Glycoprotein nonmetastatic melanoma protein b ... - The FASEB Journal

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Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome-specific and proteolytically released protein Toshihiko Hoashi,*,†,1 Shinichi Sato,† Yuji Yamaguchi,* Thierry Passeron,* Kunihiko Tamaki,† and Vincent J. Hearing*,1 *Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; and †Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan Melanosomes are organelles specialized for the production of melanin pigment and are specifically produced by melanocytic cells. More than 150 pigmentation-related genes have been identified, including glycoprotein nonmetastatic melanoma protein b (GPNMB). A recent proteomics analysis revealed that GPNMB is localized in melanosomes, and GPNMB is a membrane-bound glycoprotein that shows high homology with a well-known melanosomal structural protein, Pmel17/gp100. In this study, we show that GPNMB is expressed in melanocytes of normal human skin, as well as in human melanoma cells. GPNMB is heavily glycosylated and is enriched in mature (stage III and IV) melanosomes in contrast to MART-1 and Pmel17, which are abundant in early (stage I and II) melanosomes. MART-1 and Pmel17 play critical roles in the maturation of early melanosomes; thus, we speculate that GPNMB might be important in the functions of late melanosomes, possibly their transport and/or transfer to keratinocytes. We also demonstrate that a secreted form of GPNMB is released by ectodomain shedding from the largely Golgi-modified form of GPNMB and that the PKC and Ca2ⴙ intracellular signaling pathways regulate that shedding. We conclude that GPNMB is a melanosomal protein that is released by proteolytic ectodomain shedding and might be a useful and specific histological marker of melanocytic cells.—Hoashi, T., Sato, S., Yamaguchi, Y., Passeron, T., Tamaki, K., Hearing, V. J. Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome-specific and proteolytically released protein. FASEB J. 24, 1616 –1629 (2010). www.fasebj.org ABSTRACT

Key Words: ectodomain shedding 䡠 glycosylation 䡠 phorbol ester Melanosomes are lysosome-related organelles (LROs), which have the unique capacity to produce melanin pigment (1) and which progress through four sequential morphological steps as they mature (2). Stage I melanosomes are round, membrane-bound, and electron-lucent vesicles that are generally found in the perinuclear area where they are formed. Their transition to stage II melanosomes involves an 1616

elongation of the vesicle and the appearance within of distinct fibrillar structures. Following delivery of critical enzymes, melanins are deposited on these fibers, resulting in a progressively pigmented internal matrix, at which time the organelles are termed stage III melanosomes. In highly pigmented tissues, melanin synthesis and deposition continue until little or no internal structure is visible, at which time they are termed stage IV melanosomes. More than 150 pigmentation-related genes have been identified to date (3), many of them having specific functions in melanosomes. Of those, tyrosinase (TYR), tyrosinase related protein-1 (TYRP1), and dopachrome tautomerase (DCT) have been shown to have distinct enzymatic activities. A gene named Pmel17 was one of the earliest pigment genes cloned and is the human homologue of the mouse silver gene, the disruption of which produces a silver hair color in mice (4, 5). Pmel17/ gp100 (hereafter termed Pmel17) was also identified and cloned as a melanoma-specific antigen recognized by tumor infiltrating lymphocytes (6). Pmel17 has fibrillogenic capacities found in stage II, III, and IV melanosomes (7–11). Melanoma antigen recognized by T cell-1 (MART-1) was initially cloned by two independent groups using melanoma reactive CD8⫹ T cells (12, 13). We previously reported that MART-1 is required for the maturation of Pmel17 (14). Melanomas are among the most notorious tumors for their poor prognosis (15). Currently, S100 protein is most widely used for the diagnosis of melanoma, and it has high sensitivity, however, its specificity is relatively low (16 –18). Another melanogenic marker Pmel17, which can be detected by the HMB45 antibody, is also widely used because of its high specificity, however, its sensitivity is relatively low (16 –18). To overcome those problems, other melanogenic-related proteins, such as 1 Correspondence: V.J.H., National Institutes of Health, Laboratory of Cell Biology, Bldg. 37, Rm. 2132, Bethesda, MD 20892-4256, USA. E-mail: [email protected]; T.H., Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: [email protected] doi: 10.1096/fj.09-151019

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MART-1, TYR, and microphthalmia-associated transcription factor (MITF), are used (16 –21). Combinations of those antibodies could detect the majority of melanomas, including amelanotic melanomas, which are one subtype of melanomas (19, 20). However, some melanomas are S100-, Pmel17-, MART-1-, and/or TYRnegative (21, 22) and can be challenging to diagnose (19, 22, 23). Moreover, in other subtypes of melanomas, including desmoplastic melanomas (which are frequently misdiagnosed as scars or dermatofibromas), the standard criteria for diagnosis are still that they are S100-positive and HMB45-negative (18, 19, 23). Thus, additional specific markers that can target those melanomas are critical to develop. Glycoprotein nonmetastatic melanoma protein b (GPNMB) was initially cloned from poorly metastatic melanoma cells, and overexpression of GPNMB was shown to decrease tumor growth (24). A recent proteomics analysis revealed that GPNMB is also a melanosomal protein (25, 26), and it is regulated by MITF as is TYR, TYRP1, DCT, Pmel17, and MART-1 (27). Human GPNMB has 560 aa (24) and is a type I membrane protein predicted to consist of several domains by homology modeling (Fig. 1A). The SIG domain is a signal peptide, which is thought to determine the entry of GPNMB into the secretory pathway. PKD is a polycystic kidney disease-like domain bearing an immunoglobulinlike folding structure (28). KRG is a kringle-like domain, which is a triple disulfide-linked autonomous structural domain found throughout blood clotting and fibrinolytic proteins (29); it is generally thought the KRG domain plays a role in binding interactions with other

