognized environmental stimulator of melanogenesis, down-regulated the mRNA of BMP receptor-1B in melanocytes. Our data provide evidence of a novel regulatory pathway for melanogenesis in human skin. Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β family, signal in many cells including neural precursors. Two receptors, types 1 and 2, coordinately mediate BMP signaling, and type 1 receptor has two forms: A and B. Using RT-PCR we found that neural crest-derived human melanocytes express BMP receptor-1A, -1B, and -2. Furthermore, melanocytes and the surrounding keratinocytes express BMP-4, suggesting both autocrine and paracrine effects of this molecule. Moreover, BMP-4 supplementation of cultured human melanocytes decreases melanin synthesis, tyrosinase mRNA, and protein. The mechanism of this BMP-4 effect on tyrosinase and ultimately on melanogenesis involves modest decreases of tyrosinase transcription rate and mRNA stability. Moreover, ultraviolet irradiation, the best recognized environmental stimulator of melanogenesis, down-regulated the mRNA of BMP receptor-1B in melanocytes. Our data provide evidence of a novel regulatory pathway for melanogenesis in human skin. Melanocytes are neural crest-derived cells that migrate to the epidermis during embryogenesis and subsequently synthesize and distribute melanin to surrounding keratinocytes (reviewed in Ref. 1Halaban R. Hebert D.N. Fisher D.E. Freedberg I.M. Eisen A.Z. Wolff K. Austen K.F. Goldsmith L.A. Katz S.I. Fitzpatrick's Dermatology in General Medicine. 1. McGraw-Hill, New York2003: 127-148Google Scholar). The rate-limiting enzyme in melanin biosynthesis is tyrosinase, and subsequent reactions involving tyrosinase and other melanogenic enzymes, tyrosinase-related proteins (TRP) 1 and 2, result in melanin synthesis and deposition in specialized organelles called melanosomes (1Halaban R. Hebert D.N. Fisher D.E. Freedberg I.M. Eisen A.Z. Wolff K. Austen K.F. Goldsmith L.A. Katz S.I. Fitzpatrick's Dermatology in General Medicine. 1. McGraw-Hill, New York2003: 127-148Google Scholar). The major known stimulator of melanin biosynthesis is ultraviolet (UV) irradiation (2Gilchrest B.A. Park H.Y. Eller M.S. Yaar M. Photochem. Photobiol. 1996; 63: 1-10Crossref PubMed Scopus (316) Google Scholar). UV irradiation directly stimulates melanogenesis in pigment cells (3Friedmann P.S. Gilchrest B.A. J. Cell. Physiol. 1987; 133: 88-94Crossref PubMed Scopus (276) Google Scholar) and also induces the expression and synthesis of a variety of cytokines, primarily of keratinocyte origin, that act in a paracrine fashion to further induce melanogenesis (reviewed in Ref. 4Imokawa G. Pigment Cell Res. 2004; 17: 96-110Crossref PubMed Scopus (307) Google Scholar). In addition, several keratinocyte-derived cytokines known to inhibit human melanogenesis have been identified. These include interleukin (IL) 3The abbreviations used are: IL, interleukin; TNF, tumor necrosis factor; BMP, bone morphogenetic protein; BMP-R, BMP receptor; TGF, transforming growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CAT, chloramphenicol acetyltransferase; ELISA, enzyme-linked immunosorbent assay.-1α, a cytokine that is also produced by melanocytes (5Swope V.B. Sauder D.N. McKenzie R.C. Sramkoski R.M. Krug K.A. Babcock G.F. Nordlund J.J. Abdel-Malek Z.A. J. Investig. Dermatol. 1994; 102: 749-753Abstract Full Text PDF PubMed Scopus (53) Google Scholar), IL-6, tumor necrosis factor (TNF)-α (6Swope V.B. Abdel-Malek Z. Kassem L.M. Nordlund J.J. J Invest. Dermatol. 