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Post-Transcriptional Regulation of Melanin Biosynthetic Enzymes by cAMP and Resveratrol in Human Melanocytes Richard A. Newton1, Anthony L. Cook1, Donald W. Roberts1,2, J. Helen Leonard2 and Richard A. Sturm1

Upregulation of microphthalmia-associated transcription factor (MITF) expression has been proposed to mediate melanogenesis stimulated by cAMP, whereas downregulation of MITF has been suggested to underlie the depigmentary effects of resveratrol, a promising chemotherapeutic found in red wine. We have assessed the contribution of MITF to pigmentation regulation by treating primary cultures of normal human melanocytes with the adenylate cyclase activator forskolin and/or resveratrol, then quantifying mRNA and protein levels for MITF, tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase (DCT). The inhibition of tyrosinase activity by resveratrol was not due to alterations in MITF, but instead was explained by both direct tyrosinase inhibition and a post-transcriptional effect that reduced the amount of fully processed tyrosinase. Glycosidase digestion revealed that the basis for the tyrosinase decrease was the retention of an immature form in the ER and subsequent loss of the mature, Golgi-processed enzyme. Elevation of intracellular cAMP by forskolin markedly increased protein levels for MITF, tyrosinase and DCT, however there was no concomitant increase in tyrosinase or DCT mRNA. This indicated that elevated levels of MITF were not sufficient to promote transcription of these melanogenic genes and that the increase in their protein abundance appeared to be predominantly mediated through post-transcriptional processing events. Journal of Investigative Dermatology (2007) 127, 2216–2227; doi:10.1038/sj.jid.5700840; published online 26 April 2007

INTRODUCTION the tyrosinase-related proteins (TYRPs), TYRP1 and dopa- Melanin synthesis (melanogenesis) is stimulated in human chrome tautomerase (DCT) (Aksan and Goding, 1998; melanocytes by an increase in the intracellular cAMP Bertolotto et al., 1998b). The expression of these two concentration, which can be facilitated by activation of the enzymes is important for later steps in the synthesis of melanocortin-1 receptor or following the addition of exo- eumelanin pigments. The transcription of MITF itself is genous cAMP elevating agents such as forskolin (Busca and enhanced by the activation of a cAMP response element Ballotti, 2000). Central to this upregulation is increased contained within its promoter to provide a mechanism by activity of tyrosinase, the rate-limiting enzyme for melano- which cAMP can stimulate melanogenic enzyme expression genesis. The molecular basis for this elevated activity has (Bertolotto et al., 1998a). This cascade therefore affords a been attributed to enhanced transcription of the tyrosinase simplified scheme that has contributed to MITF being gene mediated via the binding of microphthalmia-associated considered a master regulator of human pigmentation. transcription factor (MITF) to promoter E-box motifs (Yasu- Although MITF-mediated transcriptional activation of moto et al., 1997; Bertolotto et al., 1998a). MITF has also pigmentation genes is essential for the control of melanocyte been shown to bind and transactivate promoter constructs for cellular differentiation (Vance and Goding, 2004), the upregulation of endogenous tyrosinase, TYRP1, and DCT 1Melanogenix Group, Institute for Molecular Bioscience, University of gene expression in mature melanocytes does not seem to be Queensland, Brisbane, Queensland, Australia and 2Queensland Radium satisfactorily explained by a simple direct transcriptional Institute Research Unit, Queensland Institute of Medical Research, Brisbane, control mechanism. For example, a-MSH-elicited cAMP Queensland, Australia elevation in human melanocytes can increase protein levels Correspondence: Dr Richard A. Sturm, Institute for Molecular Bioscience, The for the three melanogenic enzymes without any accompany- University of Queensland, Brisbane, Queensland 4072, Australia. E-mail: [email protected] ing change in mRNA levels, suggesting mediation via post- Abbreviations: NHM, normal human melanocytes; TYR, tyrosinase; MITF, transcriptional events (Abdel-Malek et al., 1995). Support for microphthalmia-associated transcription factor; ER, endoplasmic reticulum; the concept that as yet unidentified mechanisms underlie the DCT, dopachrome tautomerase; TYRP1, tyrosinase-related protein-1; Q-RT- upregulation of tyrosinase elicited by activation of the cAMP PCR, quantitative real-time polymerase chain reaction; OCA, oculocutaneous pathway was provided by a more recent study utilizing albinism; Endo H, endoglycosidase H; PNGase F, peptide: N-glycosidase F; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. adenovirus encoding wild-type MITF (Gaggioli et al., 2003). Received 11 September 2006; revised 4 February 2007; accepted 19 February Infection of human melanocytes with the recombinant virus 2007; published online 26 April 2007 showed that the resultant elevation of MITF levels did not

2216 Journal of Investigative Dermatology (2007), Volume 127 & 2007 The Society for Investigative Dermatology RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