proteins necessary for their regulation (30). TM is a predicted transmembrane domain. The remainder of the domains of GPNMB are annotated as NTD (Nterminal domain), GAP1, GAP2, and CTD (C-terminal domain). A dileucine-based sorting signal is located in the CTD. GPNMB has 12 potential N-glycosylation sites in its lumenal domains (Fig. 1A). An RGD motif with the capacity to bind to integrins (31) in the NTD domain appears to contribute to the adhesion to keratinocytes (32). An isoform of GPNMB (GPNMB-l) that is probably generated due to alternative splicing has been identified (32, 33) that has an additional 12-aa insert in the GAP2 region (Fig. 1A). The highest homology for GPNMB at the protein level in humans is with Pmel17 (Fig. 1B) (24), and as noted above, both GPNMB and Pmel17 are melanosomal proteins (25, 26). Pmel17 has been characterized well (7, 8, 10, 11, 14, 34 –38); thus, it should be useful to characterize GPNMB compared with Pmel17 as a control. Mature Pmel17 is processed at a furin-mediated cleavage site (CS) in GAP2 (7) and is further processed intracellularly at a metalloproteinase-sensitive cleavage site (S2), at a ␥-secretase-sensitive cleavage site (␥) and at multiple shedding sites in the RPT domain (10, 39). A major fraction of mature Pmel17 undergoes ectodomain shedding in GAP3 (34). The striking difference between GPNMB and Pmel17 is that the latter has an RPT domain, which consists of imperfect repeats of proline, serine, and threonine-rich (10), and has a fibrillogenic capacity (10, 11). Human GPNMB also shows significant homology with quail neuroretina clone 71 (40), with rat osteoac-

Figure 1. Domain mapping and proA cessing of GPNMB. A) Schematic of human GPNMB and its 8 domains as defined in the text: SIG, NTD, PKD, GAP1, KRG, GAP2, TM, and CTD. Numbers represent amino acid residues based on GPNMB. Note that the RGD motif is located in the NTD. Solid circles indicate potential N-glycosylation sites. An isoform of GPNMB has been identified (termed GPNMB-l) where the underlined 12 aa are inserted in GAP1, probably due to alternative splicing. B) Pmel17 is subdivided into B the following 10 domains as shown in A and defined in the text, including RPT. Solid circles indicate N-glycosylation sites. Dashed line indicates Furin-mediated cleavage site (CS) in GAP2, subdivided into GAP2a and C GAP2b by the CS. Metalloproteinasesensitive cleavage site (S2), ␥-secretase-sensitive cleavage site (␥), multiple shedding sites in the RPT domain (broken lines) and ectodomain shedding sites (broken lines) in the GAP3 are also shown. This schematic is based on previous publications (7, 10, 34, 35, 39). C) Scheme of the GPNMB expression vector used; pGPNMB-tag and pGPNMB represent the human GPNMB expression vectors with or without tags, respectively. HA tag was inserted just after the SIG domain; FLAG tag was inserted just after the CTD. CHARACTERIZATION OF THE MELANOCYTIC CELL MARKER GPNMB

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tivin (41), with murine osteoactivin (42– 45), with murine dendritic cell-associated heparin sulfate proteoglycan-dependent integrin ligand (46) and with murine Gpnmb (47). Murine osteoactivin has two glycosylated forms (97 and 116 kDa), which can be secreted as 90and 100-kDa forms, leaving a 20-kDa C-terminal fragment (42). In contrast, murine osteoactivin has a mature isoform (115 kDa) and an immature isoform (65 kDa). Only the mature isoform is secreted as a 115-kDa molecule as is the intracellular mature isoform. Murine Gpnmb is enriched in macrophages and is localized in the Golgi, then it is secreted during macrophage activation (47). Moreover, the secreted form of murine Gpnmb retains its C terminus and appears to be a membrane-associated form and thus does not appear to be secreted via ectodomain shedding from the plasma membrane (47). Murine Gpnmb has also been reported to be localized to lysosomes and to melanosomes (32). Recently, human GPNMB was reported to be localized at the plasma membrane (32, 33) and is strongly expressed in human keratinocytes detected by immunohistochemical staining (32). Human GPNMB appears to be secreted, although this has not been characterized well (48). Human GPNMB, murine Gpnmb, and cow Gpnmb have RGD motifs in their NTD domains, but rat Gpnmb and dog Gpnmb do not. As noted above, the RGD motif in the NTD domain has an adhesion capacity (32), which suggests that the function of the RGD motif might be species specific. Recently, a mutation of murine Gpnmb was shown to cause iris pigment dispersion in DBA/2J mice (49). However, the phenotype derived from mutations in the human GPNMB gene is currently unknown. The sum of these previous studies implies a species diversity in the function of GPNMB. In this study, we focused on characterizing human GPNMB in melanocytic cells. GPNMB was expressed in melanocytic cells with or without detectable MART-1 or Pmel17. GPNMB mRNA and protein were detected in melanocytes but not in keratinocytes or in fibroblasts of human skin. GPNMB is less stable than Pmel17 but is not significantly buried in the TX-insoluble fraction. The secreted form of GPNMB (termed sGPNMB) is largely Golgi modified and is shed by ectodomain shedding. These results show that GPNMB is a melanosomal protein and suggest that GPNMB might be a useful histological marker, as well as a serum marker for diagnosing or evaluating malignant melanoma.

grown in melanocyte growth medium, which consists of medium 254 and human melanocyte growth supplement-2 (Cascade Biologics). Melanocytes from the third to fifth passage were used in these experiments. Skin specimens were taken from three healthy volunteers, who gave written informed consent to participate in this study, which was approved by the Medical Ethical Committee of the University of Tokyo. Plasmids and transfection Total RNAs were reverse-transcribed from NHM-m using SuperScript III (Invitrogen, Carlsbad, CA, USA). cDNAs were amplified with a PCR cloning enzyme (Stratagene, La Jolla, CA, USA) using human GPNMB-specific primers. Products were ligated into the pCRII vector (Invitrogen), which was designated as pCRII-GPNMB. The GPNMB fragment was subcloned into the NheI-NotI site of the pCI mammalian expression vector (Promega, Madison, WI, USA) and was designated as pCI-GPNMB (also termed pGPNMB). A hemagglutinin (HA)-tag was inserted in pCI-GPNMB just after the N-terminal signal sequence by site-directed mutagenesis (10, 34). Briefly, pCI-GPNMB was amplified using specific primers with DNA polymerase (Stratagene). The PCR product was digested with DpnI (New England Biolabs, Ipswich, MA, USA); then it was transformed into TOP10 competent cells (Invitrogen). Moreover, a FLAG tag just after the C terminus was inserted by conventional PCR, and the vector was designated as pCI-HA-GPNMB-FLAG (also termed pGPNMB-tag). pCI-Pmel17-i containing Pmel17-i in the pCI vector was a kind gift from Dr. Michael S. Marks (University of Pennsylvania, Philadelphia, PA, USA) (7, 10, 34). All constructs were sequence verified and were transfected into HeLa cells with Lipofectamine 2000 (Invitrogen), as described previously (10, 14, 34). Transfection efficiencies were ⬃70 – 80%, assayed by monitoring fluorescent signals using the enhanced green fluorescent protein (EGFP)-C3 vector (Clontech, Mountain View, CA, USA). Antibodies and reagents

