The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells

Cell Differentiation, 23 (1988) 133-142 Elsevier Scientific Publishers Ireland, Ltd.

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The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells M a k o t o Mochii, Takashi Takeuchi, Ryuji K o d a m a , Kiyokazu Agata and Goro Eguchi The National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan (Accepted 29 October 1987)

A monoclonai antibody ( M C / I ) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that M C / 1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomai matrix. A single protein band with an apparent molecular weight of 115000 was labelled by M C / 1 in Western blotting. The 115 kDa polypeptide identified by M C / 1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable. Pigmented epithelial cell; Transdifferentiation; Melanosomal matrix protein; Monoclonal antibody

Introduction Transdifferentiation of pigmented epithelial cells (PECs) into lens cells is a conserved event in all vertebrates (Eguchi and Okada, 1973; Eguchi et al., 1974, 1979; Yasuda et al., 1978; Eguchi, 1986). There is little doubt that the in vitro system of transdifferentiation would be useful in analyzing the instability in cell differentiation at the cellular and molecular levels. Unfortunately, the efficiency of transdifferentiation from cultured PEC was very low in past studies. Itoh and Eguchi (1986a, b) established the new in vitro system, which ensures very high frequency of transdiffer-

Correspondence address: M. Mochii, The National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan.

entiation of PECs in chicken embryos. In this system, a homogeneous population of bipotent cells which are able to differentiate into both lens cells and pigmented cells was maintained in a defined culture medium containing dialyzed fetal calf serum, phenylthiourea, and testicular hyaluronidase. These bipotent cells possess a vigorous growth potential but no identifiable differentiated properties and are thus termed dedifferentiated PECs (dePECs). Almost all of the dePECs simultaneously transdifferentiate into lens cells in certain conditions, and the same population of cells is able to redifferentiate into the PECs in other conditions. The direction and the timing of differentiation from the dePECs are thus well manipulated by changing the constituents of the culture medium as well as the cell densities. This system offers us a large quantity of homogeneous

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cells that are synchronous at certain states of differentiation and makes it possible to analyze the processes of transdifferentiation at the molecular level. In the condition for maintaining a dedifferentiated state, it is known that dePEC does not synthesize 3-crystallin, a major component of the embryonic chicken lens, but starts to synthesize it rapidly after the cells are determined to differentiate into the direction of lens (Itoh and Eguchi, 1986b). Agata and Eguchi (1984)demonstrated the presence of the transcripts of &crystallin gene in the dePEC despite the absence of translated products and suggested the possibility that the expression of differentiated phenotypes of dePEC was regulated post-transcriptionally. To analyze the molecular mechanisms concerning the differentiation of dePECs, it is necessary to characterize the molecular nature of dePEC not only to the lens specificity but also to the PEC specificity, because the dePEC can differentiate into both of these cell types. For marking the specificity of PEC, we have chosen the melanosomal matrix proteins which constitute a matrix stucture of melanosome together with melanin. Several investigators have studied the melanosomal proteins of chicken, human and mouse (Doezema, 1973; Hearing and Eppig, 1974; Jimbow et al., 1982; Zimmerman, 1982), but none of the matrix proteins were well characterized. In the present work, we identified one of the melanosomal matrix proteins of chicken embryos by using hybridoma technique and analyzed the expression of this protein during transdifferentiation process of PEC into lens in vitro. Materials and Methods

Cell cultures Myeloma P3-X63-Ag8-U1 (Yelton et al., 1978) and hybridoma were cultured in a 1 : 1 mixture of Dulbecco's modified Eagle's minimal essential medium (Nissui Co., Tokyo) and Ham's F-12 medium (Nissui Co.) supplemented with 10% fetal calf serum. Procedures to obtain PEC as well as to harvest the cultured cells at the different stages of transdifferentiation were described by Itoh and Eguchi

(1986a, b). Briefly, retinal PECs isolated from 8-9-day-old chicken embryos were cultured in a basic culture medium (EF), Eagle's minimal essential medium (Nissui Co.) supplemented with 6-10% fetal calf serum. The cells cultured in EF medium do not lose their original phenotype through several passages of subculturing, but they rapidly lose it in a modified medium (EdFPH) containing dialyzed fetal calf serum (10%), phenylthiourea (0.5 mM), and testicular hyaluronidase (Boehringer-Mannheim Co., 250 units/ml). After several passages in E d F P H medium, all cells have no apparent differentiated properties but are highly proliferative. Thus, cultured cells at this stage are termed dedifferentiated PECs (dePECs). Almost all of dePECs are able to differentiate into two alternative pathways of lens cells and PECs, depending on the medium for future cultivation. In the medium EdFPHA, which is prepared by adding ascorbic acid to the EdFPH (at high cell density), dePECs would transdifferentiate into lens cells, whereas in the culture medium with the modified EF (EFA) medium containing ascorbic acid, dePECs would redifferentiate into PECs.

