PDZK1 Upregulation in Estrogen-Related Hyperpigmentation in Melasma Nan-Hyung Kim1, Kyung Ah Cheong1, Tae Ryong Lee2 and Ai-Young Lee1

ORIGINAL ARTICLE

The pathogenesis of melasma is unknown, although the potential role of estrogen has been considered. Microarray and real-time PCR analyses revealed that upregulation of PDZ domain protein kidney 1 (PDZK1) is clinically correlated with melasma. Although there has been no report that PDZK1 is involved in pigmentation and/or melanogenesis, PDZK1 expression can be induced by estrogen. In this study, the role of PDZK1 upregulation in melasma was examined, particularly in connection with estrogen, using biopsied skin specimens from 15 patients and monocultures and cocultures of melanocytes and keratinocytes with or without overexpression or knockdown of PDZK1. Estrogen upregulated PDZK1. Overexpression of PDZK1 increased tyrosinase expression and melanosome transfer to keratinocytes, whereas PDZK1 knockdown reduced estrogen-induced tyrosinase expression, through regulation of expression of estrogen receptors (ERs) ER-a and ER-b. The PDZK1-induced tyrosinase expression and melanosome transfer was regulated by ion transporters such as sodium–hydrogen exchanger (NHE), cystic fibrosis transmembrane conductance regulator (CFTR), and SLC26A3, which showed a specific association with each ER subtype. In the melanosome transfer, PDZK1 also increased phosphorylation of ezrin/radixin/moesin (ERM) and ras-related C3 botulinum toxin substrate 1, but not the expression of proteinase-activated receptor-2. Collectively, upregulation of PDZK1 could have an important role in the development of melasma in connection with estrogen through NHE, CFTR, and SLC26A3. Journal of Investigative Dermatology (2012) 132, 2622–2631; doi:10.1038/jid.2012.175; published online 14 June 2012

INTRODUCTION 2-fold or greater upregulation in the expression of PDZ domain Melasma refers to a pattern of hyperpigmentation that affects protein kidney 1 (PDZK1) in the hyperpigmented skin of skin on the upper lip, cheeks, forehead, and chin, particularly melasma patients (data not shown). PDZK1 is a member of the during the reproductive lifespan of women. Melasma differs sodium–hydrogen exchanger regulatory factor (NHERF) family, from post-inflammatory hyperpigmentation and UV-induced called NHERF-3. NHERF-1 and NHERF-2 contain two PDZ pigmentation in that it persists and is difficult to treat. As a domains, whereas NHERF-3 and NHERF-4 each have four variety of factors, including pregnancy and changes in uterine PDZ domains, which mediate the most abundant protein– and ovarian hormones, have been implicated in the pathogen- protein interactions. Studies have been rarely conducted for esis of melasma, the role(s) of estrogen and/or progesterone has NHERF-3 and NHERF-4. NHERF proteins have not been been considered. In fact, immunohistochemical staining has investigated in the skin, nor has the involvement of PDZK1 in shown an increased estrogen receptor (ER) expression in skin pigmentation. However, NHERF-1 has been identified as affected compared with unaffected skin (Lieberman and Moy, an estrogen-regulated gene in ER-containing breast cancer 2008; Jang et al., 2010). However, it is uncertain as to what cells (Ediger et al., 1999). PDZK1 expression can also be role estrogen has in melasma. Recently, microarray analysis to induced by estrogen (Ghosh et al., 2000). compare hyperpigmented to normally pigmented skin from In the regulation of ion transport in a variety of epithelia, patients with melasma was performed to identify specific including the kidney, intestine, pancreas, and the airway, factors associated with the hyperpigmentation. There was a NHERF proteins have been shown to bind to NHE, cystic fibrosis transmembrane conductance regulator (CFTR), and 1Department of Dermatology, Dongguk University Ilsan Hospital, Goyang-si, the SLC26A family. Mammalian NHE is an integral mem- 2 Gyenggi-do, South Korea and Bioscience Institute, Amore Pacific brane protein that functions to exchange one intracellular Corporation R&D Center, Yongin, Gyenggi-do, South Korea H þ for one extracellular Na þ (Nakamura et al., 2005). Correspondence: Ai-Young Lee, Department of Dermatology, Dongguk University Ilsan Hospital, 814 Siksa-dong, Ilsandong-gu, Goyang-si, The NHE1 of nine isoforms is ubiquitously expressed in Gyenggi-do 410-773, South Korea. the plasma membrane of virtually all tissues, whereas the E-mail: [email protected] or [email protected] NHE2–NHE5 isoforms have more restricted tissue distribu- Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; tions. In the skin, NHE1 has been identified in cultured ER, estrogen receptor; NHE, sodium–hydrogen exchanger; NHERF, keratinocytes and melanocytes (Sarangarajan et al., 2001; sodium–hydrogen exchanger regulatory factor; PDZK1, PDZ domain protein kidney 1 Smith et al., 2004), and NHE3 has been identified in Received 6 January 2012; revised 5 April 2012; accepted 5 April 2012; melanocytes (Sarangarajan et al., 2001). As NHE3 predomi- published online 14 June 2012 nantly mediates electroneutral sodium absorption, which