1991; 96: 180-185Abstract Full Text PDF PubMed Google Scholar), and transforming growth factor (TGF)-β1 (7Martinez-Esparza M. Jimenez-Cervantes C. Beermann F. Aparicio P. Lozano J.A. Garcia-Borron J.C. J. Biol. Chem. 1997; 272: 3967-3972Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 8Martinez-Esparza M. Ferrer C. Castells M.T. Garcia-Borron J.C. Zuasti A. Int. J. Biochem. Cell Biol. 2001; 33: 971-983Crossref PubMed Scopus (33) Google Scholar). IL-1α, IL-6, and TNF-α were reported to inhibit melanogenesis by inhibiting tyrosinase activity (6Swope V.B. Abdel-Malek Z. Kassem L.M. Nordlund J.J. J Invest. Dermatol. 1991; 96: 180-185Abstract Full Text PDF PubMed Google Scholar). They also decrease melanocyte proliferation, yet are not toxic to the cells (6Swope V.B. Abdel-Malek Z. Kassem L.M. Nordlund J.J. J Invest. Dermatol. 1991; 96: 180-185Abstract Full Text PDF PubMed Google Scholar). However, the effect of these cytokines on tyrosinase expression, protein level, and stability is not known. More recently, it was reported that TGF-β1 also inhibits tyrosinase activity in B16 melanoma cells and in black non-agouti murine melanocytes (7Martinez-Esparza M. Jimenez-Cervantes C. Beermann F. Aparicio P. Lozano J.A. Garcia-Borron J.C. J. Biol. Chem. 1997; 272: 3967-3972Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 8Martinez-Esparza M. Ferrer C. Castells M.T. Garcia-Borron J.C. Zuasti A. Int. J. Biochem. Cell Biol. 2001; 33: 971-983Crossref PubMed Scopus (33) Google Scholar). In B16 cells, TGF-β1 decreases tyrosinase expression and increases protein degradation (7Martinez-Esparza M. Jimenez-Cervantes C. Beermann F. Aparicio P. Lozano J.A. Garcia-Borron J.C. J. Biol. Chem. 1997; 272: 3967-3972Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Interestingly, in these cells TGF-β1 also decreases the protein level of TRP-1 without affecting its mRNA level (7Martinez-Esparza M. Jimenez-Cervantes C. Beermann F. Aparicio P. Lozano J.A. Garcia-Borron J.C. J. Biol. Chem. 1997; 272: 3967-3972Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), suggesting that TGF-β1 inhibits melanogenesis, at least in part, by post-translational modification of melanogenic enzymes. Bone morphogenetic proteins are secreted signaling molecules that belong to the TGF-β superfamily (reviewed in Ref. 9Botchkarev V.A. J. Investig. Dermatol. 2003; 120: 36-47Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). While originally identified as molecules that stimulate bone formation (10Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3366) Google Scholar), it is now well established that BMPs affect other tissues, inducing various effects like proliferation that oppose differentiation and apoptosis (reviewed in Refs. 11Hogan B.L. Curr. Opin. Genet. Dev. 1996; 6: 432-438Crossref PubMed Scopus (662) Google Scholar and 12Miyazono K. Kusanagi K. Inoue H. J. Cell. Physiol. 2001; 187: 265-276Crossref PubMed Scopus (458) Google Scholar). To date, more than 20 different BMP proteins have been identified, all sharing structural homology and interaction with specific BMP receptors (11Hogan B.L. Curr. Opin. Genet. Dev. 1996; 6: 432-438Crossref PubMed Scopus (662) Google Scholar, 12Miyazono K. Kusanagi K. Inoue H. J. Cell. Physiol. 2001; 187: 265-276Crossref PubMed Scopus (458) Google Scholar). BMP effects depend on many factors including tissue concentration (11Hogan B.L. Curr. Opin. Genet. Dev. 1996; 6: 432-438Crossref PubMed Scopus (662) Google Scholar, 13Whitman M. Genes Dev. 1998; 12: 2445-2462Crossref PubMed Scopus (443) Google Scholar), BMP type, presence of antagonists, embryonic stage of the target tissue, and the type of receptors expressed by the target cells (9Botchkarev V.A. J. Investig. Dermatol. 