increase expression of endogenous melanogenic enzymes. tyrosinase activity of approximately 25% evident in lysates However, this same study also provided evidence that MITF within 30 minutes of adding resveratrol to melanocyte expression does still have a crucial involvement in the cultures (Figure 1b), which indicated a possible direct regulation of human melanocyte pigmentation by demons- inhibitory effect of resveratrol. Direct inhibition of tyrosinase trating an inhibitory effect of MITF-dominant-negative re- by resveratrol was anticipated by virtue of its polyphenol combinant adenovirus on endogenous levels of tyrosinase structure and was assessed in vitro by adding resveratrol and TYRP1 proteins (Gaggioli et al., 2003). directly to untreated cell lysates and then measuring A role for MITF levels in determining the extent of tyrosinase activity (Figure 1c). DOPA oxidation activity was melanocyte pigmentation is also provided by studies utilizing decreased by 15% when resveratrol was added to melano- a variety of depigmenting agents that have demonstrated cyte lysates at 20 mg/ml (88 mM), a finding in accordance with preceding reductions in MITF expression (Jimenez-Cervantes an in vitro study of the effects of resveratrol added to a et al., 2001; Kim et al., 2002a, 2004, 2006; Choi et al., 2005). preparation of murine tyrosinase where the IC50 for resver- Resveratrol (3,5,40-trihydroxy-trans-stilbene), a polyphenol atrol-mediated inhibition of tyrosine oxidation was in excess found in red wine, has been proposed to represent another of 100 mM (Kim et al., 2002b). However, we also found that such agent because it has been shown to both decrease skin the in vitro effect of resveratrol on tyrosinase activity was pigmentation and inhibit MITF promoter activity (Lin et al., considerably more potent when determined under the 2002). Interest in this compound has shown a marked renewal conditions of the tritium release assay resulting in a 75% in recent years, being initially triggered by its identification as a decrease in activity (Figure 1c) with an apparent IC50 of chemotherapeutic agent for skin cancer (Jang et al., 1997) and approximately 2 mM (data not shown). This apparent dis- subsequently from its protective effects against heart disease cordance between the two tyrosinase assays, which contrasts and ability to extend lifespans of lower organisms (reviewed in with the excellent agreement obtained when used for the Baur and Sinclair, 2006). We have examined the depigmenta- quantitation of the cell culture effects of resveratrol, suggests tion effects of resveratrol and assessed whether MITF modula- that the direct, in vitro inhibitory action of resveratrol on tion is an essential step in the negative regulation of tyrosinase is influenced by the nature and/or concentration of melanogenesis by this agent. In addition, we have investigated substrate. For example, it is possible that resveratrol may the contribution of elevated MITF expression to the cAMP- compete more efficiently for L-DOPA binding to tyrosinase in mediated upregulation of melanogenic enzymes in primary the tritium release assay, where L-DOPA is present at a low human melanocytes. This has led to the identification of the concentration as a cofactor, rather than in the DOPA oxidase important role of post-transcriptional regulation of tyrosinase assay where it is present at a high concentration as a in governing melanin pigment synthesis. substrate. Inhibition of tyrosinase activity in NHM by resveratrol therefore has two components, a direct inhibition RESULTS of enzyme activity that is expected to be maximal in the early Resveratrol elicits a time-dependent decrease in tyrosinase stages of treatment, and a temporal-dependent, indirect activity in human melanocytes that is unaffected by co- downregulation of enzyme activity. treatment with forskolin It is well established that treatment of cultured NHM with To evaluate the ability of resveratrol to decrease melanin cAMP elevating agents can increase tyrosinase activity and synthesis we assayed the tyrosinase activity of melanocytes, subsequent melanin synthesis (Hunt et al., 1994a, b; Abdel- both in situ (Figure 1a) and from extracts of treated cultures Malek et al., 1995; Im et al., 1998). In concert with these (Figure 1b, d). Tyrosinase activity was measured because it is reports, we found that forskolin treatment of cultured NHM known to closely correlate with melanin content of cultured increased their tyrosinase activity (Figure 1d). Combined human melanocytes (Halaban et al., 1983; Wakamatsu et al., treatment with resveratrol and forskolin, however, resulted in 2006) and provides a more sensitive indicator of changes in a decrease in tyrosinase activity similar to that seen with melanin synthesis than determination of total melanin levels. resveratrol alone, whether measured from extracts of treated Treatment of normal human melanocytes (NHM) with cells (Figure 1d) or in situ (data not shown). The mechanism resveratrol (20 mg/ml) for 24 hours profoundly decreased underlying resveratrol-mediated inhibition of melanin synth- in situ tyrosinase activity without affecting cell number esis was therefore anticipated to be equally evident in cells (Figure 1a). Although concentrations lower than 20 mg/ml co-treated with forskolin. resulted in less inhibition of tyrosinase activity in NHM, a Melanogenesis is normally coupled with increased mela- significant decrease in activity was still apparent using only nocyte dendricity (Busca and Ballotti, 2000) and is exempli- 0.25 mg/ml of resveratrol (data not shown) demonstrating the fied by the ability of forskolin to increase the dendricity of potency and dose dependency of the effect. The resveratrol- NHM (Figure 1e). Quantitation of melanocyte dendricity for mediated decrease in tyrosinase activity, quantitated by both Figure 1e revealed that the effect of forskolin was mainly 3,4-dihydroxyphenylalanine (DOPA) oxidation and tritium apparent as an increased number of primary dendrites (2.54 release from [3H]-tyrosine, was strongly time-dependent, per cell) compared with the control average (2.03 per cell). requiring 18–24hours for the maximal 80% inhibition Secondary dendritic branching was also promoted by (Figure 1b). This indicated that an indirect modulation of forskolin exposure, with the proportion of melanocytes cellular processes were involved in the profound reduction in having at least one branching dendrite increased from 3 to tyrosinase activity. There was also a smaller decrease in 21%. Resveratrol has been reported to promote apoptosis of

www.jidonline.org 2217 RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

a Ctrl Rsvl

In vitro effect b NHM NHM c 3 DOPA oxidation [ H]Tyrosine assay DOPA assay [3H]Tyrosine 100 100 100 75 75 75 50 50 50 25 25 (% control) 25 TYR activity TYR activity 0 0 0 Ctrl 0.5 1 2 3 4 6 18 24 Ctrl 0.5 1 2 3 4 6 18 24 Ctrl Rsvl Time (hours) Time (hours) Dendricity deNHM DOPA oxidation

150

100 Ctrl Fsk 50 (% control) TYR activity

0 Ctrl Rsvl Fsk Rsvl +Fsk

Rsvl Rsvl+Fsk

Figure 1. Resveratrol and cAMP have opposing effects on tyrosinase activity in NHM. (a) In situ tyrosinase activity determined by incubation of cells in L-DOPA, following 24 hours treatment with either vehicle (Ctrl) or 20 mg/ml resveratrol (Rsvl). Images were captured under identical conditions using bright 3 field microscopy. Bar ¼ 40 mm. (b) Time courses for tyrosinase activity measured by both L-DOPA oxidation and tritium release from [ H]tyrosine using lysates obtained from NHM treated for the indicated periods of time with resveratrol. Error bars indicate the average7range of two melanocyte strains. (c) The direct effect of resveratrol on tyrosinase activity was determined by adding resveratrol directly to NHM lysates at an equivalent concentration to that added to cell cultures. Both the L-DOPA oxidation and tritium release assays were used to quantitate the effect. Error bars represent the mean7SEM of three independent experiments. (d) Tyrosinase activity of NHM lysates following treatment of NHM with resveratrol alone, forskolin (Fsk) alone (10 mM), or both agents. Values are given as the mean7SEM (n ¼ 4) of four different melanocyte strains, normalized for total soluble protein and expressed relative to the activity of vehicle-treated cells, as detailed in materials and methods. (e) Changes in dendritic morphology following the same treatment regime described in (d) were assessed by phase- contrast microscopy, with dendrite length quantified using ImageJ software. Bar ¼ 40 mm. All of the above results are representative of at least two independent experiments using at least two melanocyte strains.

human melanoma cells (Niles et al., 2003); however, survival resveratrol elicited a synergistic increase in dendricity (Figure of primary melanocytes was not compromised by resveratrol 1e). In particular, the percentage of melanocytes showing and surprisingly, treatment with resveratrol resulted in a secondary dendritic branching (63%) was markedly higher positive effect on melanocyte dendricity that was mainly than that seen with either forskolin (21%) or resveratrol (15%) evident as an enhancement of dendrite length (Figure 1e). alone, and contributed to the dramatic increase in total Thus, the total dendrite length per cell was 1.7-fold higher dendrite length following combination treatment (2.5-fold than in control cells despite a similar number of primary greater than control). Therefore, resveratrol has the unusual dendrites (2.1 per cell). Further, 24-hour treatment with the property of being both a positive regulator of melanocyte melanogenesis inhibiting combination of forskolin plus dendricity and a negative regulator of melanogenesis.