MATERIALS AND METHODS

AF2550, an anti-human GPNMB antibody, was purchased from R&D Systems (Minneapolis, MN, USA). ␣PEP13h and ␣PEP7h polyclonal antibodies were generated in rabbits against synthetic peptides corresponding to the carboxyl termini of human Pmel17 and TYR, respectively (8, 10, 14, 34, 36, 50). HMB45 (Dako, Carpentaria, CA, USA) and HMB50 (NeoMarkers, Fremont, CA, USA) were used to detect Pmel17. M2-9E3 (NeoMarkers) for MART-1, and TA99 (NeoMarkers) for TYRP1 were also used. Anti-HA antibody was purchased from Covance (Emeryville, CA, USA), and antiFLAG antibody was purchased from Sigma (St. Louis, MO, USA). DMSO, brefeldin-A (BFA), PMA, N-(-6-aminohexyl)-5chloro-1-napthalenesulfonamide (W-7), and staurosporine were purchased from Sigma. GM-6001 was purchased from Chemicon (Temecula, CA, USA). Endoglycosidase H (EndoH), protein glycanase F (PNGaseF) and neuraminidase were purchased from New England Biolabs.

Cell cultures and skin specimens

Subcellular fractionation

Highly pigmented MNT-1 melanoma cells, unpigmented SK-MEL-28 melanoma cells, unpigmented WM266-4 melanoma cells, and HeLa cells were obtained and cultured as described previously (10, 14, 34). Lightly pigmented and moderately pigmented normal human melanocytes, NHM-l and NHM-m, respectively, were purchased from Cascade Biologics (Portland, OR, USA). Melanocyte cultures were

We used a previously described protocol to isolate various subcellular fractions of melanosomes (8, 14, 50). Briefly, confluent monolayers of MNT-1 cells and of WM266-4 cells were harvested and washed once in 0.25 M sucrose. Specimens were then homogenized and centrifuged at 1000 g for 10 min. The supernatants were recovered and further centrifuged at 19,000 g for 30 min. The pellets were resuspended in

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2.0 M sucrose and were layered at the bottom of a 1.0 –2.0 M sucrose step gradient, which was centrifuged at 100,000 g for 1 h, and the various layers were then carefully recovered. The 1.0 M layer was subsequently layered in the middle of a 0.8 –1.4 M sucrose step gradient. That gradient was again centrifuged at 100,000 g for 1 h at 4°C, and the 0.8 and 1.0 M fractions were carefully recovered. All of these procedures were done at 4°C. Immunofluorescence microscopy Sections of paraffin-embedded skins (4 ␮m) were deparaffinized, and the expression of melanosomal proteins was detected by indirect immunofluorescence using primary antibodies, as noted in the text. Bound antibodies were visualized with appropriate secondary antibodies, Alexa Fluor 488 or 594 (Molecular Probes, Eugene, OR, USA). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) purchased from Vector Laboratories (Burlingame, CA, USA). Fluorescence was observed and analyzed using a Leica DMR B/D MLD fluorescence microscope (Leica, Wetzlar, Germany) and a Dage-MTI 3CCD 3-chip color video camera (Dage-MTI, Michigan City, IN, USA). Cells cultured in 2-well Lab-Tek chamber slides (Nalge Nunc, Rochester, NY, USA) were immunostained as described previously (10, 14, 34). The reactivities of Alexa Fluor 488 and 594 were visualized as green and red signals, respectively. All preparations were examined with a confocal microscope (LSM 510; Zeiss, Jena, Germany), equipped with HeNe (543 and 633 nm), argon, and krypton laser sources.

affinized, treated with proteinase K, and placed in 200-ml acetylation buffer (0.1 M triethylamine, pH 8.0, containing 0.25% acetic anhydride) for 15 min. After washing in 4⫻ saline sodium citrate (SSC) for 10 min, samples were incubated in prehybridization solution (2⫻ SSC and 50% deionized formamide) for 1 h at 47°C. After overnight hybridization at 47°C, samples were placed in a hybridization solution containing 10 ␮l purified digoxigenin (DIG)-labeled antisense and sense (control) riboprobes using pCRII-GPNMB as the template. Samples were then incubated in 10 mM TrisHCl, 0.5 M NaCl, and 0.25 mM EDTA (TNE) buffer, treated with RNaseA for 30 min, and returned to TNE buffer for 3 min, all at 37°C. After washing in 0.1⫻ SSC for 15 min at 47°C, samples were blocked for 30 min and were incubated with anti-DIG/HRP conjugate (Dako) for 40 min at room temperature. For detection, the tyramide signal amplification system (Dako) and VIP solution (Vector) were used according to the manufacturer’s recommendations. Samples were observed and photographed in a DMR B/D MLD fluorescence microscope (Leica). In vitro translation In vitro translation was performed using TNT T7 kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. pCI-GPNMB and pCI-Pmel17-i were used as the template and [35S]Met as the substrate.