2003; 120: 36-47Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). BMPs bind two transmembrane receptors, BMP receptor (BMP-R)1 and BMP-R2 (11Hogan B.L. Curr. Opin. Genet. Dev. 1996; 6: 432-438Crossref PubMed Scopus (662) Google Scholar). BMP-R1, when present as a monomer, binds BMP with low affinity. However, when BMP-R1 and BMP-R2 are present as a heterodimer, their affinity for BMP is substantially increased (11Hogan B.L. Curr. Opin. Genet. Dev. 1996; 6: 432-438Crossref PubMed Scopus (662) Google Scholar). Interestingly, to initiate signaling, BMP has to bind both receptors, inducing phosphorylation of an intracellular domain in BMP-R1 by BMP-R2 and activating intracellular signal transduction. Thus, both BMP-R1 and BMP-R2 are required to mediate BMP effects. Interestingly, there are several BMP-R1 receptors, BMP-R1A (Alk3), BMP-R1B (Alk6), and ActR-1 (Alk2) (14Kawabata M. Imamura T. Miyazono K. Cytokine Growth Factor Rev. 1998; 9: 49-61Crossref PubMed Scopus (456) Google Scholar) and in neural precursor cells signaling through BMP-R1A induces their proliferation while BMP-R1B induces their differentiation (15Panchision D.M. Pickel J.M. Studer L. Lee S.H. Turner P.A. Hazel T.G. McKay R.D. Genes Dev. 2001; 15: 2094-2110Crossref PubMed Scopus (289) Google Scholar), suggesting that receptor expression determines the BMP effect on cells. Noggin is a secreted BMP antagonist that competes for binding to BMP receptors (reviewed in Ref. 16Chen D. Zhao M. Mundy G.R. Growth Factors. 2004; 22: 233-241Crossref PubMed Scopus (1735) Google Scholar). Our group has recently shown that mice overexpressing noggin in the hair follicle epithelium display darker coat color than wild-type mice (17Sharov A.A. Fessing M. Atoyan R. Sharova T.Y. Haskell-Luevano C. Weiner L. Funa K. Brissette J.L. Gilchrest B.A. Botchkarev V.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 93-98Crossref PubMed Scopus (59) Google Scholar), demonstrating that BMP signaling influence murine melanogenesis in vivo. We now report that normal neonatal human melanocytes and keratinocytes express BMP-4. Melanocytes express in addition BMP-R1A, -1B, and -2. BMP-4 stimulation of melanocytes leads to decreased tyrosinase mRNA, in part by decreasing message stability and in part by repressing tyrosinase promoter activity. BMP-4 also decreases tyrosinase protein and activity and decreases melanin level. Finally, UV irradiation transiently decreases the level of BMP-R1B in melanocytes, consistent with its role as the major environmental melanogenic stimulator. Our study provides evidence of a novel signaling pathway that influences melanocyte function in human skin. Materials—Dulbecco's modified Eagle's medium, Medium 199, Leibovitz's L-15 medium, non-essential amino acids, and insulin were purchased from Invitrogen. Fetal bovine serum was purchased from Hyclone Labs, Logan, UT. Lipofectamine Plus Reagent was purchased from Invitrogen. Bio-Rad protein assay dye reagent was purchased from Bio-Rad. Dual-Luciferase Reporter Assay System was purchased from Promega, Madison, WI. Recombinant human BMP-4 was obtained from R&D Systems Inc., Minneapolis, MN. Tyrosinase antibodies were obtained from Novocastra Laboratories, Newcastle, UK and used at a dilution of 1:500. pRL-null vector containing Renilla luciferase reporter gene was purchased from Promega. Human tyrosinase cDNA (Pmel 34) was the kind gift of B. Kwon (Indianapolis, IN). The cell death detection ELISAPLUS kit was purchased from Roche Applied Science (Indianapolis, IN). Cell