2622 Journal of Investigative Dermatology (2012), Volume 132 & 2012 The Society for Investigative Dermatology N-H Kim et al. PDZK1 in Melasma

takes up Cl with Na þ in a one-to-one ratio, it may be a the expression of microphthalmia-associated transcription prerequisite to be coupled to one or several isoforms of the factor (Figure 1e). SLC26 apical anion exchanger family (Mount and Romero, 2004). The interaction of both transporters may augment pH- PDZK1 affects estrogen-induced tyrosinase expression through mediated regulation (Lamprecht and Seidler, 2006). The the regulation of ER expression reciprocal activation of transport function has identified an Estrogen has been known to stimulate the production of interaction between SLC26 and CFTR. In fact, CFTR is NHERF in ER–containing cells (Ediger et al., 1999; Stemmer- localized in the apical membrane of epithelial cells and Rachamimov et al., 2001). Although NHERFs have not been functions as a Cl channel and regulates Cl and HCO3 reported in keratinocytes or melanocytes despite the presence secretion (Grubb and Boucher, 1999), particularly in con- of ERs in those cells, treatment of melanocyte monocultures junction with SLC26 anion transporters (Ko et al., 2004; Singh and melanocyte–keratinocyte cocultures with estrogen et al., 2010). In normal human skin, CFTR expression has upregulated PDZK1 and tyrosinase expression (Figure 2a). been examined in epidermal keratinocytes, as well as in Overexpression of PDZK1 further increased estrogen-stimu- eccrine sweat glands and ducts (Sato et al., 2002), but not in lated tyrosinase expression, whereas knockdown of PDZK1 melanocytes. more than 60% at the mRNA level and 37% at the protein So far, there has been no evidence to implicate PDZK1, as level using a mixture of two different stealth small interfering well as NHE3, CFTR, and SLC26, in melasma. Although RNAs reduced tyrosinase expression to the level of the PDZK1 can be induced by estrogen (Ghosh et al., 2000), no untreated control (Figure 2b). As estrogen is known to exert study on the expression or function of NHERF proteins, biological effects that are mediated by two distinct ERs, ER-a including PDZK1, has been conducted on the skin. Never- and ER-b (Katzenellenbogen et al., 2000), the effect of PDZK1 theless, a broad role of the NHERF family in the organization on the expression of ERs was examined. The overexpression of signaling complexes and control of cell physiology has of PDZK1 significantly (Po0.05) increased both ER-a and ER- been suggested with rapid increase in the identified b expression (Figure 2c), whereas the knockdown of PDZK1 intracellular targets. In this study, we report that PDZK1 eliminated estrogen-induced expression of both ERs (Figure upregulation is involved in hyperpigmentation of melasma, 2d). At least one ER subtype was significantly upregulated which resulted from an increase in melanogenesis and in the hyperpigmented skin compared with the normally melanosome transfer to keratinocytes, and is associated with pigmented skin of 14 melasma patients, in which ER estrogen responses through ion transporters, such as NHE3, expression of the 15 patients with PDZK1 upregulation could CFTR, and SLC26A3. be measured (Figure 2e).

RESULTS PDZK1 over-expression upregulates NHE, CFTR, and SLC26A3 Upregulation of PDZK1 in hyperpigmented compared with As PDZK1 contains four protein–protein interacting PDZ normally pigmented skin of patients with melasma increases domains, known ion transporters, such as NHE, CFTR, and tyrosinase expression SLC26, were examined after overexpression of PDZK1 in Levels of PDZK1 mRNA expression were measured using melanocyte monocultures and in melanocyte–keratinocyte real-time PCR and were compared between hyperpigmented cocultures. In the case of NHE, the expression of NHE1 and and normally pigmented skin specimens biopsied from 15 NHE3, which have been detected in cultured keratinocytes patients with melasma. The level of PDZK1 mRNA was and melanocytes (Sarangarajan et al., 2001; Behne et al., higher in hyperpigmented than in normally pigmented skin, 2002; Smith et al., 2004), was examined. Overexpression of showing a statistical significance (P ¼ 0.015; Figure 1a). PDZK1 in either type of monoculture or coculture signifi- Immunohistochemical staining with an anti-PDZK1 antibody cantly (Po0.05) increased protein levels of all those showed stronger reactivity with the hyperpigmented com- molecules compared with the untreated controls (Figure 3a). pared with the normally pigmented epidermis (Figure 1b). In Immunoprecipitation using an anti-PDZK1 antibody showed the epidermis, not only keratinocytes but also melanocytes an increased binding between PDZK1 and those ion were PDZK1 positive (Figure 1b). transporters both in keratinocytes and melanocytes following Before overexpressing PDZK1 in skin cells, the expression the PDZK1 overexpression (Figure 3b). Immunohistochemistry levels of PDZK1 were examined in three different populations showed that upregulation of PDZK1 increased the staining of cultured normal human melanocytes and keratinocytes. intensity for those molecules including CFTR, with the Keratinocytes showed uniformly high levels of PDZK1 colocalization of PDZK1 and ion transporters both in expression, whereas melanocytes had lower levels of PDZK1 melanocytes and keratinocytes (CFTR shown in Figure 3c, expression with intercellular variation compared with the data from other molecules not shown). keratinocytes (Figure 1c). Overexpression of PDZK1 in melanocytes increased expression levels of tyrosinase, which NHE3, CFTR, and SLC26A3 are involved in estrogen-related was also examined in cocultures with overexpression in only tyrosinase expression keratinocytes or in keratinocytes and melanocytes (Figure 1d). The results shown above demonstrate that PDZK1 has a role PDZK1 overexpression in monocultures and/or in cocultures in estrogen-induced tyrosinase expression through ERs increased the phosphorylation of extracellular signal–regulated (Figure 2a–e). As PDZK1 interacts with CFTR, NHE3, and kinase, CRE-binding protein, and protein kinase C, as well as SLC26 in melanocytes in the presence or absence of

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a b N L Fold * 7

5 PDZK1 C-KIT PDZK1 C-KIT

1 Hoechst Bright field Hoechst Bright field Relative ratio of PDZK1/ β -actin 0 N L

Merge Merge

c d KC KC(PDZK1) KC+MC MC +MC (PDZK1) CCCCPDZK1 PDZK1 PDZK1 PDZK1 KC1 KC2 KC3 MC1 MC2 MC3 PDZK1 70 kDa PDZK1 Tyrosinase 80 kDa GAPDH β-Actin 43 kDa