RESULTS

Protein extraction and immunoblotting Cell extracts were prepared using the M-PER mammalian protein extraction reagent (Pierce, Rockford, IL, USA), 1% Triton X-100 (TX; Sigma) or 1% Nonidet P-40 (Calbiochem) in the presence of protease inhibitor cocktail (Roche, Indianapolis, IN, USA). After centrifugation at 20,000 g for 30 min, the supernatants were harvested. Protein concentrations were measured using the BCA protein assay (Pierce). Immunoblotting was performed as described previously (10, 14, 34). Metabolic labeling and immunoprecipitation Transfected HeLa cells, MNT-1 melanoma cells, or NHM-l melanocytes were pulsed for 15 or 30 min with [35S]Met/Cys (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) and then were chased for the periods indicated in the figure legends. Supernatants were harvested after a low-speed centrifugation. Cells were lysed with lysis buffer containing 1% TX. Twenty micromolar N-ethylmaleimide was added to each sample to inhibit the artificial formation of disulfide-bonds where indicated. Immunopurified samples were electrophoresed and visualized by fluorography as described previously (10, 14, 34). Glycosidase digestion Immunopurified samples or cell extracts solubilized in MPER were split into 3 or 4 equal aliquots and were digested with or without EndoH (750 U), PNGaseF (750 U), or neuraminidase (75 U) (10, 34). Digestion reactions were performed for 12 h at 37°C. Samples were electrophoresed and visualized by fluorography as described above. Tissue in situ hybridization (TISH) TISH was carried out as described previously (51). Briefly, cross sections of paraffin-embedded skins (4 ␮m) were depar-

GPNMB is expressed in melanocytic cells Prior to characterizing GPNMB expression in melanocytic cells, we tested the specificity of anti-GPNMB antibody used in this study. We constructed pGPNMB, a GPNMB expression vector, and pGPNMB-tag, which has an HA tag just after the N-terminal signal sequence and a FLAG tag just after the C terminus (Fig. 1C). HeLa cells, which have no endogenous expression of GPNMB, were transfected with pGPNMB-tag and were radiolabeled for 30 min without chase, then were harvested and immunoprecipitated using normal rabbit serum (NRS) as a negative control, or using antiGPNMB, anti-HA, or anti-FLAG antibodies. Similarly sized bands (⬃100 kDa, denoted as P1) were detected using each of the specific antibodies (Fig. 2A), which suggests that P1 is the specific band of GPNMB and probably represents its full-length form. Next, we characterized GPNMB expression in human melanocytic cells. Two independent clones of NHM-l (NHM-l1 and NHMl2) and two independent clones of NHM-m (NHM-m1 and NHM-m2) were used. Pigmented MNT-1, unpigmented SK-MEL-28, and unpigmented WM266-4 melanoma cells were also used because those cell lines have already been characterized well (7, 10, 14, 36, 50). MNT-1 cells contain all stages of melanosomes (8), and SKMEL-28 cells contain only stage I and II melanosomes (50), while WM266-4 cells contain only stage I melanosomes (14). As a control, HeLa cells transfected with an empty vector or with pGPNMB were used. Immunoblotting was performed using the anti-GPNMB antibody (Fig. 2B). HeLa cells overexpressing GPNMB revealed

CHARACTERIZATION OF THE MELANOCYTIC CELL MARKER GPNMB

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Figure 2. GPNMB is expressed in various types of melanocytic cells. A) HeLa cells transfected with the pGPNMB-tag were metabolically radiolabeled for 30 min. Cells were harvested immediately; extracts were then immunoprecipitated with NRS, anti-GPNMB, anti-HA, or anti-FLAG antibodies, separated by electrophoresis and visualized by autoradiography. B–F) HeLa cells transfected with the empty vector or with pGPNMB and melanocytic cells, two independent clones of NHM-l melanocytes, two independent clones of NHM-m melanocytes, highly pigmented MNT-1 cells, unpigmented SK-MEL-28 cells, and unpigmented WM266-4 cells, were harvested then solubilized. Samples were analyzed by immunoblotting using anti-GPNMB (B), ␣PEP13h (C), HMB45 (D), anti-TYR (E), and anti-MART-1 (F) antibodies. GPNMB is post-translationally modified in the ER (P1), then is processed to the mature form (M) in the Golgi. Pmel17 is also post-translationally modified in the ER (P1) and matures in the Golgi (to P2), then is cleaved into the M␣-M␤ complex (7, 10, 14, 34 –36). M␣ is further processed into M␣1, M␣2, and M␣3 (10). Arrows indicate specific bands.

two distinct bands: P1 as described above and a larger band (⬃110 kDa) annotated as M (for mature). The M and P1 forms are GPNMB-specific because they were detected only if GPNMB was expressed. Bands of similar size were detected using all melanocytic cells examined. As noted above, GPNMB shows high homology with Pmel17, so we also focused on Pmel17 as a control for GPNMB. Immunoblotting of the same extracts using anti-Pmel17 antibodies (␣PEP13h and HMB45). Pmel17 is a type I membrane protein and is post-translationally modified in the endoplasmic reticulum (ER; termed P1, the major ER-modified form), after which it matures in the Golgi (termed P2, the largely Golgi-modified form) (7, 36). P2 is then cleaved into the M␣ (N-terminal side) and M␤ (C-terminal side) fragments (7, 36). P1, P2, and M␤ can be detected using ␣PEP13h (Fig. 2C), which reacts with the C 1620

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terminus of Pmel17 (52). M␣ is further processed into M␣1, M␣2, and M␣3 (10), all of which can be detected using HMB45 (Fig. 2D), which reacts with the largely Golgi-modified lumenal domain of Pmel17 (7, 10, 14, 36). HMB45 reactivity corresponds to the presence of stage II melanosomes (8, 10, 14). As previously reported, WM266-4 cells express Pmel17 (detected by ␣PEP13h) but do not process it correctly (no HMB45 reactivity) and thus contain stage I melanosomes only (14). Immunoblotting using anti-TYR (Fig. 2E) and anti-MART-1 (Fig. 2F) antibodies was also performed to confirm the expression of melanosomal proteins. Also, as previously reported, unpigmented WM266-4 cells were negative for MART-1 (14), and unpigmented SK-MEL-28 cells expressed only trace amounts of TYR (14, 50). In contrast to pigmented cells, such as NHM-l, NHM-m, or MNT-1 cells, unpigmented cells lack expression of some melanosomal proteins. Importantly,


