Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding disorder, and, in some patients, ceroid storage disease and progressive lung fibrosis (Shotelersuk and Gahl, 1998). The symptoms associated with HPS reflect structural and/or functional defects in a group of related organelles including melanosomes of the melanocyte (causing albinism), dense granules of the platelet (causing a bleeding disorder), and lysosomes of reticuloendothelial cells (causing ceroid storage). The hypopigmentation associated with HPS is extremely variable and involves the skin, hair, and eyes (Shotelersuk and Gahl, 1998). The characteristic clinical symptoms associated with HPS can be due to mutations at different genetic loci including ADTB3A, which codes for a subunit of the coat protein complex AP-3 (Oh et al, 1996;Huizing et al, 2000;Shotelersuk et al, 2000). The most common variant of HPS, however, is due to mutations in HPS1, a gene that encodes a predicted polypeptide of 700 amino acids with a molecular mass of approximately 80 kDa (Huizing et al, 2000;Oh et al, 2000). The function of the HPS1 gene product is unknown, but the protein appears to be nonglycosylated, cytosolic, and partly associated with melanosomes (Oh et al, 2000). Melanocyte cultures lacking HPS1 mRNA exhibit hypomelanization due to the translocation of tyrosinase and tyrosinase-related protein 1 (TRP-1) to membranous complexes rather than premelanosomes (Boissy et al, 1998b). Based upon these results, it has been suggested that HPS1p may facilitate trafficking of melanocyte-specific gene products to the premelanosome (Boissy et al, 1998b).
This study describes ultrastructural aberrations in human melanocytes bearing mutations in HPS1. In addition, the effect of inhibition of HPS1 protein (HPS1p) expression on trafficking of melanocyte-specific gene products, tyrosinase activity, and melanin synthesis was studied by transfecting pigmented melanoma cells with HPS1 cDNA in an antisense orientation. This in vitro model of HPS1p ablation recapitulates the in vivo melanocyte defects and permits evaluation of the role of HPS1p in melanocytic cells.
Materials and methods
Skin biopsies
After informed consent, 4 mm skin biopsies were obtained from normal volunteer donors and from two patients with HPS, one homozygous for the 16 bp duplication in HPS1 typical of northwest Puerto Rican patients and one heterozygous for an A1195del mutation and an unknown mutation. Both patients have been described previously (Boissy et al, 1998b).
Cell culture
Pigmented melanoma SK-MEL-188 cells, a gift from Dr. Alan Houghton, Sloan Kettering Institute, New York, were maintained in Dulbecco's minimum essential medium (DMEM) (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco), 1% antibiotic/antimycotic solution (Gibco), and 1% MEM nonessential amino acids (Gibco).
Vector construction and transfection
A cDNA of approximately 2.3 kb encoding the full length HPS1p was excised from pcDNA3.1/His A, B, C and inserted into the EcoRI/EcoRI site of pcDNA3.1 (+) (Invitrogen, Carlsbad, San Diego) using standard molecular biology protocols. The orientations of inserts were confirmed by restriction digests and sequencing. Cells were transfected with sense or antisense HPS1 cDNA in pcDNA3.1 (+) using an Effectene reagent kit (Qiagen, Valencia, CA) according to the manufacturer's protocols. Selection of the transfectants with 1.5 mg per ml G418 (Gibco) was started on day 3 after the initial transfection. Expression of HPS1p was confirmed in the transfected cells by Western blotting. Transfection efficiency of approximately 12%-15% was achieved by this method.
Dihydroxyphenylalanine (DOPA) histochemistry and electron microscopy
Skin biopsies were cut into quarters and fixed with half-strength Karnovsky's fixative (Karnovsky, 1965) in 0.2 M sodium cacodylate buffer at pH 7.2 for 24 h at 4°C. M-188, S-188, and A-188 cells were seeded in Laboratory-Tek chamber slides (Nunc, Naperville, IL) coated with 1% pig gelatin and grown to approximately 80%-90% confluence (Boissy et al, 1998b). The cells were fixed in the wells with half-strength Karnovsky's fixative (Karnovsky, 1965) in 0.2 M sodium cacodylate buffer at pH 7.2 for 30 min at room temperature. For DOPA histochemistry, fixed tissues and cells were incubated in a 0.1% solution of L-DOPA twice for 2.5 h. The tissues and cells were washed three times in buffer and treated with 1.0% osmium tetroxide containing 1.5% potassium ferrocyanide (Karnovsky, 1971) for 30 min. The tissues and cells were washed, stained en bloc with 0.5% uranyl acetate for 30 min, dehydrated, and embedded in Eponate12. Areas of the Epon cast were cut out and mounted on Epon pegs and sectioned on an RMC MT 6000-XL ultramicrotome. Ultrathin sections were then stained with aqueous solutions of uranyl acetate (2%) and lead citrate (0.3%) for 15 min each and photographed using a JEOL JEM-100CX transmission electron microscope. All tissue processing supplies were purchased from Ted Pella (Tustin, CA).