Melanosome isolation A melanosomal fraction was obtained as described by Seiji et al. (1963) with some modifications. The redifferentiated PECs were collected on days 3-5 after replacing the medium of dePECs with EFA and were homogenized in 20 mM TrisHCI buffer, pH 7.4, containing 0.5 M sucrose, 5 mM MgCI 2 and 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 600 x g for 10 rain at 4 ° C to remove cellular debris and nuclei. The supernatant was then centrifuged at 10 000 x g for 20 rain. The pellet was resuspended in the same buffer and layered over 2 M sucrose solution in a centrifugation tube. After an ultracentrifugation at 100000 X g for 60 rain, a melanosomal fraction was recovered from the bottom of the tube. Immunization, cell fusion, and hybridoma screening The melanosomal pellet thus obtained was suspended in 20 mM Tris-HC1 buffer, pH 7.4, containing 0.5% sodium dodecyl sulfate (SDS), and diluted l : 2 0 in Dulbecco's phosphate buffered

135 saline (PBS). The diluted sample was emulsified with complete Freund's adjuvant (Difco Laboratories) and injected subcutaneously into a B A L B / c mouse. The same injection procedures were repeated three times with 2-3 week intervals. Altogether, about 100 /~g proteins were injected as an antigen into each mouse. Four days after a boosting without adjuvant, splenocytes from the immunized mouse were fused with myeloma (ratio 5 : 1) in the presence of polyethylene glycol (Merck) according to K~Shler and Milstein (1975). The hybridoma supernatants were assayed by an enzyme-linked immunosorbent assay (ELISA) according to the following procedures. The isolated melanosomes suspended in 20 mM Tris-HC1 buffer, pH 7.4, containing 0.5% SDS were centrifuged at 10000 x g for 20 min. The pellet, deprived of all the membrane components and soluble molecules, was resuspended in PBS and plated on a 96-well microtiter plate (Dynatech Laboratories, Inc.). After an incubation at 3 7 ° C for 2 h, the plate was washed with PBS. Each well of the plate was covered with hybridoma supernatant and incubated at 3 7 ° C for 1 h. After washing with PBS, the plate was then incubated with horseradish peroxidase (HRP)-conjugated antimouse IgG (Bio-Rad Laboratory) diluted 1 : 1000 in PBS containing 3% bovine serum albumin (BSA) at 37°C for 1 h. After washing, positive wells were detected in a solution composed of 0.015% 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma), 1% H202, and 5 mM citrate, pH 4.0. Positive hybridomas were cloned and were either grown in vitro or in ascite for large-scale production of antibody (MC/1).

Western transfer and imrnunoblotting Cultured cells and tissue samples were solubilized by SDS and electrophoresed on 7.5% and 10% polyacrylamide gels according to the method of Laemmli (1970). The proteins were transferred to nitrocellulose membrane (Schleicher&Schuell, BA85), and the membrane was processed according to Towbin et al. (1975) to find the samples with HRP-conjugated goat anti-mouse IgG. The binding of the monoclonal antibody to the specific proteins was visualized in 20 mM Tris-HC1 buffer (pH 7,4) containing 0.6% 4-chloro-l-naphthol

(Bio-Rad Laboratory), 20% methanol, and 0.01% H202. Proteins in the gel were stained with Coomassie brilliant blue (CBB).

lndirect immunofluorescence Tissue samples were fixed at room temperature in a mixture of 70% ethanol and 30% acetic acid and embedded in paraffin. Sections of 5-10 /~m were fixed on a glass slide. Deparaffined specimens were incubated for 1 h with hybridoma supernatant at room temperature. After washing with PBS, the sections were treated for 1 h with fluorescein isothiocyanate-conjugated anti-mouse IgG (Cappel Laboratories Inc.) diluted 1 : 50 with PBS. After washing, the specimens were mounted and observed under fluorescence microscopy. Cells cultured on plastic dishes or cover slips were fixed in cold methanol, followed by the same procedures as described above.

Immunoelectron microscopy Tissue sample was fixed in 0.1 M phosphate buffer, pH 7.4, with 2.5% glutaraldehyde and dehydrated in N,N,dimethyl formamide and infiltrated at room temperature with LR-Gold (Bio-Rad Laboratory) containing 1% dibenzoyl peroxide. The tissue was then transferred into gelatin capsule and polymerized at 60 ° C for 2 h. The ultrathin sections on formvar-coated nickel grids were blocked with normal goat serum diluted 1 : 1 0 with PBS for 10 rain and incubated with the hybridoma supernatant for 30 min at room temperature. After five washings with PBS, the sections were then incubated with 5-nm gold particle-conjugated goat anti-mouse IgG (Janssen Pharmaceuticals, Schiphol, The Netherlands) containing 10% normal goat serum for 30 rain at room temperature. After washing, the sections were stained with uranyl acetate and examined at 80 kV.