2218 Journal of Investigative Dermatology (2007), Volume 127 RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

a 1 hour 3 hours 12 hours 24 hours in a corresponding increase in MITF protein, evident as a Rsvl ––++––++ ++––++ doublet with the higher molecular weight band correspond- ing to an ERK phosphorylated form (Hemesath et al., 1998). Fsk –+ +– ++––++ ++ –– Treatment of NHM with resveratrol resulted in only a 25% P-CREB decrease in MITF transcript levels by 24 hours (Figure 2b), but with no discernable affect on protein abundance (Figure 2a) P-ERK1/2 and importantly did not reduce the elevated MITF levels elicited by forskolin. Furthermore, there was no apparent ERK1/2 effect of resveratrol on the ERK-mediated phosphorylation status of MITF that could potentially alter stability and/or DNA binding. This indicated that reduced MITF function MITF does not explain the inhibition of tyrosinase activity by resveratrol. b 400 Ctrl Fsk Tyrosinase protein, but not mRNA, is differentially regulated by Rsvl+Fsk 300 Rsvl forskolin and resveratrol As MITF is widely regarded as the key regulator of tyrosinase 200 transcription, we determined the extent to which the levels of MITF following forskolin and/or resveratrol treatment were (% control) MITF mRNA reflected in changes in the steady-state expression of 100 tyrosinase mRNA (Figure 3a). Resveratrol did not significantly alter tyrosinase transcript levels in NHM at any of the time 0 points examined, in agreement with the lack of effect of this 3 hours 12 hours 24 hours agent on MITF expression (Figure 2a). Interestingly, there was Figure 2. Effects of forskolin and resveratrol on MITF levels in NHM. no significant alteration in tyrosinase RNA expression (a) Western-blot analysis of whole-cell lysates prepared from NHM treated for following either the forskolin or forskolin plus resveratrol the indicated times with resveratrol alone, forskolin alone, or both agents. treatment regimes (Figure 3a), despite the fact that MITF Phospho-specific antibodies were used to detect phosphorylated CREB and expression is increased up to 3.5-fold in the presence of ERK (p44/p42 MAPK) because these activated forms are involved in the forskolin (Figure 2b). Tyrosinase protein was then examined transcriptional upregulation and phosphorylation of MITF, respectively. The monoclonal MITF antibody identified the protein as a doublet, with the upper to see if post-transcriptional changes could potentially band previously shown to correspond to MITF that has undergone MAPK account for the observed alterations in tyrosinase activity. phosphorylation (Hemesath et al., 1998). Similar protein levels were verified Western analysis revealed that human tyrosinase appeared using a phosphorylation-independent ERK antibody. (b) Q-RT-PCR analysis of mostly as an B80 kDa species (Figure 3b), the size of the MITF RNA levels following the same treatment regime applied for protein mature, fully Golgi-processed form (Halaban et al., 1997). analysis. Transcript abundance was normalized against 18S ribosomal RNA Exposure of NHM to resveratrol led to a time-dependent and expressed relative to the 3 hours control. Each experiment utilized two melanocyte strains and are presented as the mean7SEM (n ¼ 4) of two decrease in abundance of mature tyrosinase and, crucially, independent experiments. this was also seen following co-treatment with forskolin (Figure 3b, c). Therefore, a post-transcriptionally mediated decrease in mature tyrosinase protein can account for resveratrol-elicited reductions in tyrosinase activity. Forskolin MITF levels are increased by forskolin but are unaltered by treatment alone markedly increased the level of mature resveratrol tyrosinase after 24 hours indicating that a post-translation To ascertain whether decreased MITF expression could regulatory mechanism could potentially explain the elevation underlie the depigmentation effects of resveratrol, the levels in tyrosinase activity produced by this agent. However, the of MITF mRNA and protein were determined over the time extent of this increase in fully processed tyrosinase only frame that inhibition of tyrosinase activity occurred (Figure 2). seemed to be partially reflected as elevated tyrosinase activity In addition, the phosphorylation status of cAMP-responsive (Figure 1c). This may reflect the fact that alterations in the element-binding protein (CREB) and extracellular-regulated catalytic activity of tyrosinase cannot be explained simply by kinase (ERK) were also measured (Figure 2a) because these changes in tyrosinase abundance (Iozumi et al., 1993) activated forms can modulate the levels of MITF in because mechanisms regulating the activity of tyrosinase melanocytes via increased transcription or decreased stabi- within the melanosome, the site of melanin synthesis, are also lity, respectively (Vance and Goding, 2004). Forskolin crucial (Fuller et al., 2001). treatment of NHM induced a rapid increase in phosphory- lated CREB that was temporally associated with increased Retention of immature tyrosinase in the endoplasmic reticulum transcription of MITF (Figure 2b), in agreement with the by resveratrol prevailing concept that activated CREB confers cAMP Examination of the relative abundance of the different forms responsiveness to the MITF promoter (Price et al., 1998; of tyrosinase detected by Western analysis indicated potential Busca and Ballotti, 2000). This increase in RNA was reflected effects of resveratrol on the trafficking of tyrosinase. Thus, in

www.jidonline.org 2219 RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

a Ctrl Endo H PNGase F Fsk Rsvl – – ++–– ++ –– ++ 125 Rsvl+Fsk Rsvl Fsk – + + –– ++ –– ++ – 100 100 TYR 70 75 55 50 40 (% control) TYR mRNA GAPDH 25 35