Culture—Primary melanocyte cultures were prepared from newborn foreskin as described (18Gilchrest B.A. Albert L.S. Karassik R.L. Yaar M. In Vitro Cell Dev. Biol. 1985; 21: 114-120Crossref PubMed Scopus (43) Google Scholar). Primary cultures of melanocytes were subcultured and maintained in Medium 199 supplemented with 2% fetal bovine serum, 10 μg/ml insulin, 10–9 m triiodothyronine, 10 μg/ml transferrin, 10 ng/ml epidermal growth factor, 10 μg/ml myoinositol, 1.4 × 10–6 m hydrocortisone, 10 ng/ml basic fibroblast growth factor, 80 μm dibutyryl cAMP, and bovine hypothalamic extract (100 μg/ml). MM4 human melanoma cells were obtained from Dr. U. Stierner (Gothenburg, Sweden) and maintained in Dulbecco's modified Eagle's medium (55%), L15 (27%), fetal bovine serum (15%), nonessential amino acids (1%), glutamine (2 mm), and insulin (10 μg/ml) (19Park H.Y. Perez J.M. Laursen R. Hara M. Gilchrest B.A. J. Biol. Chem. 1999; 274: 16470-16478Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Keratinocytes were established from neonatal foreskin or adult skin as described (20Gilchrest B.A. J. Investig. Dermatol. 1979; 72: 219-223Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 21Yaar M. Gilani A. DiBenedetto P.J. Harkness D.D. Gilchrest B.A. Exp. Cell Res. 1993; 206: 235-243Crossref PubMed Scopus (35) Google Scholar). For experimental use, first subcultures of the primary keratinocyte cultures were used. Cells were grown as previously described (21Yaar M. Gilani A. DiBenedetto P.J. Harkness D.D. Gilchrest B.A. Exp. Cell Res. 1993; 206: 235-243Crossref PubMed Scopus (35) Google Scholar) in serum-free Medium 199 (Invitrogen) containing 0.09 mm CaCl2 with 10 μg/ml insulin (Sigma), 10 ng/ml epidermal growth factor (Invitrogen), 10–9 m triiodothyronine (Sigma), 10 μg/ml transferrin (Sigma), 1.4 × 10–6 m hydrocortisone (Calbiochem-Behring Corp., La Jolla, CA), 2 μg/ml bovine serum albumin (Sigma), 10 μg/ml choline chloride (Sigma), and 10 μg/ml inositol (Sigma). Melanin Content—1 × 105 cells were spun at 2500 rpm for 15 min, and the pellet was then dissolved in 0.5 ml of 1 n NaOH. Melanin concentration was calculated by A475 and compared with a standard curve of synthetic melanin as previously described (22Eller M.S. Yaar M. Gilchrest B.A. Nature. 1994; 372: 413-414Crossref PubMed Scopus (174) Google Scholar). Tyrosinase Activity—Tyrosinase activity was measured up to 3 days after BMP-4 stimulation as described (23Pomerantz S.H. Biochem. Biophys. Res. Commun. 1964; 16: 188-194Crossref PubMed Scopus (129) Google Scholar). In brief, 5 × 105 cells were sonicated in 80 mm PO4 (pH 6.8) containing 1% Triton X-100, and tyrosinase was extracted for 60 min at 4 °C. 2–5 μg of cellular proteins were incubated with 31.25 μm l-tyrosine, 3.125 nml-dihydroxyphenylalanine, and 5 μCi of l-[3,5-3H]tyrosine (40–60 Ci per mmol) for 30–60 min at 37 °C. The reaction was stopped by addition of 500 μl of 10% trichloroacetic acid and 250 μl of 0.2% bovine serum albumin. Trichloroacetic acid-soluble material was reacted with Norit A, and released 3H2O was measured using a scintillation counter. The activity was expressed as counts per minute of 3H2O released per microgram protein per hour minus the nonspecific incorporation of radioactivity, determined by using lysate boiled with β-mercaptoethanol for 30 min (background, generally less than 5–10% of the sample). DNA Transfection—Plasmids containing the mouse 6.1-kb tyrosinase promoter (pTYRCAT6) as well as deletion mutations of the promoter (60 bp, 280 bp and 3.8 kb) (24Kluppel M. Beermann F. Ruppert S. Schmid E. Hummler E. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3777-3781Crossref PubMed Scopus (106) Google Scholar, 25Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar) and the empty vector (pBLCAT3) were transfected into MM4 cells together with a plasmid containing the Renilla luciferase reporter gene (pRL-null Vector). The expression of Renilla luciferase was used as an internal control to normalize the expression of firefly luciferase. Twenty-four hours before transfection, cells were plated at a density of 3 × 105/60-mm culture dish. Prior to transfection, medium was changed to Dulbecco's modified Eagle's medium, and 4 μg of pTYRCAT6 or pBLCAT3 together with 0.2 μg of pRL-null vector were suspended in 12 μl of Lipofectamine Reagent and 16 μl of Plus Reagent; the suspension was added to the medium for 3 h. Twenty-four hours after transfection, cells were supplemented with BMP-4 (25 ng/ml) or vehicle alone. Two days after BMP-4 supplementation, total cellular proteins were harvested and equal amounts of proteins were analyzed using the Dual-Luciferase Reporter Assay system. Fluorescence intensity was determined using a luminometer (Turner Biosystems Model TD-20/20). Northern Blot Analysis—Total RNA was isolated from cells with the Tri-Reagent kit (Invitrogen) following the protocol of the manufacturer. RNA concentrations were determined by absorbance at 260 nm. The purity of the preparations was determined by the 260/280 absorbance ratio, which was consistently ≥1.8. 20 μg/lane of denatured RNA samples were separated on 1% agarose gel containing 2.2 m formaldehyde as previously described (26Verdier-Sevrain S. Yaar M. Cantatore J. Traish A. Gilchrest B.A. Faseb. J. 2004; 18: 1252-1254Crossref PubMed Scopus (97) Google Scholar). RNA was transferred onto nylon membranes (Hybond, Amersham Biosciences) and was immobilized by shortwave UV irradiation (UV-Stratalinker 1800, Stratagene, La Jolla, CA). Blots were prehybridized for 2 h at 45 °C in a solution containing formamide, Denhart's solution, dextran sulfate, and denatured salmon sperm DNA. Hybridization was carried out overnight at 45 °C in the same solution containing 32P-labeled tyrosinase cDNA. Autoradiography with XAR film (Eastman Kodak, Rochester, NY) and development after overnight exposure at –70 °C was performed as described previously (27Yaar M. Eller M.S. DiBenedetto P. Reenstra W.R. Zhai S. McQuaid T. Archambault M. Gilchrest B.A. J. Clin. Investig. 1994; 94: 1550-1562Crossref PubMed Scopus (128) Google Scholar). mRNA Stability—Melanocytes were stimulated with BMP-4 (25 ng/ml) or with diluent as above. At the time of BMP-4 treatment actinomycin D (5 μg/ml) was added to cultures. Total cellular RNA was harvested before and at different intervals after stimulation, processed for northern blotting and hybridized to tyrosinase cDNA. Tyrosinase mRNA half-life was determined after densitometric analysis of band intensity. Western Blot Analysis—Total cellular proteins were collected in radioimmune precipitation assay buffer consisting of 0.25 m Tris-HCl, pH 7.5, 0.375 m NaCl, 2.5% sodium deoxycholate, 1% Triton X-100, 25 mm MgCl2, 1 mm phenylmethyl sulfonyl fluoride, and 0.1 mg/ml aprotinin as described (26Verdier-Sevrain S. Yaar M. Cantatore J. Traish A. Gilchrest B.A. Faseb. J. 2004; 18: 1252-1254Crossref PubMed Scopus (97) Google Scholar). Protein concentrations were determined by the Bradford method, and 50–100 μg protein/lane were processed for Western blot analysis as described (26Verdier-Sevrain S. Yaar M. Cantatore J. Traish A. Gilchrest B.A. Faseb. J. 2004; 18: 1252-1254Crossref PubMed