KC KC+MC MC C PDZK1 C PDZK1 C PDZK1 44 kDa 44 kDa p-ERK 42 kDa P-ERK/ERK p-ERK 42 kDa P-CREB/CREB 44 kDa 44 kDa 4 3 T-ERK 42 kDa P-PKC/PKC T-ERK 42 kDa C 2.5 * 3 * 43 kDa * 43 kDa * PDZK1 p-CREB 2 * * * * p-CREB 1.5 2 * * T-CREB 43 kDa T-CREB 43 kDa 1 1 80 kDa 0..5 p-PKC 80 kDa

p-PKC increment of Ratios Ratios of increment of Ratios 0 0 PKC 80 kDa C C PKC 80 kDa β PDZK1 PDZK1 MITF 60 kDa -actin p-ERK/ERK p-PKC/PKC MITF/ β-Actin 43 kDa p-CREB/CREB

Figure 1. Increased PDZK1 expression in hyperpigmented skin of melasma patients increases tyrosinase expression. (a) Real-time PCR results for relative ratios of PDZ domain protein kidney 1 (PDZK1) mRNA expression in hyperpigmented (L) compared with normally pigmented (N) skin biopsied from 15 patients. (b) Immunohistochemical staining with anti-PDZK1 and anti-c-Kit antibodies. Nuclei were counterstained with Hoechst 33258 (bar ¼ 0.1 mm). (c) Reverse transcription–PCR analysis of PDZK1 expression in cultured normal human melanocytes (MC) and keratinocytes (KC). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard. (d, e) Western blot analysis of PDZK1, tyrosinase, extracellular signal–regulated kinase (ERK), CRE- binding protein (CREB), protein kinase C (PKC), and microphthalmia-associated transcription factor (MITF) expression in monocultures of keratinocytes or melanocytes and in mixed cell cultures (KC þ MC) 24 hours after the transfection with either PDZK1 (PDZK1) or control vector (C). Data in the graphs represent means±SD of five independent experiments (*Po0.05).

keratinocytes (Figure 3a–c), the interaction of PDZK1 with specific agonists of ER-a (propyl pyrazoletriol) and ER-b NHE3, CFTR, and SLC26A3 in estrogen-induced tyrosinase (diarylpropionitrile). Treatment with propyl pyrazoletriol expression was examined. The expression of those transporter significantly (Po0.05) increased the expression of NHE3 proteins increased with the concentration of estrogen (Figure along with ER-a, without any change in ER-b or CFTR 4a). The estrogen-induced upregulation of tyrosinase in the expression. On the other hand, treatment with diarylpropio- presence or absence of PDZK1 was significantly (Po0.05) nitrile significantly (Po0.05) increased CFTR expression inhibited by 5-ethylisopropyl amiloride and the CFTR without any change of NHE3 levels (Figure 4d). The Inhibitor-172 (3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxy- relationship between each ER subtype and NHE3 or CFTR phenyl)methylene]-2-thioxo-4-thiazolidinone), specific inhi- expression was also examined in skin specimens from 12 of bitors of NHE3 (Figure 4b) and CFTR (Figure 4c), respectively, the 15 melasma patients with PDZK1 upregulation. A parallel without any change in PDZK1 expression. The association of increased and decreased expression between NHE3 and ER-a the ion transporter with ER subtypes was also examined using was detected in 5 and 2 of the 12 patients, respectively. In the

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case of CFTR, which was measured in 12 patients, 7 and 3 expected from the effect of stem cell factor on melanocyte patients showed a parallel increase and decrease with ER-b, dendricity (Grichnik et al., 1998) and the role of ERM respectively (Figure 4e). phosphorylation on the stem cell factor–induced action on melanocytes (Jeon et al., 2009). Therefore, western blot PDZK1 and ion transporter are involved in estrogen-induced assays for ERM, Rac1, and PAR2 were performed, which melanosome transfer showed a significant (Po0.05) increase of ERM and Rac1 Considering the important role of melanosome transfer to phosphorylation, but not PAR-2 expression (Figure 5b). neighboring keratinocytes in skin pigmentation, the effect of PDZK1 upregulation on melanosome transfer was examined DISCUSSION in a melanocyte–keratinocyte coculture system. FACS analy- Our quantitative real-time PCR analysis identified a clinical sis using TYRP-1 and K14 antibodies showed that over- correlation between the upregulation of PDZK1 and melasma expression of PDZK1 significantly (Po0.05) increased the (Figure 1a), although the correlation was not analyzed proportion of TYRP-1 and K14 double-positive cells, whereas according to individual variables, such as age of patients, specific inhibitor of ERs, NHE3, and CFTR reduced PDZK1- disease duration, sun exposure, and serum levels of estrogen. stimulated melanosome transfer to the level of the untreated PDZK1 could be associated with NHERF1 (Gisler et al., 2003; control (Figure 5a). In the melanosome transfer, the role of Broere et al., 2009); however, there was no significant proteinase-activated receptor (PAR)2 has been identified upregulation of NHERF-1 in the same patients (data not (Seiberg et al., 2002). In addition, ras-related C3 botulinum shown). Immunohistochemistry of melasma skin and western toxin substrate 1 (Rac1) is known to be involved in dendrite blotting analysis of cultured cells showed that both keratino- formation (Scott and Cassidy, 1998). The involvement of cytes and melanocytes were sources of PDZK1 expression ezrin/radixin/moesin (ERM) in melanosome transfer might be in the epidermis (Figure 1b and c), which supported the

a KC+MC MC b KC+MC Estrogen 0 10 100 (nM) Estrogen 0 10 100 (nM) Control PDZK1 Fold

f Estrogen 0 10 100 0 10 100 (nM) 3 Tyrosinase Tyrosinase * * * Tyrosinase 2 * PDZK1 PDZK1 * β-Actin ve ratio o 1 β -Actin MITF 0

tyrosinase/actin Relati 0 10 100 0 10 100 (nM) β-Actin Control PDZK1 KC+MC Fold Tyrosinase Control siRNA PDZK1 siRNA 2 PdZK1 * * 1.5 Estrogen 0 10 100 0 10 100 (nM) 1.5 * * # # 1 Tyrosinase 1 # # 0.5 PDZK1 0.5