0 Figure 4. Endo H sensitivity of tyrosinase from resveratrol and forskolin 3 hours 12 hours 24 hours treated NHM. Cell extracts obtained following 24 hours treatment with either resveratrol, forskolin, or both agents were subsequently incubated in the b 1 hour 3 hours 12 hours 24 hours presence or absence of the glycosidases, Endo H and PNGase F. Western-blot Rsvl –– ++ ––++ – – ++ –– ++ analysis for tyrosinase was then used to monitor the extent of digestion. Fsk –––++ ++ –––– ++ ++ – Arrows at approximately 80 and 60 kDa indicate bands corresponding to the kDa mature and PNGase F digested forms of tyrosinase, respectively. An Endo 100 H-sensitive band is coincident with the 70 kDa marker. Samples were run on 80 TYR 70 60 two gels (indicated by gap) and blots processed in parallel under identical 55 conditions. Vertical lines indicate removal of two intervening lanes. Identical GAPDH 40 35 results were obtained in a second independent experiment. c Ctrl 200 Fsk Rsvl+Fsk 150 Rsvl The treatment of NHM with proteasomal inhibitors has previously been shown to induce the accumulation 60 kDa 100 tyrosinase protein, indicating that a portion of recently synthesized tyrosinase is targeted for proteolytic degradation (% control) TYR protein 50 (Halaban et al., 1997). The loss of a similarly sized tyrosinase species following resveratrol treatment may therefore repre- 0 Ctrl 1 3 12 24 131224 1 3 12 24 sent accelerated transfer of newly synthesized tyrosinase to hours hours hours the proteasomal complex. The 60 kDa band was not seen in Figure 3. Effects of forskolin and resveratrol on tyrosinase levels in NHM. the control extracts of Figure 4, which may be a consequence (a) Q-RT-PCR analysis of tyrosinase RNA levels following treatment of NHM of the lower protein concentration present in these lysates. with resveratrol alone, forskolin alone, or both agents, for the indicated times. Glycosidase digestion was utilized to further evaluate Error bars indicate the mean7SEM (n ¼ 4), relative to the 3 hours control, aberrant trafficking of tyrosinase following treatment with and were obtained from two melanocyte strains from two independent resveratrol. Digestion with Endo H cleaves oligosaccharides experiments. (b) Western-blot analysis of tyrosinase protein levels in whole- cell lysates prepared from NHM treated as described above. Similar protein added in the endoplasmic reticulum (ER) but not those levels were verified using an antibody against GAPDH. Samples were run on matured in the Golgi, whereas peptide: N-Glycosidase F two gels (indicated by gap) and blots processed in parallel under identical (PNGase F) cleaves both immature and mature oligosacchar- conditions. Vertical lines indicate that single lanes either side of the forskolin ides. Inspection of Figure 4 reveals that the majority of lanes were removed during preparation of the Figure. (c) Fully processed tyrosinase protein extracted from resveratrol-treated NHM tyrosinase, represented by the uppermost band at approximately 80 kDa, was was Endo H-sensitive and hence was ER-retained protein. quantified by densitometry of Western-blot autoradiographs. Error bars This reflects the relative proportions of the 70 and 80 kDa indicate the mean7SEM (n ¼ 4), relative to the untreated controls, and were obtained using three melanocyte strains from two independent experiments. forms that correspond to Endo H-sensitive and -resistant populations, respectively. In contrast, tyrosinase from NHM treated with either vehicle of forskolin alone had mainly the Golgi processed, Endo H-resistant form, consistent with the addition to the dramatic reduction in fully processed 80 kDa high proportion of mature tyrosinase detected by Western species, evident at 12 and 24 hours following resveratrol, analysis. Digestion with PNGase F generated a 60 kDa band there also appeared to be a small increase in intensity for a representing deglycosylated tyrosinase and demonstrated that broad band detected at approximately 70 kDa at these time the total amount of tyrosinase was increased by forskolin points (Figure 3b). This species has previously been shown to whereas it appeared to be reduced by resveratrol (Figure 4). correspond to an immature, endoglycosidase H (Endo H)- sensitive glycoform of tyrosinase (Halaban et al., 1997) and DCT protein is also differentially regulated by forskolin and indicated that resveratrol may cause aberrant processing of resveratrol tyrosinase. Furthermore, it was also apparent that both 12- Although changes in the abundance of tyrosinase can and 24-hours treatment with resveratrol resulted in the loss of account for the observed changes in tyrosinase activity seen a tyrosinase immunoreactive band at around 60 kDa (Figure with these agents, it was also necessary to evaluate whether 3b), a size expected for the deglycosylated, core polypeptide. there were also alterations in the two TYRPs, TYRP1 and

2220 Journal of Investigative Dermatology (2007), Volume 127 RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

abCtrl Fsk DCT protein 200 Rsvl+Fsk 1 hour 3 hours 12 hours 24 hours Rsvl 150 Rsvl – – + + – + + – + + – ++ – Fsk – + + – + + – + – ++ + – – 100

50 DCT (% control) DCT mRNA 0 GAPDH 3 hours 12 hours 24 hours c Ctrl d Fsk Rsvl+Fsk TYRP1 protein 125 Rsvl 1 hour 3 hours 12 hours 24 hours 100 Rsvl – – ++ – ++ – + + – ++ – 75 Fsk ––+ + – + + – + – ++ + – 50 TYRP1 (% control) 25 TYRP1 mRNA 0 GAPDH 3 hours 12 hours 24 hours

e SLC45A2 OCA2 SLC24A5 Ctrl 250 250 250 Fsk 200 200 200 Rsvl+Fsk Rsvl 150 150 150 100 100 100 50 50 50

mRNA (% control) 0 0 0 3 hours 12 hours 24 hours 3 hours 12 hours 24 hours 3 hours 12 hours 24 hours

Figure 5. DCT, TYRP1, SLC45A2, OCA2, and SLC24A5 expression in NHM following treatment with resveratrol, forskolin, or both agents, for the indicated times. Q-RT-PCR and Western immunoblot analysis was used to determine the levels of mRNA and protein, respectively, for DCT (a, b) and TYRP1 (c, d). (e) Q-RT-PCR was also used to quantify expression of the genes SLC45A2, OCA2, and SLC24A5 that encode the proteins MATP, P-protein, and NCKX5, respectively. Transcript abundance was normalized against 18S ribosomal RNA and expressed relative to the 3 hours control. Error bars indicate the mean7SEM (n ¼ 4) of two independent experiments, each utilizing two melanocyte strains.