Scopus (97) Google Scholar). Antibody reactions were performed with mouse monoclonal anti tyrosinase antibody followed by HRP-tagged goat anti-mouse IgG (secondary antibody, Bio-Rad). The secondary antibodies were used at 1:2000 dilution. Antibody binding was detected by the ECL detection kit (Amersham Biosciences), followed by autoradiography (Kodak X-Omatic AR). PCR Amplification—Total melanocyte RNA was harvested as above. cDNA was generated by reverse transcription as described (27Yaar M. Eller M.S. DiBenedetto P. Reenstra W.R. Zhai S. McQuaid T. Archambault M. Gilchrest B.A. J. Clin. Investig. 1994; 94: 1550-1562Crossref PubMed Scopus (128) Google Scholar). Briefly, 2 μg of total RNA in a 20-μl volume was reverse-transcribed using oligo pd(N)6 (Amersham Biosciences) as a primer. Reverse transcription was carried out for 1 h at 37°C. A 1-μl aliquot of this reverse transcription product (0.1μg of cDNA) was then amplified with 15 pmol of each forward and reverse primers complementary to the human published sequences (28Mohan R.R. Kim W.J. Chen L. Wilson S.E. Investig. Ophthalmol. Vis. Sci. 1998; 39: 2626-2636PubMed Google Scholar). BMP-R1A: upstream primer, GGACATTGCTTTGCCATCATA and downstream primer, CAGACCCACTACCAGAACTTT; BMP-R1B: upstream primer, GTTGTAAATGCCACCACCATT and downstream primer, GTCTGGTTTCTTGTCTTTTAT; BMP-R2: upstream primer, TGGCTGAACTTATGATGATTT and downstream primer, TGTTGGTGGAGAGGCTGGTGA; BMP-4, upstream primer, ACCTGAGACGGGGAAGAAAA and downstream primer, TTAAAGAGGAAACGAAAAGCA; Tyrosinase: upstream primer, TCAGACCCAGACTCTTTTCA and downstream primer, GAACCTGGACATTACTTTGA; GAPDH: upstream primer, GAAGGTGAAGGTCGGAGTCA and downstream primer, TTGATTTTGGAGGGATCTCG. Initially, 37 cycles of amplification were performed on cDNA as well as on RNA from each sample to assure no contamination of RNA samples with genomic DNA. Subsequently, all PCR reactions were terminated at the exponential phase of the amplification (BMP and BMP-Rs 29 cycles, tyrosinase 35 cycles, and GAPDH 26 cycles), which was found to be sufficient for optimal detection of BMP receptors, BMP-4, tyrosinase, and GAPDH and allowed analysis of mRNA transcript modulation. Denaturation was performed at 94 °C for 30 s, primer annealing at 58 °C for 1 min, and DNA polymerization at 72 °C for 1 min in a thermal cycler (MJ Research Inc., Waltham, MA). PCR products were separated over a 1% agarose gel in 1× TAE and stained with ethidium bromide. Quantitative Real-Time PCR (QRT-PCR)—The mRNA expression of BMP-R1A and -R1B was also analyzed using MyiQ™ optical module Real-Time PCR system (Bio-Rad) and iQ SYBR® Green Supermix containing the following primers: BMP-R1A: upstream primer, TTGTCCTGTGTCCTTAGTGATG and downstream primer, AGTAAATGGCTTGTATGAGATTGG; BMP-R1B: upstream primer, GCAGCACAGACGGATATTG and downstream primer, GGCAGTGTAGGGTGTAGG; β-actin: upstream primer, GCATTGTTACAGGAAGTCC and downstream primer, CATTACATAATTTACACGAAAGC. The PCR reaction was performed in a 50-μl volume containing 2–4 ng of cDNA. The final primer and probe concentrations were optimized for each primer/probe combination. Two-step PCR cycling was carried as follows: 95 °C 5 min × 1 cycle, 95 °C 0.5 min, 55 °C 0.5 min × 40 cycles. Apoptosis Analysis—DNA fragmentation as a measure of apoptosis was determined using the cell death detection ELISAPLUS kit and following the manufacturer's instructions (29Reinartz J. Bechtel M.J. Kramer M.D. Exp. Cell Res. 1996; 228: 334-340Crossref PubMed Scopus (33) Google Scholar). Briefly, melanocytes were stimulated with BMP-4 (25 ng/ml), and cells were harvested 24 and 48 h after stimulation. 