0 β-Actin Relative ratio of of ratio Relative NC oligo1 oligo2 oligo1+2 Tyrosinase/actin 0

mRNA expression expression mRNA 0 10 100 0 10 100 (nM) Relative ratio of PDZK1 PDZK1 of ratio Relative Control PDZK1

ER-α KC+MC c Fold ER-β d KC+MC MC Control siRNA PDZK1 siRNA Fold ER-α C PDZK1 C PDZK1 2.5 * * 3 Estrogen 0 10 100 0 10 100 (nM) * ER-β ER-α 66 kDa 2 2.5 * * ER-α 2 1.5 * * * β 1.5 ## # # ER- 56 kDa 1 ER-β 1 0.5 0.5 β-Actin β-Actin 0 0 Ratios of increment Ratios of increment PDZK1 PDZK1 010100010100(nM) KC+MC MC Control siRNAPDZK1 siRNA PDZK1 e ER-α 10 ER-β 9 8 7 6 5 4 3 2

Ratios of increment 1 0 1234567891011121314 Patients

Figure 2. PDZK1 affects estrogen-induced tyrosinase expression. (a, b) Western blotting of PDZ domain protein kidney 1 (PDZK1) or tyrosinase expression after treatment of indicated concentrations of estrogen only or with PDZK1 overexpression or knockdown. (c, d) Western blotting of estrogen receptor (ER)-a and ER-b in total cell lysates, prepared 24 hours after PDZK1overexpression, or estrogen with or without PDZK1 knockdown. Data in the graphs (b–d) represent means±SD of five independent experiments (*Po0.05 vs. untreated control, #Po0.05 vs. samples treated with the corresponding concentration of estrogen). (e) Real-time PCR of ER-a and ER-b in the hyperpigmented compared with normally pigmented skin from 14 of the 15 patients. ERK, extracellular signal–regulated kinase; KC, keratinocytes; MC, melanocytes; MITF, microphthalmia-associated transcription factor; siRNA, small interfering RNA.

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a KC+MC MC KC+MC, MC C Fold Fold C PDZK1 C PDZK1 KC+MC, PDZK1 4 2.5 MC PDZK1 Tyrosinase * * 2 * * * * 3 * * * CFTR 1.5 * 150 kDa 2 NHE3 100 kDa 1 Ratios of increment 1 0.5 NHE1 100 kDa Relative ratio of ratio Relative tyrosinase/actin 0 0 SLC26A3 85 kDa CCPDZK1 PDZK1 CFTR NHE3 NHE1 SLC26A3 β-Actin KC+MC MC

b MC KC IP: PDZK1 Total lysate IP: PDZK1 Total lysate PDZK1C CPDZK1 PDZK1C C PDZK1 PDZK1 PDZK1 CFTR CFTR SLC26A3 SLC26A3 NHE3 NHE1 NHE1 β-Actin

c C PDZK1

PDZK1 CFTR PDZK1 CFTR

Hoechst Merge Hoechst Merge

PDZK1 CFTR PDZK1 CFTR

KC+MC

Hoechst Merge Hoechst Merge

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in vitro system in this study using keratinocyte–melanocyte about the interaction, western blotting analysis (Figure 3a) cocultures and melanocyte monocultures. Increased expres- and immunoprecipitation with and without PDZK1 over- sion of tyrosinase (Figure 1d), as well as of tyrosinase expression (Figure 3b) revealed that three transporter proteins transcription regulator, microphthalmia-associated transcrip- (CFTR, NHE3, and SLC26A3) interacted with PDZK1 in tion factor, and stimulation of extracellular signal–regulated epidermal keratinocytes and melanocytes as they do in other kinase, CRE-binding protein, and protein kinase C phos- organs (Gisler et al., 2003; Rossmann et al., 2005). In phorylation with the PDZK1 upregulation (Figure 1e) addition, double staining in cultured cells with and without suggested a role of these pathways in tyrosinase and PDZK1 overexpression showed the interactions and coloca- microphthalmia-associated transcription factor upregulation. lization between transporter proteins and PDZK1 (data not Estrogen stimulates PDZK1 expression in ER-containing shown except CFTR in Figure 3c). Estrogen also acted on the cells (Ediger et al., 1999; Stemmer-Rachamimov et al., 2001); expression of three transporter proteins (Figure 4a) similar to however, the estrogen-induced upregulation of PDZK1 has that of PDZK1 (Figure 2a), although each specific inhibitor of not been reported in connection with melanogenesis. In this those transporter proteins inhibited the estrogen-induced study, we showed that estrogen-stimulated expression of expression of tyrosinase in the presence or absence of PDZK1 PDZK1 paralleled tyrosinase expression (Figure 2a), although overexpression without a change in the expression of PDZK1 PDZK1 overexpression did not change the cell number either (Figure 4b and c), which indicates that the transporter in melanocytes or keratinocytes (data not shown). The proteins are downstream of PDZK1 in the signaling pathway estrogen-induced tyrosinase expression was increased further of estrogen-induced expression of tyrosinase. The role of ion by PDZK1 overexpression and was inhibited by the knock- transporters in the estrogen-induced expression of tyrosinase down of PDZK1 (Figure 2b), which suggests that estrogen is is further supported by the results of ER agonist treatment; the involved in regulating melanogenesis through PDZK1. It ER-a agonist increased NHE3 expression and the ER-b agonist could be expected that the action of estrogen would intensify changed CFTR. As the ER-a agonist did not induce a in the skin with upregulated PDZK1, even though serum significant change in CFTR and the ER-b agonist did not estrogen levels were the same. Of course, it does not mean affect NHE3 expression (Figure 4d), there seems to be a that innate pigmentation is linked to estrogen. If it did, young specific association between each ER subtype and an ion women would tend to be darker as they ascend through transporter. In fact, the expression of NHE3 is reduced in ER- puberty and men would have fairer skin compared with a-knockout mice (Lee et al., 2001; Zhou et al., 2001), women. The result that overexpression of PDZK1 (Figure 2c), whereas CFTR expression is increased (Lee et al., 2001). As as well as estrogen treatment (Figure 2d), increased the estrogen exerts its actions through ER-b in ER-a-knockout expression of both ER-a and ER-b, whereas knockdown of mice (Shughrue et al., 1997), the modulation of CFTR PDZK1 reduced the estrogen-induced upregulation of ERs expression through ER-b could be explained. Interestingly, down to the untreated control levels (Figure 2d) indicated the concordance in the expression between ion transporter and function of estrogen induced through the expression of its each ER subtype, particularly between CFTR and ER-b, was receptors (Katzenellenbogen et al., 2000). ER expression in suggested in a patient with melasma (Figure 4e). patients with PDZK1-upregulated melasma (Figure 2e) In addition to melanogenesis, the FACS analysis results supported the potential association between PDZK1 and showed that PDZK1 upregulation stimulated melanosome ERs, although the expression levels between PDZK1 and ER-a transfer to keratinocytes. With the reduction of PDZK1- and/or ER-b were not correlated in some patients. It is unclear induced melanosome transfer by inhibitors of ERs and by ion why the levels were not correlated. Other types of ERs transporter (Figure 5a), sequential processes of estrogen (GPER1/GPR30 (Nilsson et al., 2011)) or other factors (anti- through PDZK1 and then ERs into ion transporters could be estrogens, protein kinase activators, and growth factors) may suggested for PDZK1-induced melanosome transfer. Whereas be involved in the regulation of ER biological activity PAR-2 is involved in melanosome transfer (Seiberg et al., (Katzenellenbogen, 1996). The increase in ER expression in 2000), Rac1 and ERM instead of PAR-2 are involved in concert with PDZK1 overexpression in cultured cells (data PDZK1-stimulated melanosome transfer (Figure 5b). not shown) further supported the fact that PDZK1 regulated In summary, this study demonstrates a significant role for ER expression. the upregulation of PDZK1 in the development of hyperpig- NHERF family proteins mediate the most abundant mentation in the skin of melasma patients, particularly protein–protein interactions and bind to NHE, CFTR, and associated with estrogen, through identification of the clinical the SLC26A family. Although no report has been available correlation and the mechanism of action with its interacting