DCT. These two enzymes are important for the stimulation of transcript abundance after 24 hours; however, only a 25% eumelanin production, which is likely to be either due to a reduction was seen when forskolin was also present (Figure direct catalytic role and/or an ability to stabilize tyrosinase 5c). Treatment with forskolin alone resulted in a small activity (for review, see Slominski et al., 2004). As was seen increase in TYRP1 mRNA at 12 and 24 hours that approached for tyrosinase, the transcript levels for both DCT (Figure 5a) 25% above control levels. These changes in RNA expression and TYRP1 (Figure 5c) showed little correspondence with were reflected by the modest increase in the protein level at changes in MITF expression (Figure 2), again implicating the 24 hours, as determined by Western analysis (Figure 5d). involvement of additional regulatory factors and/or post- Protein levels of the melanosomal protein MART-1 were also transcriptional control. examined in two melanocyte strains following the same DCT showed extensive differential regulation following treatment regime and were not altered by resveratrol (data not resveratrol and/or forskolin treatment (Figure 5a, b). This was shown) providing further evidence that the effects of particularly apparent at the protein level, 12 and 24 hours resveratrol were not due to a generalized effect on after treatment, with forskolin stimulating a profound increase melanosomal protein stability. in DCT, and resveratrol markedly decreasing protein abun- dance (Figure 5b). Whereas the decrease in DCT protein siRNA-mediated knockdown of MITF demonstrates that MITF could at least partially be accounted for by reduced levels of expression is required for the maintenance of melanogenic mRNA, altered transcription did not seem to be a potential enzyme transcription explanation for the forskolin-stimulated increase in protein The preceding data indicated that elevation of MITF expression. This again suggested that post-transcriptional expression does not necessarily directly regulate melano- regulation might be a crucial mediator of cAMP-induced genic enzyme levels in cultured NHM. However, MITF may increases in melanogenic enzymes. still make an important contribution to the maintenance of Analysis of TYRP1 mRNA levels in NHM revealed that melanogenic enzyme transcription. We therefore utilized a resveratrol treatment caused an approximate 50% decrease in siRNA against MITF to evaluate the requirement of this

www.jidonline.org 2221 RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

a MITF TYR TYRP1 GAPDH siRNA 1.0 Negative 1.0 1.0 1.0 control 0.8 MITF 0.8 0.8 0.8 0.6 0.6 0.6 0.6

0.4 0.4 0.4 0.4

0.2 0.2 0.2 0.2 Normalized mRNA level 0.0 0.0 0.0 0.0

bcdNegative + – Negative TYR activity control control + – siRNA

siRNA MITF – + Negative siRNA MITF – + control

MITF MITF 95 95 TYR 1.0 TYR 54 0.8 54 95 0.6 95 TYRP1 TYRP1 54 0.4 54 0.2 GAPDH GAPDH

12 Relative tyrosinase activity 0.0 12

Figure 6. Effect of MITF inhibition on melanogenic enzyme levels in NHM. The cells were transfected with siRNAs that were either directed against MITF or consisted of a commercial negative control sequence. 48 hours later, cells were assessed for expression of MITF, tyrosinase, TYRP1, and GAPDH by (a) Q-RT-PCR measurement of mRNA, and (b) Western-blot analysis of protein. (c) Cellular lysates containing tyrosinase were also prepared from identically treated cells then digested with PNGase F to facilitate comparison of core deglycosylated tyrosinase protein levels by Western analysis. (d) Cellular lysates were also assessed for relative tyrosinase activity using the DOPA oxidation assay. Error bars represent the mean7SEM (n ¼ 3) of three independent experiments.

transcription factor for the expression of tyrosinase and that showed only minor variation (Figure 6b). Therefore, TYRP1 in NHM. The effectiveness of the MITF siRNA in NHM some level of MITF expression appears to be required for the was shown by MITF mRNA levels being dramatically reduced maintenance of melanogenic enzyme transcription in NHM. to 25% of control levels (Figure 6a), concomitant with a substantial decrease in MITF protein (Figure 6b). Tyrosinase DISCUSSION and TYRP1 mRNA expression levels in NHM following A number of chemical agents can lead to depigmentation of siRNA-mediated knockdown of MITF was reduced to human skin, an effect that is commonly due to phenol-based approximately 50 and 40% of that seen in negative controls, structures causing direct inhibition of tyrosinase enzymatic respectively (Figure 6a). Similarly, mRNA levels for SLC24A5, activity (Ando et al., 2007; Solano et al., 2006). An SLC45A2, and OCA2 were also decreased in NHM trans- additional, indirect depigmenting mechanism invokes a key fected with siRNA molecules targeting MITF (data not regulatory role for MITF in governing levels of tyrosinase, shown). These decreases were substantially greater than that with compounds such as sphingosine-1-phosphate and seen for glyceraldehyde-3-phosphate dehydrogenase sphingosylphosphorylcholine shown to decrease tyrosinase (GAPDH) mRNA (Figure 6a). abundance via a preceding reduction in MITF (Kim et al., Tyrosinase and TYRP1 mRNA expression level changes 2003; Kim et al., 2006). Such a mechanism was also were at least partially replicated at the protein level (Figure proposed to underlie the inhibition of melanogenesis by 6b, c), with TYRP1 protein obviously decreased. For resveratrol (Lin et al., 2002). This suggestion was largely tyrosinase, the decrease in protein appeared to be mainly based on the findings that resveratrol was able to decrease the confined to the lower molecular weight bands (Figure 6b), activity of an MITF promoter-luciferase reporter construct and with decreased protein levels only readily apparent following reduce endogenous MITF mRNA levels in NHM. We also PNGase F-mediated digestion to the deglycosylated core found that resveratrol treatment was able to decrease MITF tyrosinase polypeptide (Figure 6c). However, little difference mRNA in NHM, however, this reduction was only by 25% in tyrosinase activity was detected using the DOPA oxidation and was not accompanied by any alteration in MITF protein assay (Figure 6d), consistent with this catalytic measure abundance or tyrosinase transcription. Furthermore, we reflecting changes in the fully processed form of tyrosinase found that resveratrol, in the presence of forskolin, actually