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all melanocytic cells examined in this study expressed detectable levels of GPNMB. Pmel17 expression correlates well with pigmentation (5), and that was confirmed in this study. Interestingly, the more highly pigmented NHM-m melanocytes and MNT-1 melanoma cells expressed relatively low levels of GPNMB; thus, GPNMB expression might be negatively correlated with pigmentation in contrast to Pmel17. GPNMB is expressed in epidermal melanocytes These results demonstrate that GPNMB is expressed in melanocytic cells in vitro. A recent study showed that human keratinocytes express considerable amounts of GPNMB but that GPNMB is not detected in epidermal cell suspensions (32). To evaluate GPNMB expression in human skin, we examined GPNMB mRNA expression using GPNMB-specific TISH. In normal skin, melanocytes located at the dermal:epidermal border were readily detected using a GPNMB-specific riboprobe (Fig. 3A). In contrast, epidermal keratinocytes and dermal fibroblasts did not show a detectable signal for GPNMB. Next, we performed immunohistochemistry using the anti-GPNMB antibody and costaining with anti-MART-1 or anti-TYR antibodies to identify melanocytes (Fig. 3B). Melanocytes at the dermal:epidermal border express GPNMB, although TYR expression was relatively weak. Staining of keratinocytes or fibroblasts with the GPNMB antibody was at background level, as expected from the TISH results. Moreover, the GPNMBpositive cells also expressed MART-1 and/or TYR. Interestingly, GPNMB expression seemed to be detected more sensitively than MART-1 or TYR. Taken together, GPNMB mRNA and GPNMB protein were

detected in melanocytes but not in keratinocytes or in fibroblasts of human skin. GPNMB is relatively enriched in mature stage III/IV melanosomes A proteomics analyses revealed that GPNMB is localized in early melanosomes (25) and also in late stages of melanosomes (26), and we wanted to confirm that subcellular localization using immunohistochemistry. HMB50, an antibody that recognizes Pmel17, reacts with the lumenal structure of stage I, II, III, and IV melanosomes, although it preferentially reacts with stage II melanosomes (7, 53, 54) while TA99, an antibody that recognizes TYRP1, reacts with stage III and IV melanosomes (53, 55). The anti-GPNMB and HMB50 antibodies colocalized in the same melanosomes in MNT-1 cells and in SK-MEL-28 cells (Fig. 4A). SK-MEL-28 cells have only early melanosomes (stage I and II) (50), thus GPNMB does localize to early melanosomes. Since anti-GPNMB and TA99 antibodies also colocalize in the same melanosomes in MNT-1 cells, this suggests that GPNMB also localizes to stage III and IV melanosomes. We used a previously established subcellular fractionation technique to purify melanosomes at various stages from pigmented MNT-1 cells (8) and from unpigmented WM266-4 cells (14), and we performed immunoblotting of those fractions using the anti-GPNMB antibody (Fig. 4B). Interestingly, GPNMB is relatively enriched in stage III and IV melanosomes in MNT-1 cells. GPNMB is similarly distributed in the fractions of WM266-4 cells, probably because WM266-4 cells have only stage I melanosomes (14). Taken together, GPNMB

Figure 3. GPNMB mRNA and protein are expressed in epidermal melanocytes in vivo. A) Skin biopsies were examined by TISH using a GPNMB-specific riboprobe, as reported in Materials and Methods. B) Skin biopsies were subjected to immunohistochemistry using anti-TYR, anti-GPNMB, and anti-MART-1 antibodies. GPNMB was detected with Alexa Fluor 594 (red). TYR or MART-1 was detected with Alexa Fluor 488 (green). Nuclei were counterstained with DAPI. Broken line indicates basal membrane zone. Scale bars ⫽ 100 ␮m. CHARACTERIZATION OF THE MELANOCYTIC CELL MARKER GPNMB

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Figure 4. GPNMB is preferentially detected in stage III and IV melanosomes. A) MNT-1 cells and SK-MEL-28 cells were fixed and immunostained with anti-GPNMB and HMB50 antibodies, then reacted with Alexa Fluor 594 (red) and 488 (green), respectively. MNT-1 cells were also fixed and immunostained with anti-GPNMB and TA99 antibodies, then reacted with Alexa Fluor 594 and 488, respectively. Scale bars ⫽ 20 ␮m. B) Subcellular fractions purified from MNT-1 cells (top) and from WM266-4 cells (bottom) were analyzed by immunoblotting using the anti-GPNMB antibody.

is localized in melanosomes, especially in stage III and IV melanosomes. Stability of GPNMB is lower than that of Pmel17 The stability of Pmel17 is lower than that of TYR or TYRP1 (7, 36), and we next characterized the relative stability of GPNMB. We radiolabeled pigmented MNT-1 melanoma cells with [35S] for 30 min, chased them for up to 6 h, and then immunoprecipitated the cell lysates using antiGPNMB or anti-Pmel17 (HMB50) antibodies (Fig. 5A). GPNMB initially appeared as the P1 form immediately (no chase), which then matured to the M form within 1 h; the P1 form decayed quickly at 1 h and was virtually undetectable at 3 h. The M form appeared at 1 h and then quickly decayed and was nearly undetectable at 3 h. In contrast, Pmel17 initially appeared as P1, then gradually decayed but was still detectable at 6 h; P2 appeared at 1 h; then it was cleaved and formed the M␣-M␤ complex. Because the molecular masses of P1 and M␣ are very similar, their bands overlap. At 6 h, the M␣-M␤ complex was undetectable, and only P1 was detectable, as reported previously (7, 36). Thus, compared with the stability of Pmel17, the stability of GPNMB is relatively lower. To more precisely evaluate 1622