Figure 3. PDZK1 over-expression upregulates NHE, CFTR, and SLC26A3. (a) Western blot analysis of NHE1, NHE3, CFTR, and SLC26A3 expression in melanocytes and cocultures (KC þ MC) 24 hours after transfection of PDZ domain protein kidney 1 (PDZK1). Data in the graphs represent means±SD of five independent experiments (*Po0.05). (b) Immunoprecipitation (IP) of total cell lysates of melanocytes and keratinocytes using an anti-PDZK1 antibody with or without PDZK1overexpression, followed by reactivity with antibodies as noted. (c) Immunohistochemical staining of cells with or without PDZK1overexpression using anti-PDZK1 and anti-CFTR antibodies. Nuclei were counterstained with Hoechst 33258 (bar ¼ 0.1 mm). CFTR, cystic fibrosis transmembrane conductance regulator; ERK, extracellular signal–regulated kinase; KC, keratinocytes; MC, melanocytes; MITF, microphthalmia-associated transcription factor; NHE1, sodium–hydrogen exchanger 1.

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KC+MC MC PDZK1 transfection – – – ++ + Fold Tyrosinase Estrogen (100 nM) – + +++– NHE3 Estrogen 0 10 100 0 10 100 (nM) 3 EIPA (5 μM) – – + ––+ * * CFTR Tyrosinase * * 2 ** # # NHE3 NHE3 # # SLC26A3 β-Actin 1 β -Actin PDZK1 Ratios of increment 0 C C ES ES c ES+EIPA ES+EIPA PDZK1 transfection – – – +++ Fold Tyrosinase Estrogen (100 nM) – + + – + + 3 * PDZK1 CFTR * 172lnh (5 μM) – – + ––+ 2.5 Tyrosinase 2 * * * * # CFTR 1.5 # # # 1 β-Actin 0.5 Ratios of increment PDZK1 0 C C ES ES

ES+172lnh ES+172lnh PDZK1

d PPT DPN Fold 010 100 100 1,000 (nM) ER-β 3 ER-α Fold ER-α CFTR NHE3 * 2 * * NHE3 * * 2 * * 1.5 * ER-β 1 1 Ratios of increment

CFTR Ratios of

increment 0.5

SLC26A3 0 0 C 10 100 (nM)(C 100 1,000 nM) Tyrosinase PPT DPN β-Actin

e 10 9 PDZK1 8 ER-α 7 NHE3 β 6 ER- CFTR 5 4 3

Ratios of increment 2 1 0 1 234567891011 12 Patients

Figure 4. NHE3, CFTR, and SLC26A3 involvement in estrogen-induced tyrosinase expression. (a) Western blotting of NHE3, CFTR, and SLC26A3 expression after indicated concentrations of estrogen (ES) treatment. (b, c) Western blotting of tyrosinase and NHE3 or CFTR expression after treatment of 100 nM estrogen with or without a specific inhibitor of NHE3 (5-ethylisopropyl amiloride, EIPA), or CFTR (172Inh) with or without PDZ domain protein kidney 1 (PDZK1) overexpression (*Po0.05 vs. untreated control, #Po0.05 vs. samples treated with the corresponding concentration of estrogen). (d) Western blotting after indicated concentrations of specific agonists of ER-a (propyl pyrazoletriol, PPT) or ER-b (diarylpropionitrile, DPN) (*Po0.05). Data in the graphs (b–d) represent means±SD of five independent experiments. (e) Real-time PCR analysis of the association of ER-a with NHE3 mRNA expression and of ER-b with CFTR expression in 12 melasma skin samples with PDZK1 upregulation. (Line indicates expression level of each molecule in normal skin.) CFTR, cystic fibrosis transmembrane conductance regulator; ER, estrogen receptor; ERK, extracellular signal–regulated kinase; KC, keratinocytes; MC, melanocytes; NHE3, sodium–hydrogen exchanger 3.