2222 Journal of Investigative Dermatology (2007), Volume 127 RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

elevated levels of MITF mRNA and protein, despite the fact compound with considerable structural similarity to resver- that the inhibition of tyrosinase activity by this treatment atrol, 2,20-dihydroxy-5,50-dipropyl-biphenyl, that appears to combination is virtually identical to that seen with resveratrol downregulate melanogenesis in mouse B16 melanoma cells alone. Therefore downregulation of endogenous MITF in a similar manner (Nakamura et al., 2003). Thus, 2,20- expression did not account for resveratrol-mediated depig- dihydroxy-5,50-dipropyl-biphenyl treatment for 24 hours mentation and a new mechanistic explanation was required. strongly suppressed tyrosinase activity, an effect that was Our data demonstrated that at least two mechanisms also explained via ER-retention of tyrosinase and a subse- underlie the inhibition of tyrosinase activity following quent acceleration of tyrosinase degradation (Nakamura resveratrol treatment of NHM. The first mechanism caused et al., 2003). Although 2,20-dihydroxy-5,50-dipropyl-biphenyl a reduction of tyrosinase protein levels that was shown to did not show any direct inhibition of tyrosinase, it may share correlate with the major decrease in tyrosinase activity seen with resveratrol a structural ability to interact with tyrosinase after 18–24hours. Secondly, resveratrol had the capacity to to hinder correct folding/processing of the protein. Misfolding directly inhibit tyrosinase activity when added to cell of mutant tyrosinase from albino melanocytes underlies the extracts, an effect that has been previously described (Kim subsequent increase in ER retention and degradation of et al., 2002b). Although the decrease in tyrosinase protein tyrosinase (Halaban et al., 2000). The importance of folding can account for the time-dependent reduction in tyrosinase in tyrosinase maturation has also been demonstrated in activity measured in lysates from treated NHM, it is unclear amelanotic melanomas by the ability of the tyrosinase to what extent the direct inhibition mechanism contributes to substrates, DOPA and tyrosine, to facilitate the correct reduced tyrosinase activity in situ. The influence of direct folding of the enzyme to the mature, proteolytically resistant inhibition, however, would be expected to decline with time form (Halaban et al., 2001). Also crucial for tyrosinase because studies on resveratrol uptake in cultured human trafficking is the pH within the ER-Golgi compartments, with hepatic cells demonstrated that intracellular levels peak amelanotic melanoma cells shown to have enhanced within 10 minutes then gradually decrease (Lancon et al., tyrosinase maturation and to pigment following treatment 2004). The high-affinity interaction between resveratrol and with agents that increase the luminal pH of subcellular tyrosinase revealed by the in vitro studies may also be organelles (Halaban et al., 2002). In this regard, it of interest relevant for the molecular mechanism mediating long-term that resveratrol was able to decrease expression of two genes, downregulation of tyrosinase protein. SLC45A2 and SLC24A5, which encode transmembrane Resveratrol treatment of NHM substantially decreased the proteins implicated in the regulation of ion transport (for amount of tyrosinase protein without affecting steady-state review, see Sturm, 2006). Therefore, resveratrol, either levels of tyrosinase RNA confirming that regulation of directly or indirectly, may alter the processing environment tyrosinase transcription does not mediate depigmentation with concomitant ER retention of tyrosinase. by resveratrol. The basis for the decrease in tyrosinase protein In contrast to tyrosinase, the profound decrease in DCT was revealed by Western analysis and glycosidase digestion, protein elicited by resveratrol was also accompanied by a with extracts from resveratrol treated NHM shown to consist corresponding reduction in mRNA, implicating transcription mostly of ER-retained, immature tyrosinase, whereas control control. The decrease in DCT following resveratrol treatment NHM contained mainly the mature, Golgi-processed form. could contribute to skin depigmentation and may also have Therefore resveratrol treatment disrupted the trafficking of relevance for potential chemotherapeutic applications. tyrosinase from the ER to the Golgi leading to a dramatic Although we did not observe an effect of resveratrol on the decrease of functional tyrosinase. Interestingly, retention of proliferation of primary melanocytes, it has been shown to tyrosinase in the ER can also occur for mutant forms of induce apoptosis in melanoma cells (Niles et al., 2003) and tyrosinase that cause type I oculocutaneous albinism increase sensitivity to anticancer therapy (Fulda and Debatin, (Halaban et al., 2000). Furthermore, the loss of pigmentation 2004; Yang et al., 2005). The chemotherapy sensitization that is often seen in human melanomas has also been shown effects towards melanoma may relate, in part, to decreased to be the result of aberrant ER retention, with tyrosinase again DCT levels, because expression of DCT has been associated mainly evident as a 70 kDa Endo H-sensitive, immature form with resistance to chemo- and radiotherapy (Chu et al., 2000; (Halaban et al., 1997). This ER-trapped form is also subject to Pak et al., 2004). increased proteolytic degradation (Watabe et al., 2004) Stimulation of pigmentation following activation of the accounting for the overall decrease in tyrosinase protein cAMP signal transduction cascade is at least partially evident following resveratrol exposure. attributable to an upregulation of melanogenic enzymes Depigmentation due to ER-retained tyrosinase has recently and studies with gene reporter assays have implicated a key also been demonstrated in melanoma cells that have been role for MITF in such induction via transcriptional control of engineered to display enhanced sensitivity to oxidative stress melanogenic genes (Bertolotto et al., 1998a, b; Price et al., via downregulation of the L-ferritin gene (Maresca et al., 1998; Busca and Ballotti, 2000). However, the forskolin- 2006). Resveratrol, however, is a free radical scavenger and elicited upregulation of tyrosinase and DCT proteins in NHM thus would be expected to decrease oxidative stress. This described in this study was not associated with an increase in suggests that the antioxidant properties of resveratrol are tyrosinase or DCT transcription, despite a robust increase in unlikely to account for its effects on tyrosinase maturation. MITF expression. Previous evidence for post-transcriptional Intriguingly, there also appears to be another polyphenol control of melanogenic enzyme levels in NHM was provided