Western blotting analysis
Total cellular protein, from untransfected SK-MEL-188 (M-188) cells and M-188 cells transfected with HPS cDNA in either sense (S-188) or antisense (A-188) orientation, was extracted using RIPA buffer. Equal amounts of protein were fractionated on a 10% sodium dodecyl sulfate polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane and incubated with 10% nonfat dry milk in phosphate-buffered saline (PBS) and Tween-20 (PBST, pH 7.4 with 0.2% Tween-20) for 1 h at room temperature. The membrane was probed with two polyclonal rabbit antibodies. One, called p-80, was generated against the peptide sequence DDIQPSPRRARSSQN, corresponding to residues 253–267 of the human HPS1p (1:1000) (Dell'Angelica et al, 2000). The other, called HPS-C, was generated against the carboxy terminus of the HPS1p (1:1000) in 2% nonfat milk/PBST solution at 4°C for 3 h. The bands of interest were visualized by indirect immuno-enzymatic staining using an alkaline phosphatase labeled secondary antiserum followed by BCIP/NBT substrate (Kirkegaard and Perry, Gaithersburg, MD).
Tyrosine hydroxylase activities
To quantitate tyrosinase activity, two assay protocols were employed. In an in situ assay, intact cells were incubated in medium containing 1.0 
Ci per ml [3H]tyrosine (specific activity, 54.2 Ci per mmol) for 24 h. In an in vitro assay, solubilized cell lysates (in triplicate) were incubated in 1 ml of reaction mixture (in triplicate) containing 80–100 g of protein, 1.0 M L-DOPA, and 1.0 Ci [3H]tyrosine at 37°C for 1 h. Each reaction was stopped by addition of an equal volume of 10% (wt/vol) solution of activated charcoal in 0.2 N citric acid. The samples were centrifuged at 1500g in a Beckman 5000 centrifuge for 5 min and the supernatants were passed over a Dowex ion-exchange column followed by a wash of 0.1 N citric acid. After addition of 10 ml of scintillation fluid (Ultima Gold, Packard Biosciences, Groningen, The Netherlands), the radioactivity of the eluate was counted in a Packard 1900 CA liquid scintillation analyzer. Tyrosinase activity was expressed as dpm per mg protein (in vitro assay) and dpm per cell (in situ assay).
Melanin content
Equal numbers of M-188, S-188, and A-188 cells were washed in ice-cold PBS and sonicated in 0.5% Nonidet P-40/PBS solution. After determination of protein content using the bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL), aliquots of the cell lysates were solubilized in 0.5 ml of 0.1 N NaOH. The optical density of the supernatant at 475 nm was compared with a standard curve by using known concentrations of synthetic melanin, and results were expressed as micrograms of melanin per milligram of protein.
Immunofluorescence
Cell cultures were plated on gelatin-coated Laboratory-Tek (Nunc) chamber slides at 104 cells per 0.9 cm2 well and processed for indirect immunofluorescence the next day. The cells were fixed in 5% formalin for 10 min, permeabilized with 100% methanol for 3 min, rinsed in PBS containing 1% bovine serum albumin (BSA) (Sigma, St. Louis, MO) three times, blocked with 10% normal goat serum in PBS/BSA, and incubated in primary antibody (as described below) for 1 h. Specimens were rinsed with PBS/BSA and incubated in goat antispecies-specific IgG (secondary antibody) conjugated to CyTM2 or CyTM3 affinity purified antiserum (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h. The slides were washed and mounted using Fluoromount (Southern Biotechnology Associates). Fluorescence images were acquired on a Zeiss LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY) using identical parameters for untransfected and transfected cells. Primary antibodies consisted of hPEP7 (1:100) (a gift from Drs King and Oetting, Minnesota, MN), reactive against human tyrosinase, and MEL-5 (1:50) (Signet Pathological Laboratories, Dedham, MA), reactive against human TRP-1 (Boissy et al, 1998a).