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a C C+lCI C+PHTPP C+EIPA C+172Inh

K14

TYRP-1 15.6±0.36 12.65±2.7 14.62±2.18 11.34±0.78 16.34±0.2

K14

TYRP-1 23.4±2.20 15.42±1.25 17.37±1.6 13.35±2.1 17.45±1.09 PDZK1 PDZK1+lCI PDZK1+PHTPP PDZK1+EIPA PDZK1+172lnh

1.8 1.6 * 1.4 # # 1.2 # # 1 0.8 0.6 0.4 0.2 Ratios of increment 0 C PDZK1 CCCCPDZK1 PDZK1 PDZK1 PDZK1 ICI PHTPP EIPA 172lnh b C PDZK1 80 kDa p-ERM 75 kDa ERM 80 kDa 3 75 kDa 2.5 * p-RAC 2 * 25 kDa 1.5 RAC 25 kDa 1 PAR-2 0.5

50 kDa Ratios of increment 0 β-Actin 43 kDa C

p-ERM/ERM p-RAC/RAC PAR-2/actin

Figure 5. PDZK1 and ion transporter are involved in estrogen-induced melanosome transfer with ezrin/radixin/moesin (ERM) and Rac1 phosphorylation. (a) FACS analysis using anti-TYRP-1 and anti-K14 antibodies in cocultures. Both melanocytes and keratinocytes, which were transfected with or without PDZ domain protein kidney 1 (PDZK1), were treated with inhibitor of both estrogen receptor (ER)-a and ER-b (ICI), inhibitor of ER-b (PHTPP), or a specific inhibitor of NHE3 (5-ethylisopropyl amiloride, EIPA) or CFTR (172Inh) (*Po0.05 vs. untreated control, #Po0.05 vs. samples with overexpression of PDZK1). (b) Western blot assays of ERM and Rac1 phosphorylation and PAR-2 expression in the cocultures with or without PDZK1 overexpression (*Po0.05). Data in the graphs represent means±SD of five independent experiments. CFTR, cystic fibrosis transmembrane conductance regulator; ER, estrogen receptor; ERK, extracellular signal–regulated kinase; NHE3, sodium–hydrogen exchanger 3; PAR-2, proteinase-activated receptor 2; Rac1, ras-related C3 botulinum toxin substrate 1.

molecules. These results indicate the potential application of comparisons. The locations of the hyperpigmented lesions were the PDZK1 and/or ion transporters for the targeted therapy in forehead or upper cheeks, which were shaded by scalp hairs. melasma. Control uninvolved skin was taken from the retroauricular area. These specimens were used for real-time PCR and immunohisto- MATERIALS AND METHODS chemistry. Patients Fifteen female patients diagnosed with melasma (36 to 61 years, with Normal human epidermal cell culture a mean age of 48 years) were included in the study. The Institutional Skin specimens obtained from cesarean sections and circumcisions Review Board of the Dongguk University Ilsan Hospital approved were used for establishing cells in culture. The epidermis was this study, and the study was conducted according to the separated from the dermis, and suspensions of individual epidermal Declaration of Helsinki Principles. After obtaining informed written cells were prepared. consent from each patient, pairs of normally pigmented and Keratinocytes were suspended in EpiLife medium (#M-EPI-500- hyperpigmented skin specimens were taken by biopsy for direct CA; Invitrogen, Carlsbad, CA) supplemented with bovine pituitary