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the stability of GPNMB, we radiolabeled pigmented MNT-1 melanoma cells with [35S] for 15 min and chased them for up to 2 h over 30-min intervals (Fig. 5B). In this experiment, in vitro translation was also performed using GPNMB and Pmel17-specific cDNAs (note that glycosylation does not occur in the in vitro translation method used). Nascent GPNMB was observed as a ⬃70 kDa band (termed P0). The P1 form appeared at 0 h; then, it was undetectable at 1.5 h. The M form appeared at 0.5 h and was the strongest at 1 h, after which it quickly decayed and was nearly undetectable at 1.5 h. Nascent Pmel17 was also observed as a ⬃85 kDa band (also termed P0). P1 appeared at 0 h and was still clearly detectable at 2.0 h. P2 appeared at 0.5 h but was undetectable at 1.5 h. The M␣-M␤ complex appeared at 0.5 h and was still clearly detectable at 2.0 h. These results clearly show that the stability of GPNMB is lower than that of Pmel17. GPNMB is not buried in TX-insoluble fractions As noted above, the stability of Pmel17 is lower than that of TYR or TYRP1 (7, 36), a phenomenon that is interpreted as follows: The mature form of Pmel17 (M␣) is buried in the TX-insoluble fraction to form


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ble fractions of MNT-1 cells solubilized with M-PER, 1% TX, or 1% Nonidet P-40 based lysis buffers and then performed immunoblotting using anti-GPNMB, ␣PEP13h, and HMB45 antibodies. Both the P1 and the M forms of GPNMB were strongly detected in the detergent-soluble fractions (Fig. 6A). In contrast, the P1 and M␤ bands of Pmel17 were detected mainly in the detergent-soluble fractions, while M␣ was detected in the TX-insoluble fraction (Fig. 6B), as reported previously (10, 35). Thus, TX-insolubility does not explain the lower stability of GPNMB, and these results suggest that GPNMB is not significantly buried in the TX-insoluble fraction as is Pmel17. Ectodomain of GPNMB is secreted into the medium

Figure 5. GPNMB is quickly degraded. A) MNT-1 cells were metabolically radiolabeled for 30 min, then chased for specific periods as noted. Cell lysates were immunoprecipitated with anti-GPNMB or HMB50 antibodies, separated by electrophoresis, and visualized by autoradiography. B) MNT-1 cells were metabolically radiolabeled for 15 min, then chased for specific periods as noted. Cell lysates were analyzed as described above. In vitro translation of GPNMB and Pmel17 was performed and is also shown.

fibers and seems to disappear in the TX-soluble fractions (7, 10, 35). We used the M-PER cell lysis buffer because we had previously found that TX-insoluble fractions are effectively solubilized in that buffer (10, 36); thus, we used that same approach to examine the solubility of GPNMB. We prepared soluble and insolu-

Recently, we reported that the ectodomain of Pmel17 is released by regulated ectodomain shedding (34). GPNMB also has been reported to be detectable in the medium; however, it has not been characterized well (48). To characterize this process, we radiolabeled HeLa cells transfected with GPNMB, and harvested cell lysates immediately after radiolabeling and medium after a 3-h chase. The labeled extracts were then immunoprecipitated with NRS, or with anti-GPNMB, anti-HA, or anti-FLAG antibodies (Fig. 7A). The P1 form was readily detected in the cell lysates using the anti-GPNMB antibody. The anti-GPNMB and the anti-HA antibodies also recognized slightly slower migrating bands than P1 in the medium, but the NRS or antiFLAG antibodies did not. These results clearly indicate that sGPNMB is specifically detected in the medium. sGPNMB consists of the lumenal domains of GPNMB but does not contain the cytoplasmic tail since it was not recognized by the anti-FLAG antibody. This also suggests that the epitope recognized by the antiGPNMB antibody might lie in the lumenal domains of GPNMB and not in the cytoplasmic tail (Fig. 1A). We then tried to determine whether endogenous

Figure 6. GPNMB is not significantly buried in the TX-insoluble fraction. MNT-1 cells were solubilized with M-PER, 1% Nonidet P-40 or 1% TX containing lysis buffer. Soluble fractions (S) and insoluble fractions (I) were analyzed by immunoblotting using anti-GPNMB (A), ␣PEP13h (B, left), and HMB45 (B, right) antibodies. CHARACTERIZATION OF THE MELANOCYTIC CELL MARKER GPNMB

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Figure 7. sGPNMB is secreted into the medium. A) HeLa cells overexpressing GPNMB were metabolically radiolabeled for 30 min, then chased for 0 h (cell lysates) or 3 h (medium). Cell lysates (C) and medium (M) were immunoprecipitated with the antibodies noted and then were analyzed as described above. B) NHM-l melanocytes were metabolically radiolabeled for 30 min, then chased for specific periods as noted. Cell lysates (C) and medium (M) were immunoprecipitated with the anti-GPNMB antibody, separated by electrophoresis, and visualized by autoradiography. C) Pulse-chase experiment performed as described for B in the presence of BFA at 10 ␮g/ml.

GPNMB is secreted. We radiolabeled NHM-l melanocytes with [35S] for 30 min followed by chases for up to 6 h, then immunoprecipitated the cell lysates and medium using the anti-GPNMB antibody (Fig. 7B). The P1 form quickly decayed as expected and was nearly undetectable at 1 h. Moreover, the M form was also detected immediately, i.e., with no chase. These results are probably due to the higher levels of GPNMB expression by melanocytes than seen in MNT-1 cells (cf. Fig. 2B). sGPNMB was faintly detected at 1 h and was clearly seen at 3 h. sGPNMB migrated between the P1 and M bands, suggesting that it might be the shed product from the M form. The effects of BFA on many types of cells have been well documented. Treatment with BFA results in the retrograde transport of cis-, medial- and trans-Golgi but not trans-Golgi network (TGN) enzymes back to the ER, which inhibits protein flow through the secretory pathway (56). Treatment with BFA increases the stability of Pmel17 and inhibits its secretion (7, 54). We evaluated the effect of BFA on the stability and the secretion of GPNMB. The M form of GPNMB (⬃110 kDa) was clearly detected at all time points (Fig. 7C) but interestingly, sGPNMB was totally inhibited by treatment with BFA. These results suggest that the degradation of GPNMB occurs in a post-Golgi compartment and that GPNMB is secreted through the early secretory pathway.