Results

lmmunofluorescent studies A paraffin section of an eye dissected from 5-day-old chicken embryos was stained by M C / 1 . Specific stains were observed in the layer of pig-

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Fig. 1. Indirect immunofluorescence of histological sections probed with M C / 1 antibody. (A, B) Staining of the pigmented epithelium in sections of an eye of a 5-day-old chicken embryo. (C) Staining of the pigmented epithelium under higher magnifications. (D) The same section as (C) with transmitted light; see correspondence of sites of granular staining to the intracellular distribution of pigment granules. (E) Staining of dermal melanocytes around a feather bulb. (F) The same section as (E) with transmitted light. L, lens; PE, pigmented epithelium; NR, neural retina. Bar, 40 #m.

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Fig. 2. Immunofluorescent localization of M C / 1 antigens in a cultured PEC. PEC cultured on a cover slip was probed with M C / 1 and then viewed with fluorescence (A) and transmission (B) microscopy. Only melanosomes were stained with M C / 1 . Bar, 10 ~tm.

mented epithelial cells (PECs) but not in the neural retina, lens or choroidal layer (Fig. 1A, B). At higher magnifications, bright granular staining in the cytoplasm of PECs was evident (Fig. 1C), and these labeled regions corresponded to the cell site, where pigments were localized under a transmission microscope (Fig. 1D). PECs at various stages from 4-day-old embryos to newborn chickens were stained by MC/1 similarly. Intensity of staining appears to increase with development. Skin sections of an 18-day-old embryo were also examined. In the embryo at this stage, melanocytes derived from the neural crest are present in the skin especially around the feather bulbs. These melanocytes are strongly stained by MC/1 as in the PECs (Fig. 1E, F). Thus, the PEC and the neural-crest-derived melanocyte share common antigen molecule(s) which are recognized by MC/1. Immunofluorescence of cultured PEC showed that the intracellular distribution of granules stained by MC/1 is identical to that of the melanosomes (Fig. 2A, B). It was obvious that MC/1 antibody reacts with some component(s) of melanosomes.

Immunoelectron microscopy Although binding of MC/1 to the SDS extracts of melanosomes by the ELISA method suggested that MC/1 antibody recognizes some components of a melanosomal matrix, a histochemical ex-

amination was performed using the immuno-gold technique to confirm this. Embryonic tissues with melanosomes were embedded in LR-gold. Polymerization was performed chemically in the presence of dibenzoyl peroxide, because routine polymerization procedure by irradiation interfered with pigments in the tissue. Ultrathin sections were probed with MC/1 antibody and followed by the treatment with anti-mouse IgG linked with 5-rim gold particles. Both premelanosomes containing several fibrous structures and apparently matured melanosomes were stained (Fig. 3). Most gold particles were distributed on the peripheral region of the matrix of melanosomes at all stages of maturation, including premelanosomes. The gold particles of the inner region of the melanosomal matrix were rare, especially if heavily melanized. Gold labelling on other organelles in the PEC than melanosomes was indistinguishable against the background.

Immunoblotting Specificity of the MC/1 antibody was examined using the immunoblotting technique. Each sample was electrophoresed on SDS polyacrylamide gel, blotted to nitrocellulose membrane, and probed with MC/1. A single protein band with an apparent molecular weight of about 115000 was detected in PEC, and this polypeptide was concentrated in the melanosomal fraction, although there were some other minor bands which may be proteolytic products of this 115 kDa one (Fig. 4).

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redifferentiation as defined by Itoh and Eguchi (1986a, b) were harvested for analysis. The dedifferentiated PECs (dePECs), which were maintained and grown in EdFPH-medium, were divided into two aliquots; the one was transferred to EdFPH-medium containing ascorbic acid under high cell density (a condition allowing transdifferentiation into lens), and the other to EF-medium containing ascorbic acid (permissive to redifferentiation). In the former condition, the lentoid cells were abundant on day 7; whereas in the

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Fig. 3. lmmunogold electron microscopy. LR-Gold section of PEC of 9-day-old embryo was probed with MC/1 antibody and followed by 5 nm gold conjugated anti-mouse IgG. Distribution of gold particles on peripheral regions of the mamx in the various stages of maturation of premelanosome and the matured melanosome. Bar. 0.4 Fm-

Various tissues isolated from 8-day-old embryos were also examined. The 115 kDa protein was detected only in the pigmented epithelial cells, whereas no proteins were detected in the lens, neural retina, brain, heart, liver or gizzard to react with M C / 1 (Fig. 5).