1 × 104 cells were used as antigen source in a sandwich ELISA assay with a primary anti-histone antibody coated to the microtiter plate and a secondary anti-DNA antibody coupled to peroxidase. For positive control, we used the squamous carcinoma line SCC 12F (30Rheinwald J.G. Jermaine E. Beckell M.A. Harris C.C. Autrup H.N. Human Carcinogenesis. Academic Press, New York1983: 85-96Google Scholar), 24 and 48 h after irradiation with 30 mJ/cm2 (metered at 285 ± 5 nm). UV Irradiation—A solar simulator (Spectral Energy Corporation, Westwood, NJ) housing an appropriately filtered 1 kilowatt xenon arc lamp (XMN 1000–21; Optical Radiation Corp., Azusa, CA) adjusted to 2 × 10–4 watts cm–2 was used to irradiate melanocytes in phosphate-buffered saline through the Petri dish plastic cover. This system delivers a spectral output virtually identical to that of terrestrial sunlight (31Werninghaus K. Handjani R.M. Gilchrest B.A. Photodermatol. Photoimmunol. Photomed. 1991; 8: 236-242PubMed Google Scholar). Dosage was metered at 285 ± 5 nm with a research radiometer (Model IL1700A; Inter-national Light, Inc., Newburyport, MA) fitted with a UVB probe (detector SSE 240, diffuser W, filter UVB). Dose was calculated to contain 20 mJ/cm2 UVB, a dose that is physiologically relevant (3Friedmann P.S. Gilchrest B.A. J. Cell. Physiol. 1987; 133: 88-94Crossref PubMed Scopus (276) Google Scholar) and closely conforms to terrestrial sunlight (31Werninghaus K. Handjani R.M. Gilchrest B.A. Photodermatol. Photoimmunol. Photomed. 1991; 8: 236-242PubMed Google Scholar). Sham-irradiated melanocytes were handled identically but were placed under aluminum foil cover during the irradiation. After irradiation, cells were provided their original medium and were harvested at different intervals after irradiation. Densitometric Analysis—Autoradiograms of Western and Northern blots and PCR reactions were scanned into a computer (PC Dell™). Band intensity was determined after background subtraction using Bio-Rad Gel Doc 1000/2000 imaging densitometer. Normal Human Melanocytes Express BMP Receptor and BMP-4 mRNAs—Total cellular RNA was isolated from melanocyte and keratinocyte cultures. Using sequence-specific primers to BMP-R1A, -1B, and -2 and the PCR technique, only samples of reverse-transcribed melanocyte RNA showed bands at the expected molecular weights for BMP-R1A, -1B, -2, and BMP-4 and keratinocytes for BMP-4 when analyzed by ethidium bromide-stained agarose gel (Fig. 1). The bands were sequenced and identified as BMP-R1A, -1B, -2, and BMP-4, respectively, by comparison to the working draft sequence of the human genome (BLAST). BMP-4 Decreases Melanin Synthesis—Preliminary experiments suggested that 25 ng/ml of BMP-4 decrease melanogenesis. Therefore, subconfluent melanocyte cultures were supplemented with BMP-4 (25 ng/ml). Seventy-two hours after supplementation, proteins were harvested and total melanin/cell was determined. BMP-4 decreased melanin content by 47 ± 15% within 72 h (p < 0.04, n = 5) (Fig. 2A). Furthermore, tyrosinase activity, a reflection of melanin synthesis rate (1Halaban R. Hebert D.N. Fisher D.E. Freedberg I.M. Eisen A.Z. Wolff K. Austen K.F. Goldsmith L.A. Katz S.I. Fitzpatrick's Dermatology in General Medicine. 1. McGraw-Hill, New York2003: 127-148Google Scholar), was similarly inhibited by BMP-4, yielding a 34.0 ± 9.5% (mean ± S.E.) decreased activity within 72 h after stimulation compared with diluent-treated cells (p < 0.05, n = 3) (Fig. 2B). BMP-4 Down-regulates Tyrosinase mRNA and Protein Levels and Decreases Tyrosinase mRNA Stability—Because tyrosinase is considered the rate-limiting enzyme in melanin biosynthesis (1Halaban R. Hebert D.N. Fisher D.E. Freedberg I.M. Eisen A.Z. Wolff K. Austen K.F. Goldsmith L.A. Katz S.I. Fitzpatrick's Dermatology in General Medicine. 