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extract, bovine insulin, hydrocortisone, human epidermal growth G-30 (forward) and 50-GTTCCCACTAACCTTCCTTTTC-30 (reverse); factor, and bovine transferrin (#S-001-5; Invitrogen). Keratinocytes NHE350-GGAGTCCTTCAAGTCGACCA-30 (forward) and 50-GGGA from passages 3 or 4 were used in these experiments. Melanocytes TGCTGCTGTTTCTCC-30 (reverse); CFTR 50-GAAGTAGTGATGG were suspended in Medium 254 (Invitrogen) supplemented with AGAATGTAACAGC-30 (forward) and 50-GCTTTCTCAAATAATTCC bovine pituitary extract, fetal bovine serum, bovine insulin, CCAAA-30 (reverse); and glyceraldehyde 3-phosphate dehydrogen- hydrocortisone, basic fibroblast growth factor, bovine trans- ase 50-TCCACTGGCGTCTTCACC-30 (forward) and 50-GGCAGAGA ferrin, heparin, and phorbol 12-myristate 13-acetate (Invitrogen). TGATGACCCTTT-30 (reverse). Melanocytes at passage numbers between 7 and 15 were used for these experiments. Western blot analysis All procedures were performed three times for each of the three Equal amounts of extracted proteins (20 mg) were resolved using 10% cell lines, i.e., a total of nine procedures for each experiment. SDS-PAGE and were then transferred to nitrocellulose membranes. The membranes were incubated with antibodies to phospho- PDZK1 overexpression and knockdown extracellular signal–regulated kinase, extracellular signal–regulated For PDZK1 overexpression, transfection of cells with pcDNA3.1- kinase, phospho-CRE-binding protein, CRE-binding protein, phos- MYC containing the PDKZ1 gene was carried out using Lipofecta- pho-protein kinase C, protein kinase C, phospho-ERM, ERM, mine 2000 according to the manufacturer’s protocol. For PDZK1 phospho-Rac1, Rac1 (rabbit polyclonal; Cell Signaling Technology, downregulation, cultured cells in six-well plates were transfected Beverly, MA), tyrosinase, SLC26A3 (goat polyclonal; Santa Cruz with two different stealth small interfering RNAs (1, 2) and a negative Biotechnology, Santa Cruz, CA), ER-a, ER-b, NHE3, CFTR, micro- control stealth small interfering RNA (Invitrogen) using Lipofecta- phthalmia-associated transcription factor (rabbit polyclonal; Santa mine LTX (Invitrogen) and 60 nM small interfering RNA, according to Cruz Biotechnology), PDZK1 (mouse monoclonal; Santa Cruz the manufacturer’s instructions. The cells were used for the Biotechnology), and further with anti-rabbit or anti-mouse horse- experiments 48 hours after the transfection. radish peroxidase–conjugated antibodies (Santa Cruz Biotechnol- ogy), and were then treated with an enhanced chemiluminescence Treatment with estrogen with or without specific inhibitors solution (Thermo, Rockford, IL). The signals were captured on an against ion transporters, ER agonists, or ER antagonists Image Reader (LAS-3000; Fuji Photo Film, Tokyo, Japan). To monitor The transfected cells were incubated for 24 hours in media contain- the amount of protein loaded in each lane, the membranes were ing 10 nM or 100 nM 17b-Estradiol (E2), with or without 5 mM CFTR reprobed with mouse monoclonal anti-b-actin antibody (Sigma) and 172Inh (a CFTR inhibitor), and 5 mM 5-ethylisopropyl amiloride (a were processed as described above. Protein bands were analyzed by NHE inhibitor; Sigma Diagnostics, St Louis, MO). densitometry. The cultured cells were incubated in media without supplements, after which they were treated with 10 nM or 100 nM propyl Immunohistochemistry pyrazoletriol (an ER-aagonist), 100 nM or 1,000 nM diarylpropionitrile For immunofluorescence staining, after deparaffinization and (an ER-b agonist; Tocris Bioscience, Ellisville, MO), 1 mM PHTPP (an rehydration, the sections were preincubated with 3% BSA. For ER-b antagonist), or 2.5 mM ICI 182,780 (an ER-a,b antagonist; Sigma double staining, the cultured cells or sections were reacted sequen- Diagnostics) for 24 hours. tially with the anti-PDZK1 antibody and 1:200 Alexa Fluor–labeled goat anti-mouse IgG (488; Molecular Probes, Eugene, OR), and then Reverse transcription–PCR with anti-c-kit, ER-a, ER-b, and CFTR antibodies and Alexa Total RNAs from cells cultured under each condition were obtained Fluor–labeled goat anti-rabbit IgG (594; Molecular Probes). Nuclei with TrizolReagent (Invitrogen). Reverse transcription of each RNA were counterstained with Hoechst 33258 (Sigma). The stained was performed using a First Strand cDNA Synthesis Kit for RT-PCR specimens were evaluated using an image analysis system (Dp (AMV; Boehringer, Mannheim, Germany). Human glyceraldehyde Manager 2.1; Olympus Optical, Tokyo, Japan). 3-phosphate dehydrogenase was used as the internal standard. The primer sequences for PDZK1 were as follows: forward 50-ATGA Immunoprecipitation CCTCCACCTTCAACCCC-30 and reverse 50-ACATCTCTGTATCT Cells were solubilized with lysis buffer and centrifuged at 10,000g TCAGAAT-30. The primer sequences for glyceraldehyde 3-phosphate for 10 minutes at 4 1C. Lysates were immunoprecipitated with the dehydrogenase were as follows: forward 50-TCCACTGGC-GTC anti-PDZK1 antibody, and were then separated by SDS-PAGE. TTCACC-30 and reverse 50-GGCAGAGATGATGACCCTTTT-30. The Western blotting was performed using antibodies against CFTR, DNA fragments produced by PCR were separated by electrophoresis NHE1, NHE3, and SLC26A3. on 2% agarose gels. Melanosome transfer Real-time PCR Flow cytometry was used for quantitative analysis of melano- Levels of mRNAs relative to glyceraldehyde 3-phosphate dehydro- some transfer to keratinocytes, as previously described (Lin et al., genase were measured by quantitative real-time PCR using the Light 2008). In brief, the cells were fixed with 2% paraformaldehyde for Cycler real-time PCR (Roche, Mannheim, Germany). The primers 10 minutes, washed with phosphate-buffered saline containing BD used were as follows: PDZK1 50-GGCAGGCTCAGAACAGAAAG-30 PhosFlow Perm/Wash Buffer I (BD Biosciences, San Jose, CA) for (forward) and 50-CTACCAGATCATTGTTCTTC-30 (reverse); ER-a50- 10 minutes, and then incubated with both FITC-conjugated goat anti- TGTGCAATGACTATGCTTCA-30 (forward) and 50-GCTCTTCCTC TYRP-1 (1 g per 1 106 cells; Santa Cruz Biotechnology) and CTGTTTTTA-30 (reverse); ER-b50-GTATGCGGAACCTCAAAAGA phycoerythrin-conjugated mouse anti-Cytokeratin 14 (1:10; BD