Results
Ultrastructural analysis of melanocytes in the basal layer of HPS1 skin
Skin biopsies obtained from a normal individual and from two patients with HPS1 were processed for DOPA histochemistry and electron microscopy. Approximately 10% of melanocytes in the basal epithelial layer from both the HPS1 patients exhibited an occasional large complex with DOPA-positive cisterna and 50 nm vesicles throughout the periphery of the HPS melanocytes Figures 1a, b. These structures were absent from the control samples Figure 1c.
Figure 1.
HPS melanocytes exhibit DOPA-positive membranous complexes.Ultrastructure of melanocytes in the basal layer of the epidermis from an HPS patient homozygous for the 16 bp duplication in HPS1 (a), an HPS patient heterozygous for an A1195del mutation (b), and a control individual (c). Punch biopsies were processed for DOPA histochemistry and electron microscopy. Melanocytes of the HPS patients exhibited complexes with DOPA-positive cisterna (arrows) and 50 nm vesicles (arrowheads) throughout the periphery of the HPS melanocytes that were absent from the control. K, keratinocyte. Scale bar: (a, c) 2.0 m; (b) 0.7 m.
Western blot analysis
The absence of HPS1p from M-188 cells transfected with the HPS1 antisense cDNA was confirmed by Western blot analysis using two polyclonal antibodies, identified as p-80 and HPS-C. An expected band of approximately 79 kDa in size corresponding to HPS1p was detected in lanes containing M-188 and S-188 cell extracts using either p-80 or HPS-C antiserum Figure 2. HPS1p expression was significantly reduced in the pigmented melanoma SK-MEL-188 cells transfected with antisense HPS1 cDNA (A-188 lane in Figure 2).
Figure 2.
Expression of the HPS1 gene product in pigmented melanoma cells by Western blot analysis. SK-MEL-188 (M-188) cells were transfected with human HPS1 cDNA inserted into an EcoRI/EcoRI site of the pcDNA3.1 expression vector in the sense (S-188) and antisense (A-188) orientations. Following selection with G-418, the cells were analyzed for expression of HPS1p by Western blot analysis using HPS-C and p-80 antisera specific for HPS1p. As expected, both the M-188 and S-188 lanes contain a band of approximately 79 kDa using either the HPS-C or HPS-p80 antiserum. In contrast, this band was significantly reduced in the lane containing A-188 cell extract.
Full figure and legend (18K)Tyrosine hydroxylase activity and melanin content
After transfection, cells were pelleted prior to subculturing. The decrease in melanin in the A-188 cell cultures became visible at passage 6. At this passage, the in vitro tyrosinase activities of cell lysates from M-188, S-188, and A-188 cells were not significantly different Figure 3a. In contrast, tyrosinase activity assessed in intact cultures of A-188 cells was 30% lower compared to activities determined in intact M-188 and S-188 cells Figure 3b. The decrease in the tyrosinase activity in intact A-188 cells corresponded to a decrease in melanin synthesis as observed in the cell pellet Figure 4a and cell suspension Figure 4b along with a 50% decrease in soluble melanin content in these cells Figure 4c.
Figure 3.
In vitro and in situ determination of tyrosine hydroxylase activities of tyrosinase in SK-MEL-188 (M-188) cells transfected with human HPS1 cDNA in the sense (S-188) or antisense (A-188) orientations reveal altered activity. In vitro (cell lysate) and in situ (intact cell) tyrosine hydroxylase activities of tyrosinase in SK-MEL-188 cells transfected with the human HPS1 cDNA in the sense (S-188) and antisense (A-188) orientations were determined as described in Materials and Methods. The in vitro determinations in M-188, S-188, and A-188 were not significantly different (A). Tyrosine hydroxylase activities in A-188 cells were significantly lower (p <0.05), however, compared to M-188 and S-188 as determined by the in situ method (B). Tyrosine hydroxylase activities were expressed in dpm per mg protein (in vitro method) and dpm per cell (in situ method).
Full figure and legend (74K)Figure 4.
Melanin content and cellular localization of tyrosinase and TRP-1 was assessed in pigmented melanoma cells (M-188) untransfected and transfected with sense (S-188) or antisense (A-188) human HPS1 cDNA. Approximately equal numbers of cells were centrifuged to form a pellet (a, top panel) and cell suspension (b, bottom panel) photographed to demonstrate large (M-188, S-188) and small (A-188) amounts of melanin pigmentation. Soluble melanin content, expressed in g melanin per mg protein (c), was also determined. There was a significant reduction in the amount of pigmentation in A-188 cells (a, b) and of soluble melanin in A-188 (c) compared to M-188 and S-188 cells (p <0.05). Cellular localization of tyrosinase and TRP-1 in S-188 and A-188 cells was determined by immunofluorescent techniques. In contrast to M-188 (M column, frames d, g, j) and S-188 (S column, frames e, h, k), both tyrosinase (frames d, e) and TRP-1 (frames g, h) in A-188 cells (A column, frames f, i, l) were localized to distinct large granular structures throughout the cytoplasm and dendrites (frames f, i). Images in frames j, k, and l are the merged images of frames d, e, f with frames g, h, I, respectively, to document colocalization of tyrosinase and TRP-1.