www.jidonline.org 2223 RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

in a report where a-MSH stimulated an increase in tyrosinase, Post-transcriptional regulation of melanogenic enzyme TYRP1, and DCT protein without altering respective mRNA expression has previously been primarily associated with the levels, as detected by Northern analysis (Abdel-Malek et al., inhibition of melanin synthesis, as evident in amelanotic 1995). The inability of elevated MITF protein to stimulate melanoma and classic oculocutaneous albinism. The loss of tyrosinase and DCT transcription in this investigation may be tyrosinase activity and ER retention of misfolded tyrosinase a consequence of the endogenous context of the tyrosinase that underlies such loss of pigmentation has now also been promoter, a concept supported by a study using adenovirus shown to be the main mechanism responsible for the encoding wild-type MITF which demonstrated that exogen- profound loss of pigmentation elicited by the chemopreven- ous MITF was not sufficient to increase levels of endogenous tative agent resveratrol. Our data also indicated that post- tyrosinase and TYRP1 protein in NHM (Gaggioli et al., 2003). transcriptional upregulation of melanogenic enzyme levels This latter study also utilized adenovirus expressing dominant represents an important regulatory control point for melanin negative MITF to demonstrate that MITF was, however, still synthesis following activation of the cAMP pathway in NHM. required for maintaining endogenous tyrosinase gene expres- As upregulation of tyrosinase and DCT is an important sion. Our study employed siRNA-mediated knockdown of component of the photoprotective delayed tanning response, MITF to similarly examine the relationship between MITF and further understanding of the molecular details of this post- constitutive enzyme levels, and we also observed a sig- transcriptional control may have significant therapeutic nificant decrease in tyrosinase and TYRP1 expression in implications. NHM following a 75% reduction in MITF mRNA levels. Our data therefore support the concept that ‘MITF is required, but MATERIALS AND METHODS is not sufficient to induce the expression of melanogenic Materials genes’ (Gaggioli et al., 2003). Antibodies against the following human proteins were used: An alternative mechanistic basis for the observed post- tyrosinase (monoclonal T311, Upstate, Charlottesville, VA), DCT transcriptional upregulation of melanogenic proteins by (TRP2, D-18, Santa Cruz Biotechnology, Santa Cruz, CA), TYRP1 cAMP is therefore required. Insight into the molecular (monoclonal B8G3 (Takahashi and Parsons, 1990), a gift from P. scheme responsible for the increase may be provided by Parsons), MITF (Ab-2, monoclonal D5) and MART-1 (NeoMarkers, the observation that the two melanogenic proteins with well- Melbourne, Australia), phospho-CREB (Upstate), phospho-ERK characterized enzyme activity, tyrosinase, and DCT, were (phospho-p44/42 MAPK, monoclonal E10, Cell Signaling Technol- strongly increased by forskolin, in contrast to TYRP1, a ogy, Beverly, MA), ERK (p44/42 MAPK, Cell Signaling Technology), protein that may not have an enzymatic role (Boissy et al., and GAPDH (Genzyme, Cambridge, MA). Horseradish peroxidase- 1998). An attractive possible mechanism is that the cAMP conjugated secondary antibodies against rabbit or mouse IgG were signaling pathway can influence the enzyme environment to purchased from Upstate. The horseradish peroxidase-conjugated modulate activity and subsequent processing and/or traffick- anti-goat secondary antibody, forskolin, resveratrol, and activated ing. We therefore investigated this possibility by quantitating charcoal were all obtained from Sigma-Aldrich (Sydney, NSW, the expression of the genes SLC45A2, OCA2, and SLC24A5 Australia). L-Tyrosine-3,5-3H (33.9 Ci/mmol) was purchased from that encode transmembrane proteins implicated in the Perkin Elmer (Melbourne, VIC, Australia). regulation of ion transport and/or the processing and trafficking of melanogenic enzymes (Sturm, 2006). The most Cell culture dramatic effect of forskolin was a twofold increase in Queensland foreskin primary melanocyte strains were isolated and expression of the SLC45A2 gene, encoding the MATP protein, propagated from anonymous neonatal foreskin samples as described which is likely to reflect the ability of SLC45A2 to be previously (Leonard et al., 2003) and maintained in RPMI-1640 transcriptionally modulated by MITF (Du and Fisher, 2002), media containing 3 mM HEPES buffer supplemented with 8% heat- (our data, not shown). As MATP plays a crucial role in inactivated fetal bovine serum, penicillin/streptomycin (40 mg/ml), tyrosinase trafficking and stability (Costin et al., 2003), the 1mM pyruvic acid, 0.2 mM. nicotinamide, 10 ng/ml TPA, 5 ng/ml upregulation of this protein provides a promising candidate to bFGF, and 0.6 mg/ml cholera toxin. One week before, and for the facilitate the increased tyrosinase abundance. However, it is duration of, experimentation, cholera toxin was removed from the difficult to reconcile the greater upregulation of DCT than melanocyte growth media to facilitate investigation of cAMP- TYRP1 by such a MATP-mediated mechanism because DCT mediated responses. Stimulation of NHM was carried out in culture trafficking is the least affected melanogenic enzyme in mice media that had been present for 3 days since seeding. The with defective MATP function (Costin et al., 2003). Forskolin melanocyte strains used were all from Caucasian donors with a also induced a 1.5-fold increase in expression of the OCA2 darkly pigmented and lightly pigmented strain used for all gene encoding the P-protein. The P-protein has been shown experiments. A further melanocyte strain of intermediate pigmenta- to be crucial for normal post-translational processing and tion was also used for tyrosinase, DCT, and TYRP1 Western trafficking of tyrosinase in the ER (Chen et al., 2002). immunoblot analysis. The study was conducted according to the However, it has also been reported that melanocytes cultured Declaration of Helsinki Principles, and ascertainment of foreskin from p-null mice can still elevate tyrosinase levels in response tissue samples was approved by the University of Queensland to IBMX, a cAMP phosphodiesterase inhibitor, although it Medical Research Ethics Committee and QIMR Human Research was not established whether this was a transcriptional or Ethics Committee. Experiments were conducted with parental post-translational effect (Chen et al., 2004). consent to the use of tissue for research.

2224 Journal of Investigative Dermatology (2007), Volume 127 RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

Tyrosinase activity assays and L-DOPA staining transfection, the liposomes were aspirated and the medium replaced To prepare cell extracts following drug treatment, cells were washed with growth medium lacking antibiotics and the culture continued in ice-cold phosphate-buffered saline (PBS), lysed in 0.1 M for a further 24 hours before preparation of total protein lysates, phosphate buffer (pH 6.8) containing 1% Triton X-100 and complete DOPA oxidation assay extracts, or total RNA extracts as appropriate. protease inhibitor cocktail (Roche Diagnostics, Sydney, NSW, Australia) for 20 minutes at 41C, then clarified by centrifugation Gene expression quantitation (13,000 g, 5 minutes). The tyrosinase activity of 100 ml aliquots of RNA extraction, DNase treatment and quantitative real-time poly- extract was then determined by either measuring the rate of merase chain reaction (Q-RT-PCR) procedures were carried out oxidation of L-DOPA (Takahashi and Parsons, 1992) or the release essentially as previously described (Newton et al., 2005; Roberts of tritium from tyrosine-3,5-3H (Pomerantz, 1966). For the DOPA et al., 2006). Ten percent of cDNA synthesis reactions from 0.3 mg oxidation assay, 100 ml duplicates were incubated with 100 ml total RNA were used in 25 ml Q-RT-PCR reactions containing L-DOPA (3 mg/ml in 0.1 M phosphate buffer, pH 6.8) at 371C. The TaqMan gene expression assays (Applied Biosystems, Foster absorbance at 490 nm was measured every 30 minutes for at least City, CA, USA) for either tyrosinase (Hs00165976_m1), DCT