glycosylation. NHM-l melanocytes were radiolabeled and then chased. Cell lysates and medium were immunoprecipitated and digested with or without EndoH (which removes ER-modified N-glycans) or PNGaseF (which removes all N-glycans). The M form of GPNMB was largely EndoH resistant but PNGaseF sensitive in NHM-l melanocytes (Fig. 8A). The P1 form of GPNMB was EndoH sensitive and PNGaseF sensitive. These results were confirmed by immunoblotting of MNT-1 melanoma cells and HeLa cells overexpressing GPNMB. Neuraminidase removes terminal sialic acids of N-glycans or O-glycans (58) and the M form of GPNMB was slightly sensitive to neuraminidase digestion, which implies that it is heavily sialylated. These results indicate that the P1 form of GPNMB is an ER-modified form and that the M form is a largely Golgi-modified form. Interestingly, sGPNMB in the medium was largely EndoH resistant but PNGaseF sensitive. The molecular mass of sGPNMB was slightly lower than that of the M form. Moreover, deglycosylated sGPNMB clearly migrated faster than the deglycosylated M form (or P1 form of GPNMB). In accordance with these results, we conclude that sGPNMB consists of the ectodomains of GPNMB, and the results also strongly suggest that sGPNMB is the ectodomain of the M form. sGPNMB is enhanced by PMA or CaM inhibition but is inhibited by a metalloproteinase inhibitor

Secreted GPNMB is largely Golgi modified ER-modified (8, 36) and largely Golgi-modified forms of Pmel17 (7) have been reported to be trafficked to melanosomes but only the largely Golgimodified form of Pmel17 forms the fibrillar matrix of stage II melanosomes (9, 10, 57). Next, we tried to clarify which form of GPNMB is secreted in terms of 1624

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In previous studies, GPNMB was localized to the plasma membrane of melanocytic cells (32, 33, 48), and melanocytes are associated with keratinocytes via the RGD motif of GPNMB (32). Recently, we reported that the secreted form of Pmel17 is released from the plasma membrane (34), which raises the question of where the ectodomain release occurs in GPNMB. Deglycosylated


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um

cells, or transfected HeLa cells. These results suggest that the release (secretion) of GPNMB likely occurs in the extracellular milieu but not in intracellular organelles. The release of extracellular domains has recently been described as ectodomain shedding (59, 60). What regulates the ectodomain shedding of GPNMB? Is it a proteolytic event? In some cases, ectodomain shedding is regulated by matrix metalloproteinases, can be enhanced by PMA or by calmodulin (CaM) inhibitors (CaMIs), and can also be inhibited by BFA (60 – 62). We radiolabeled NHM-l melanocytes and chased them for 4 h in the presence of various reagents, then attempted to detect sGPNMB in the medium (Fig. 8B). Significant inhibition of sGPNMB was observed in the presence of GM-6001, which is a broad spectrum metalloproteinase inhibitor (48). The release of sGPNMB was significantly enhanced by W-7, a CaMI and by PMA but was down-regulated by staurosporine, a protein kinase C (PKC) inhibitor. Taken together, the ectodomain shedding of sGPNMB seems to be a proteolytic event dependent on matrix metalloproteinases and enhanced by PMA or CaMI, and the shedding might occur from the plasma membrane rather than in the cytoplasm.

DISCUSSION

Figure 8. sGPNMB is largely Golgi modified, and its secretion is regulated ectodomain shedding. A) NHM-l melanocytes were radiolabeled and chased for 0 h (cell lysates) or for 4 h (medium), then were immunoprecipitated with the NKI/ beteb antibody. Immunopurified samples were split into 3 portions; one was digested with EndoH (E), another was digested with PNGaseF (P), and the third was the untreated control. Digestion reactions were performed for 12 h at 37°C, then samples were electrophoresed and visualized by autoradiography. Bottom: MNT-1 cells and HeLa cells overexpressing GPNMB were solubilized and then digested as described above. One sample was digested with neuraminidase (N). Samples were analyzed by immunoblotting using the antiGPNMB antibody. B) NHM-l melanocytes were radiolabeled and chased in the presence of 100 nM GM-6001, DMSO, 200 nM PMA ⫹ 100 ␮M staurosporine, 200 nM PMA, and 50 ␮M W-7. Medium was immunoprecipitated with the anti-GPNMB antibody, separated by electrophoresis, and visualized by autoradiography.

sGPNMB clearly has a molecular mass distinct from the deglycosylated M or P1 forms (Fig. 8A). Moreover, deglycosylated sGPNMB bands were not detected in cell lysates of NHM-l melanocytes, MNT-1 melanoma

This study shows that GPNMB is expressed in melanocytic cells, including normal human melanocytes, as well as human melanoma cells. As noted above, antiS100 and HMB45 antibodies are widely used for melanoma detection (16 –18, 63). Recently, other melanogenic related proteins, such as MART-1, TYR, and MITF, have been used (16 –20), but some melanomas are S100-, Pmel17-, MART-1-, and/or TYR-negative (19, 21–23). Therefore, other melanoma-specific markers are awaited to avoid misdiagnosis. In this study, we showed that WM266-4 cells, which are MART-1 negative and HMB45 negative, and SK-MEL-28 cells, which express trace amounts of TYR, express detectable amounts of GPNMB. In addition, SK-MEL-28 cells and WM266-4 cells, both of which are amelanotic, express relatively higher levels of GPNMB than do pigmented MNT-1 cells. This implies that MART-1-, Pmel17-, and/or TYR-negative melanomas express detectable amounts of GPNMB. Regarding specificity, GPNMB is specific to melanocytes in human skin. From these results, GPNMB might prove to be a useful specific marker of melanocytic cells, which should be helpful to diagnose melanomas in the clinic. We are now further exploring that possibility. GPNMB has been reported to be detected in histiocytic lymphomas and in renal carcinomas, as well as in melanocytic cells (24), although a recent study reported that renal carcinomas do not express detectable levels of GPNMB (33). Moreover, another recent report showed that almost all keratinocytes express GPNMB at levels stronger than melanocytes, although