Immunofluorescence
At passage 6, intracellular localization of tyrosinase and TRP-1 in SK-MEL-188 (M-188) as well as in those transfected with sense (S-188) and antisense (A-188) HPS1 cDNA was performed by immunofluorescence analysis using antibodies. There were no significant differences in the patterns of expression of either tyrosinase or TRP-1 in M-188 Figures 4d, g, j and S-188 Figures 4e, h, k cells. In contrast, in A-188 cells, tyrosinase and TRP-1 were localized to distinct large granular structures throughout the cytoplasm and dendrites Figures 4f, i, l.
DOPA histochemistry and electron microscopy
Ultra structural analysis of SK-MEL-188 (M-188) cells transfected with sense (S-188) or antisense (A-188) HPS1 cDNA was performed using a combination of DOPA histochemistry and electron microscopy. DOPA reaction products were present in the trans-Golgi network and melanosome-like vesicles of M-188 and S-188 cells Figures 5a, b. In A-188 cells, DOPA reaction products were observed within large membranous complexes of the cell body and dendrites as well as in the normal intracellular location Figures 5c, d. These large membranous complexes were morphologically similar to those described in human HPS1 melanocytes in vivo Figure 1 and in vitro (Boissy et al, 1998b).
Figure 5.
Electron microscopy of M-188, S-188, and A-188 cells processed by DOPA histochemistry for the localization of functional tyrosinase. SK-MEL-188 (M-188) cells transfected with sense (S-188) or antisense (A-188) human HPS1 cDNA were processed for electron microscope analysis. DOPA reaction products were present in the trans-Golgi network (block arrows) and melanosomes (block arrowheads) of M-188 (a), S-188 (b), and A-188 cells (c, d). The A-188 cells (c, d), however, also exhibited the presence of DOPA reaction products in large membranous complexes (block arrows in c and d), in contrast to M-188 and S-188 cells. Scale bar: (a, b, c) 0.6 m; (d) 0.45 m.
Discussion
The HPS represents a collection of autosomal recessive genetic disorders, characterized clinically by oculocutaneous albinism and a mild to severe bleeding diathesis due to absence of platelet dense granules. Some patients accumulate electron dense ceroid lipofuscin in various tissues, and some develop pulmonary fibrosis or granulomatous colitis (Huizing et al, 2000). The genetic cause of one type of HPS was first identified as a mutation in the HPS gene (currently classified as HPS1), characterized by a 16 bp duplication in a subpopulation of Puerto Rico inhabitants, resulting in absence of HPS1p (Oh et al, 1996). Since then, numerous mutations in various ethnic groups have been identified (Huizing et al, 2000). Although mutations in the HPS1 gene affect skin pigmentation, the role of HPS1p in melanogenesis remains unclear. We have previously suggested that HPS1p is involved in mediating proper intracellular trafficking of melanocyte-specific gene products to the target organelle, the melanosome (Boissy et al, 1998b). This was based on the observation that melanocyte cultures established from patients carrying mutations in HPS1 demonstrated altered intracellular trafficking of tyrosinase and TRP-1 to large membrane complexes. Recently, it was shown that in melanocytic cells HPS1p exists as a > 500 kDa complex consisting of a 200 kDa component localized to the peri-nuclear area and associated with small noncoated vesicles as well as early stage melanosomes (but not with mature melanosomes) (Oh et al, 2000). The authors suggested that HPS1p functions in the early stages of melanosome biogenesis. Concrete evidence regarding the exact function of HPS1p in melanocytes and its role in melanogenesis is still elusive, however. This study was an attempt to characterize the role of HPS1p in mediating intracellular trafficking of tyrosinase/TRP-1, as well as melanocytic function, by knockout of HPS1p expression in a pigmented melanoma cell line.