2 hours and the tyrosinase activity calculated as OD490/min/mg of (Hs00157244_m1), TYRP1 (Hs00167051_m1), MITF (Hs00165 lysate protein then expressed relative to the activity of vehicle- 156_m1), GAPDH (Hs99999905_m1), OCA2 (Hs00609341_m1), treated cells. To assess direct inhibitory effects on tyrosinase activity, SLC45A2 (Hs00211813_m1), or SLC24A5 (Hs01385408_m1), compounds were added to cell lysates at an equivalent concentra- together with TaqMan PCR master mix. Transcript levels were tion to that used for treating whole-cell cultures, incubated for normalized against 18S ribosomal RNA that was quantified using 5 minutes at room temperature, mixed with L-DOPA solution and a Pre-Developed TaqMan assay for 18S (Applied Biosystems) and incubated for 2 hours at 371C, as described above. The protein 50-fold diluted cDNA. Q-RT-PCR was performed in 96-well plates concentration of extracts was determined by a BCA assay (Pierce, on an ABI Prism 7500 Sequence Detector system using a two-step Rockford, IL, USA). The tritium release assay of Pomerantz thermal cycling protocol of 40 cycles of 951C for 15 s and 601C for (Pomerantz, 1966) was performed essentially as first described. 1 minute. Briefly, 100 ml of cell extract was added to a 900 ml reaction mixture containing 0.1 M phosphate buffer (pH 6.8) and 0.1 mM phenyl- Western immunoblot analysis methanesulfonylfluoride. For experiments examining the in vitro Following appropriate periods of treatment at 371C, culture plates effects of resveratrol, the drug or DMSO was also added at this stage were placed on ice, rinsed with ice-cold PBS, SDS extraction buffer to give the desired final concentration and incubated at room added and Western blotting performed as described previously temperature for 5 minutes. L-DOPA (0.03 mM final concentration) (Newton et al., 2005). PageRuler prestained protein standards and 1 mCi tyrosine-3,5-3H were then added and the reaction (MBI Fermentas, Quantum Scientific, Brisbane, QLD, Australia) were incubated for 1 hour at 371C. The reaction was stopped by adding used for molecular weight estimation. Membranes were blocked in 50 ml 1 M HCl then mixed with approximately 100 mg activated Tris-buffered saline (TBS) (pH 7.6), 0.05% Tween 20, 5% skim charcoal for 1 hour at room temperature, centrifuged briefly and milk powder for 1 hour at room temperature. Primary antibodies 200 ml supernatant mixed with 1.8 ml scintillation fluid, and were used at the following dilutions: anti-tyrosinase, 1:4,000; anti- counted. Background counts were assessed using 100 ml 0.1 M DCT, 1:500; anti-TYRP1, 1:20; anti-MITF, 1:1,000; anti-GAPDH, phosphate buffer in place of cell extract. 1:10,000; anti-phospho-CREB, 1:1,000; anti-phospho-ERK, 1:2,000; In situ L-DOPA reactivity of NHM was assessed using cultures anti-ERK, 1:1,000; anti-MART-1, 1:1,000. Dilutions were all in fresh fixed with 4% paraformaldehyde in PBS for 40 min at room 5% skim milk blocking buffer, apart from ERK where BSA replaced temperature. Following permeabilization in 0.1% triton X-100 in milk. Incubations were overnight at 41C, except for TYRP1 which PBS for 2 min, cells were washed with PBS and incubated in 0.1% had 1.5 hours at room temperature. After washing in TBS, 0.1% L-DOPA for 3 hours at 371C. Cells were then rinsed in PBS and Tween 20 (3 10 minutes at room temperature), the appropriate images captured using an Olympus CKX41 inverted microscope horseradish peroxidase-conjugated secondary antibody was incu- equipped with an Olympus Camedia C-5060 digital camera. bated with the blot for 1 hour at room temperature (1:10,000 in 3% blocking buffer). Following washing, as above, the blot was exposed siRNA transfection to Supersignal West Pico (Pierce) for detection. The relative intensity A predesigned siRNA directed against MITF (Ambion, Austin, TX, ID of bands was determined by scanning films on a GS800 calibrated No. 115142) was used to downregulate MITF, and a commercial densitometer (Bio-Rad, NSW, Australia), followed by volumetric negative control sequence (Ambion, Catalogue No. 4611) was used quantitation using Quantity One software (Bio-Rad). The signal to monitor for off-target effects. NHMs were seeded in complete obtained from reprobing blots with GAPDH was used to monitor for growth medium in 10 cm dishes (5 105 cells) or 6-well plates equal total protein levels. (2 105 cells per well) and allowed to adhere for 48 hours. Transfection of siRNAs was carried out essentially as described Glycosylation analysis previously (Cook et al., 2005). Briefly, siRNA molecules were Protein was extracted from NHM using the same lysis buffer transfected at a concentration of 100 pmol using 5 ml Lipofectamine employed for the tyrosinase assay and the protein concentration 2000 (Invitrogen, Melbourne, VIC, Australia) per 1 ml consisting of determined by the BCA procedure. 10 mg aliquots of lysate were then 50% OptiMEM and 50% growth medium without antibiotics. After digested with 500 U Endo H or PNGase F (New England Biolabs, 6 hours, an equal volume of growth medium lacking antibiotics was Beverly, MA), or incubated with reaction buffer alone, in accordance added and the cells cultured overnight. Twenty-four hours post- to the conditions specified by the manufacturer. After incubation,

www.jidonline.org 2225 RA Newton et al. Post-Transcriptional Regulation of Tyrosinase

samples were mixed with SDS extraction buffer (5 ), heat Fuller BB, Spaulding DT, Smith DR (2001) Regulation of the catalytic activity denatured, and detected by Western immunoblotting using anti- of preexisting tyrosinase in black and Caucasian human melanocyte cell cultures. Exp Cell Res 262:197–208 tyrosinase, as described above. Gaggioli C, Busca R, Abbe P, Ortonne JP, Ballotti R (2003) Microphthalmia- associated transcription factor (MITF) is required but is not sufficient to CONFLICT OF INTEREST induce the expression of melanogenic genes. Pigment Cell Res The authors state no conflict of interest. 16:374–82 Halaban R, Cheng E, Svedine S, Aron R, Hebert DN (2001) Proper folding and ACKNOWLEDGMENTS endoplasmic reticulum to golgi transport of tyrosinase are induced by its We thank Professor Peter Parsons for the gift of the TYRP1 antibody, B8G3. substrates, DOPA and tyrosine. J Biol Chem 276:11933–8 RAS is a Senior Research Fellow of the National Health and Medical Research Halaban R, Cheng E, Zhang Y, Moellmann G, Hanlon D, Michalak M et al. Council of Australia. DWR was supported by a University of Queensland (1997) Aberrant retention of tyrosinase in the endoplasmic reticulum Confirmation Scholarship. The work was funded by project grants NHMRC- mediates accelerated degradation of the enzyme and contributes to the 301173 and ARC-DP0451738. The Institute for Molecular Bioscience dedifferentiated phenotype of amelanotic melanoma cells. Proc Natl incorporates the Centre for Functional and Applied Genomics as a Special Acad Sci USA 94:6210–5 Research Centre of the Australian Research Council. Halaban R, Patton RS, Cheng E, Svedine S, Trombetta ES, Wahl ML et al. (2002) Abnormal acidification of melanoma cells induces tyrosinase retention in the early secretory pathway. 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