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GPNMB could not be detected in epidermal cell suspensions (32). To evaluate the expression of GPNMB more precisely in human skin, we performed immunohistochemistry, as well as GPNMB-specific TISH (shown here for the first time for human skin samples). GPNMB mRNA and protein were detected in melanocytes but not in keratinocytes or in fibroblasts of human skin. Thus, keratinocytes do not express detectable levels of GPNMB in situ, at least under basal conditions. However, GPNMB expression levels in keratinocytes and in melanocytes can be altered by ultraviolet irradiation (unpublished results). To clarify this, further studies will be required. GPNMB was found in melanosomes using a proteomics approach (25, 26), and in a related study, murine Gpnmb was localized to melanosomes in murine melanoma cells (32). In this study, GPNMB expression was relatively stronger in unpigmented melanoma cells (SK-MEL-28 cells or WM266-4 cells) or in lightly pigmented melanocytes (NHM-l cells). GPNMB is localized specifically in melanosomes, especially stage III and IV melanosomes. We conclude that GPNMB might play some role in late melanogenesis (in stage III or IV melanosomes) rather than in early melanogenesis (in stage I and II melanosomes). However, the expression level of GPNMB is not directly associated with pigmentation. We showed that GPNMB is less stable than Pmel17, that the degradation of GPNMB occurs in a post-Golgi compartment and that GPNMB is secreted through the early secretory pathway. However, GPNMB is not significantly buried in the TX-insoluble fractions, as is Pmel17 (10, 35). The meaning of the decreased stability of GPNMB is unclear, and GPNMB might be degraded or secreted quickly. Ectopic GPNMB cannot form fibers like Pmel17 (data not shown), probably because GPNMB does not have the RPT domain, which is required for the fibrillogenic capacity (11). Since GPNMB might not have amyloid characteristics, it might not be buried in the TX-insoluble fractions. The shedding of extracellular domains of transmembrane proteins by this kind of specific proteolytic process in the juxtamembrane is a well-recognized phenomenon. The shedding of membrane proteins is apparently a universal characteristic of eukaryotic cells

(60). However, shedding is restricted to a limited subset of membrane proteins, which are usually type I, type II, or glycosylphosphatidylinositol-anchored membrane proteins (60), which have the following four features: First, one characteristic of the activity of membrane secretases is their highly regulated nature. Second, the activation of intracellular second messenger systems, such as phorbol esters, which are well-characterized activators of PKC or intracellular Ca2⫹ pathways, induce shedding (60, 64). Third, shed proteins might also contain as yet undefined recognition motifs in their extracellular domains that signal stalk cleavage (65). Fourth, in many cases, candidate shedding proteases are unusual disintegrin Zn-metalloproteases (66). In this study, we conclude that GPNMB undergoes ectodomain shedding by which sGPNMB is released. Moreover, the shedding seems to occur mainly at the plasma membrane and is less likely to occur within the cells. GPNMB ectodomain shedding is enhanced by PMA, as occurs for many membrane-localized molecules (67), and that effect could be neutralized by staurosporine. An important intracellular mediator in the actions of Ca2⫹ is CaM (61), which is involved in regulating the shedding of several membrane proteins (62). The relationship between pigmented melanocytes and CaM has been poorly investigated (68). We showed that a CaMI stimulates the shedding of GPNMB. The ectodomain shedding of CD44 has been reported to be stimulated by a CaMI, and the CaMI-induced shedding is mediated by a disintegrin and metalloproteinase (ADAM)10, whereas CD44 shedding induced by PMA is mediated by ADAM-17 (69). Thus, stimulation of the secretion of GPNMB by PMA and by CaMI might be via different pathways. Taken together, GPNMB shedding is a regulated proteolytic process induced by PMA or CaMI. The precise mechanisms responsible for the shedding of GPNMB is as yet unclear. Although further studies are required, GPNMB is released via a particular proteolytic pathway (60, 66). Ectodomain shedding was initially believed to be a cell-surface event; however, there is increasing evidence that ectodomain cleavage can also take place in intracellular compartments, such as exosomes (70), which in tumors could be a source of tumor antigens (71).

Figure 9. Schematic of the maturation of GPNMB. GPNMB is translated as the nascent form P0. The P1 form is modified in the ER and is further modified in the Golgi to the M form. sGPNMB is produced by regulated proteolytic ectodomain shedding. Open arrowheads indicate ER-modified N-glycans; checkmarks indicate Golgi-modified Nglycans. Note that some checkmarked glycans might still be the high-mannose type or the hybridtype N-glycan.

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The trafficking routes that Pmel17 uses to get to melanosomes has been under intense discussion (7, 8, 35, 36, 72). One proposed pathway is that the ERmodified form of Pmel17 is trafficked from the ER or the cis-Golgi to stage I melanosomes and is then further processed, leading to the formation of the fibrillar matrix in stage II melanosomes (8, 36). Another proposed sorting route is that the largely Golgi-modified form of Pmel17 is trafficked from the TGN to early endosomes/stage I melanosomes and then finally to stage II melanosomes (7). In our study, the M form of GPNMB was shown to be the Golgi-modified form, thus GPNMB seems to be trafficked from the TGN to melanosomes (a schematic is shown in Fig. 9). However, in the subcellular fractionation experiments, the P1 (ER-modified) form of GPNMB was also detected in the fractions (Fig. 4B). It is not possible to completely avoid contamination of ER-resident proteins in the fractionated samples (9), and thus, we can not exclude the possibility that the P1 form is also trafficked to melanosomes. Moreover, how Pmel17 is trafficked to the plasma membrane is also under discussion (51, 53, 54). One proposed pathway is that ER-modified Pmel17 escapes from the Golgi stack and then is trafficked to the plasma membrane (51, 54). Another proposed pathway is that Pmel17 is trafficked to the plasma membrane through the TGN (53). In this study, we convincingly show that sGPNMB is Golgi modified and is derived from the M form, which means that sGPNMB is a late Golgi-modified form and that the P1 form might not be secreted even if it is trafficked to the plasma membrane. In conclusion, S100 and Pmel17 are widely used as histological melanocytic tumor markers (16 –18), and there are several melanoma serum markers used in the clinic (16, 73, 74). GPNMB is an excellent candidate for an additional specific histological, as well as a serum melanocytic marker, and we are now further exploring this possibility. The authors gratefully thank Dr. Michael S. Marks (University of Pennsylvania, Philadelphia, PA, USA) for kindly providing the Pmel17-i vector. This work was largely supported by a Shiseido grant for science research (T.H.) and in part by the Intramural Research Program of the U.S. National Institutes of Health, National Cancer Institute.

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