We initiated the study by characterizing the ultrastructural characteristics of the melanocytes in intact skin of two HPS patients, one homozygous for the 16 bp duplication and the other heterozygous for an A1195del and an unidentified mutation. Electron microscope analysis of the skin revealed large complexes with DOPA-positive cisterna and 50 nm vesicles throughout the periphery of the HPS melanocytes. This is consistent with our previous report demonstrating large membrane-bound complexes containing DOPA reaction products and DOPA-positive rings delineated on both sides by limiting membranes in melanocyte cultures from HPS patients (Boissy et al, 1998b). The findings confirm that the ultrastructural abnormality previously observed in HPS1 melanocytes recapitulates the in vivo situation. The presence of numerous giant melanosomes in a subset of Japanese patients carrying mutations in HPS1 has been reported (Horikawa et al, 2000).
Significant reduction in HPS1p expression in pigmented melanoma cells by transfection of HPS1 cDNA in the antisense orientation provided an in vitro model of HPS1 to facilitate characterization of the role of HPS1p in intracellular trafficking of tyrosinase/TRP-1. Significant reduction in HPS1p expression in the pigmented melanoma cells (M-188) transfected with HPS1 antisense cDNA was confirmed by Western blot analysis. There was no significant difference in the tyrosine hydroxylase activities in cell lysates of M-188, S-188, and A-188 cells, suggesting that lack of HPS1p does not influence the level of tyrosinase protein expression in melanocytic cells. This is consistent with previous reports demonstrating normal tyrosine hydroxylase activities in lysates of HPS1 melanocytes (Boissy et al, 1998b). In contrast, the tyrosine hydroxylase activity of intact A-188 cells was significantly reduced compared to that of M-188 and S-188 cells. This is consistent with previous reports demonstrating decreased tyrosine hydroxylase activities in intact HPS1 melanocyte cultures (Boissy et al, 1998b). As the melanosome is the primary site possessing the biochemical conditions necessary for eliciting tyrosine hydroxylase activities associated with tyrosinase, the decrease in in situ tyrosine hydroxylase activity can be attributed to a decreased presence of tyrosinase in the melanosome. In addition, a significant decrease in melanin content in the A-188 cells (lacking expression of HPS1p) was observed. The differences in tyrosine hydroxylase activities associated with in vitro and in situ determinations, along with the decrease in melanin content in cells not expressing HPS1p, suggest aberrant targeting/localization of tyrosinase to the melanosome.
Immunohistochemical analysis of melanocyte cultures established from HPS1 patients demonstrated that both tyrosinase and TRP-1 were localized in a coarse granular pattern with large aggregates throughout the cell body that appear ultrastructurally as large membranous complexes (Boissy et al, 1998b). Consistent with this observation, knockout of HPS1p (A-188 cells, Figure 4) resulted in confinement of tyrosinase and TRP-1 expression to large membranous complexes throughout the cell body and dendrites. Electron microscope analysis of the A-188 cells demonstrated that absence of HPS1p resulted in the deposition of DOPA reaction products (thereby localizing tyrosinase) in large membrane-bound structures with limiting membranes. It has recently been speculated that melanocyte-specific proteins are trafficked from the trans-Golgi network to the melanosome via a "coated endosome" (of endosomal lineage) that served as an intermediate repository for these proteins before their eventual sorting to the melanosome (Raposo et al, 2001). The large membranous complexes observed in A-188 cells could be viewed as a structure similar to the "coated endosome". Absence of normal HPS1p would prevent the trafficking of tyrosinase to the melanosome and consequently accumulates in these membrane complexes. Alternatively, these membranous complexes may represent residual bodies (of lysosomal lineage) where degradation is occurring. The large membranous complexes reported herein (Figure 1, Figure 5) as well as in cultured HPS1 melanocytes (Boissy et al, 1998b) contain other inclusions in addition to DOPA-positive elements that consist of 50 nm vesicles, compact aggregates of membranes, and mitochondria. These heterogeneous aggregates are characteristic of developing residual bodies (i.e., secondary lysosomes) that occur during autography. We speculate that the undirected tyrosinase in HPS1 melanocytes may be subsequently trafficked to the lysosome system for degradation. Precise confirmation of the large membranous complexes is warranted, however. Nevertheless, we can conclude that, with significant reduction in the expression of HPS1p, tyrosinase is trafficked to intracellular sites that are distinctly different from melanosomes.
Biochemical and ultrastructural data obtained from this study recapitulate previous observations in skin biopsies as well as cultures of melanocytes established from HPS1 patients. Thus, knockout of the HPS1p from the pigmented melanoma cell line represents a viable experimental model that can be utilized to evaluate the role of HPS1p in intracellular trafficking of melanocyte-specific gene products, tyrosinase and TRP-1 in particular, in the modulation of pigmentation and melanocytic function.
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