2630 Journal of Investigative Dermatology (2012), Volume 132 N-H Kim et al. PDZK1 in Melasma

Biosciences) for 30 minutes at room temperature. The cells were Katzenellenbogen BS, Choi I, Delage-Mourroux R et al. (2000) Molecular then washed thoroughly with phosphate-buffered saline and mechanisms of estrogen action: selective ligands and receptor pharma- cology. J Steroid Biochem Mol Biol 74:279–85 prepared for flow cytometry. Labeled cells were analyzed with a Cytomics FC500 flow cytometer (Beckman Coulter, Hialeah, FL) Ko SB, Zeng W, Dorwart MR et al. (2004) Gating of CFTR by the STAS domain of SLC26 transporters. Nat Cell Biol 6:343–50 using the CXP software (Beckman Coulter). The melanosome transfer Lamprecht G, Seidler U (2006) The emerging role of PDZ adapter proteins for efficacy was determined as the number of Cytokeratin 14–positive regulation of intestinal ion transport. Am J Physiol Gastrointest Liver and TYRP-1-positive cells divided by the total number of Cytokeratin Physiol 291:G766–77 14–positive cells. Lee KH, Finnigan-Bunick C, Bahr J et al. (2001) Estrogen regulation of ion transporter messenger RNA levels in mouse efferent ductules are Statistical analysis mediated differentially through estrogen receptor (ER) alpha and ER beta. Biol Reprod 65:1534–41 Statistical significance was evaluated using one-way analysis of Lieberman R, Moy L (2008) Estrogen receptor expression in melasma: results variance analysis with the Bonferroni’s multiple comparison test. from facial skin of affected patients. J Drugs Dermatol 7:463–5 The Wilcoxon signed rank test was used to analyze statistical Lin HC, Shieh BH, Lu MH et al. (2008) A method for quantifying melanosome significance between hyperpigmented and normally pigmented skin transfer efficacy from melanocytes to keratinocytes in vitro. Pigment Cell specimens. A P-value of less than 0.05 is considered to be Melanoma Res 21:559–64 statistically significant. All results are presented as means±SD of Mount DB, Romero MF (2004) The SLC26 gene family of multifunctional the combined data from replicate experiments. anion exchangers. Pflugers Arch 447:710–21 Nakamura N, Tanaka S, Teko Y et al. (2005) Four Na+/H+ exchanger isoforms CONFLICT OF INTEREST are distributed to Golgi and post-Golgi compartments and are involved The authors state no conflict of interest. in organelle pH regulation. J Biol Chem 280:1561–72 Nilsson BO, Olde B, Leeb-Lundberg LM (2011) G protein-coupled oestrogen receptor 1 (GPER1)/GPR30: a new player in cardiovascular and ACKNOWLEDGMENTS metabolic oestrogenic signalling. Br J Pharmacol 163:1131–9 This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. 2011-0026205). Rossmann H, Jacob P, Baisch S et al. (2005) The CFTR associated protein CAP70 interacts with the apical Cl/HCO3 exchanger DRA in rabbit small intestinal mucosa. Biochem 44:4477–87 REFERENCES Sarangarajan R, Shumaker H, Soleimani M et al. (2001) Molecular and functional characterization of sodium–hydrogen exchanger in skin as Behne MJ, Meyer JW, Hanson KM et al. (2002) NHE1 regulates the stratum well as cultured keratinocytes and melanocytes. Biochim Biophys Acta corneum permeability barrier homeostasis. Microenvironment acidifica- 1511:181–92 tion assessed with fluorescence lifetime imaging. J Biol Chem 277: 47399–406 Sato F, Soos G, Link C et al. (2002) Cystic fibrosis transport regulator and its mRNA are expressed in human epidermis. J Invest Dermatol 119: Broere N, Chen M, Cinar A et al. (2009) Defective jejunal and colonic salt 1224–30 absorption and alteredNa(+)/H (+) exchanger 3 (NHE3) activity in NHE regulatory factor 1 (NHERF1) adaptor protein-deficient mice. Pflugers Scott GA, Cassidy L (1998) Rac1 mediates dendrite formation in response to Arch 457:1079–91 melanocyte stimulating hormone and ultraviolet light in a murine melanoma model. J Invest Dermatol 111:243–50 Ediger TR, Kraus WL, Weinman EJ et al. (1999) Estrogen receptor regulation of the Na+/H+ exchange regulatory factor. Endocrinology 140:2976–82 Seiberg M, Paine C, Sharlow E et al. (2002) The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions. Exp Ghosh MG, Thompson DA, Weigel RJ (2000) PDZK1 and GREB1 are Cell Res 254:25–32 estrogen-regulated genes expressed in hormone-responsive breast cancer. Cancer Res 60:6367–75 Shughrue P, Scrimo P, Lane M et al. (1997) The distribution of estrogen receptor-beta mRNA in forebrain regions of the estrogen receptor-alpha Gisler SM, Pribanic S, Bacic D et al. (2003) HPDZK1: a major scaffolder in knockout mouse. Endocrinology 138:5649–52 brush borders of proximal tubular cells. Kidney Int 64:1733–45 Singh AK, Riederer B, Chen M et al. (2010) The switch of intestinal Slc26 Grichnik JM, Burch JA, Burchette J et al. (1998) The SCF/KIT pathway plays exchangers from anion absorptive to HCO formula secretory mode is a critical role in the control of normal human melanocyte homeostasis. dependent on CFTR anion channel function. Am J Physiol Cell Physiol J Invest Dermatol 111:233–8 298:C1057–65 Grubb BR, Boucher RC (1999) Pathophysiology of gene-targeted mouse Smith DR, Spaulding DT, Glenn HM et al. (2004) The relationship between models for cystic fibrosis. Physiol Rev 79:193–214 Na(+)/H(+) exchanger expression and tyrosinase activity in human Jang YH, Lee JY, Kang HY et al. (2010) Oestrogen and progesterone receptor melanocytes. Exp Cell Res 298:521–34 expression in melasma: an immunohistochemical analysis. J Eur Acad Stemmer-Rachamimov AO, Wiederhold T, Nielsen GP et al. (2001) NHE-RF, Dermatol Venereol 24:1312–6 a merlin-interacting protein, is primarily expressed in luminal epithelia, Jeon S, Kim NH, Kim JY et al. (2009) Stem cell factor induces ERM proteins proliferative endometrium, and estrogen receptor-positive breast carci- phosphorylation through PI3K activation to mediate melanocyte nomas. Am J Pathol 158:57–62 proliferation and migration. Pigment Cell Melanoma Res 22:77–85 Zhou Q, Clarke L, Nie R et al. (2001) Estrogen action and male fertility: Katzenellenbogen BS (1996) Estrogen receptors: bioactivities and interactions roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in with cell signaling pathways. Biol Reprod 54:287–93 reproductive tract function. Proc Natl Acad Sci USA 98:14132–7