1. McGraw-Hill, New York2003: 127-148Google Scholar), we investigated BMP-4 effect on tyrosinase mRNA and protein levels. Subconfluent melanocytes were stimulated with 25 ng/ml BMP-4, and the expression of tyrosinase mRNA and protein was determined by Northern and Western blot analyses using tyrosinase cDNA (Fig. 3A) and anti-tyrosinase antibodies (Fig. 3B), respectively. The 2.4-kb band that represents tyrosinase mRNA was strongly expressed in melanocytes treated with diluent alone. However, tyrosinase mRNA level was lower in cultures treated with BMP-4 (42 ± 16%, and 44 ± 17% (mean ± S.E.) at 24 and 48 h, respectively, p < 0.02). Similarly, the 66–68-kDa band representing tyrosinase protein was reduced by BMP-4. In diluent-treated cultures, tyrosinase protein increased with time, consistent with culture maturation. However, tyrosinase levels in BMP-4-treated cultures were lower: 42 ± 20 and 53 ± 29% (mean ± S.E.) of diluent controls at 24 and 48 h, respectively, (p < 0.05), as determined by densitometry. These data suggest that the inhibitory effect of BMP-4 on melanin synthesis is mediated at least in part through inhibition of tyrosinase expression and synthesis. To determine if BMP-4 transcriptionally regulates tyrosinase, BMP-4 effect on a 6.1-kb mouse tyrosinase promoter as well as on three deletion mutations (3.8 kb, 280 bp, and 60 bp) was examined (24Kluppel M. Beermann F. Ruppert S. Schmid E. Hummler E. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3777-3781Crossref PubMed Scopus (106) Google Scholar, 25Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar) by transfecting MM4 melanoma cells that, like melanocytes, express BMP-R1A, -1B, and -2 (data not shown). There was no consistent effect of BMP-4 on the activity of the 3.8-kb, 280-bp, and 60-bp promoters (data not shown). However, BMP-4 decreased the activity of the 6.1-kb promoter by 27.4% ± 10.0% (mean ± S.E.) (p < 0.03, Student's paired t test, n = 10) (Fig. 4A). Although a small effect, it is significant, suggesting that BMP-4 effect on tyrosinase is in part transcriptionally mediated. To determine the BMP-4 effect on tyrosinase mRNA stability, melanocytes were stimulated with BMP-4 or diluent in the presence of actinomycin D. BMP-4 decreased tyrosinase mRNA half-life from ≫6 h to 4 h (Fig. 4B). BMP-4 Does Not Induce Melanocyte Apoptosis—To rule out the possibility that BMP-4 effect is the result of cell damage leading to apoptosis of more pigmented cells, melanocytes stimulated with BMP-4 (25 ng/ml) were harvested 48 and 72 h after stimulation and apoptosis was determined. There was no significant difference in the level of cytoplasmic DNA-histone between diluent-treated and BMP-4-treated cells (p = 0.12), while SCC12F cells irradiated with a UV dose of 30mJ/cm2 displayed a significantly higher level of the apoptotic products (p < 0.02), comparable to the manufacturer's positive control (Fig. 5). UV Irradiation Decreases the Expression of BMP-R1B—Because UV irradiation is the major environmental stimulus for melanogenesis (tanning), and UV irradiation of epidermal melanocytes alone can lead to increased melanin synthesis (3Friedmann P.S. Gilchrest B.A. J. Cell. Physiol. 1987; 133: 88-94Crossref PubMed Scopus (276) Google Scholar), we investigated the effect of solar-simul
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