Antigen expression during in vitro transdifferentiation Expression of antigen molecules to react with M C / 1 was studied in cultured PEC by immunoblotting (Fig. 6). Cultured cells at each stage of (1) dedifferentiation, (2) transdifferentiation, and (3)

Fig. 4. Immunoblotting analysis of MC/1 antigen. PEC (lanes 2, 4) and a purified melanosomal fraction (lanes 3,5) were electrophoresed on SDS-polyacrylamide gel, blotted to a nitrocellulose sheet and probed with MC/1 antibody. 1, 2, 3, CBB staining; 4,5, immunoblotting. Lane 1 contains molecular weight markers as follows: myosin, 205 kDa; fl-galactosidase, 116 kDa; phosphorylase b, 97 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa, from the top to the bottom.

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Fig. 6. Expression of MC/1 antigen in cultured PECs as studied by immunoblotting (lanes 1-5) with CBB staining (lanes 6-10). Each lane contains about 50 /ig of proteins: PECs of 9-day-old embryos; dedifferentiated PECs (lanes 2,7); redifferentiated PECs (lanes 3,4 and 8,9; respectively 2 and 7 days after replacing the culture permissive to redifferentiation); transdifferentiated lens cells (lanes 5, 10).

cal to the time when pigment granules first appeared by microscopic observation.

Discussion

B Fig. 5. Tissue-specificexpression of MC/1 antigen. Proteins of various embryonic tissues were electrophoresed and visualized by CBB staining (A) and by immunoblotting (B). Each lane contains about 50 ~g of proteins: lens (lane 2); neural retina (lane 3); pigmented epithelium (lane 4); brain (lane 5); heart (lane 6); liver (lane 7); gizzard (lane 8). Lane 1 contains the same molecular weight markers as in Fig. 4 (lane 1). latter, pigment granules were microscopically visible on day 2 and gradually increased to attain the m a x i m u m pigmentation by day 7. The 115 k D a protein was detected in ceils at rededifferentiated stage and redifferentiated PECs, but it was not detected in the transdifferentiated lens cells or the dedifferentiated PECs (dePECs) by immunoblotting (Fig. 6). After transferring de PECs into the medium permissive for redifferentiation, this protein was first detected on day 2 which was identi-

F r o m the results of immunohistochemical studies and immunoblotting analysis, it is highly probable that the polypeptide recognized by M C / 1 monoclonal antibody is a melanosomal matrix protein in chicken PECs. Several investigators reported melanosomal proteins in chicken, human, and mouse (Doezema, 1973; Hearing and Eppig, 1974; Jimbow et al., 1982; Zimmerman, 1982) and recently, some monoclonal antibodies were reported which recognize melanosome-related molecules in mouse melanomas (Akutsu and Jimbow, 1986; Hayashibe et al., 1986). In chicken PECs, Hearing and Eppig (1974) solubilized a number of m e m b r a n e proteins and a single-matrix protein with a molecular weight of 70 000 from purified melanosomes. Z i m m e r m a n (1982) reported six matrix proteins with a molecular weight ranging from 12000 to 31 000. Thus, it is conceivable that these previously reported matrix proteins and the 115 k D a one described here are independent molecules although the possibility remains that

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the small polypeptides described by others may be proteolytic products of the present 115 kDa matrix protein. MC/1 antigen is distinguishable from tyrosinase. Tyrosinase activity is visualized in the SDS polyacrylamide gel by the DOPA reaction as a single band with a molecular weight of about 60 000. However, on the same gel, the reaction of 115 kDa with MC/1 antibody was detected as an independent band by the Western blotting method (data not shown). The present 115 kDa polypeptide Js the only protein which has immunologically been identified to be localized in the melanosomal matrix. Various stages of maturation of the premelanosomes were observed by electron microscopy (Fig. 3). Some helical fibers appear in their early stages, and they become larger during melanosome maturation (Drochmans, 1966). The fibrous structures are thought to be the cores of melanin accumulation. If 115 kDa molecule were a constituent of the fibrous core, the labelling by MC/1 would be distributed along the fibers in the immunoelectron microscopic observation, and the binding of this antibody would be blocked by accumulated melanins after the maturation. However, this is not the case. Immunogold analysis demonstrated that 115 kDa protein is located on the peripheral region of the matrix at all stages of maturation of the premelanosomes as well as in completely melanized melanosomes. Perhaps 115 kDa molecule is not a constituent of the fibrous cores, but connects the melanin or tyrosinase to the core during the maturation process of the melanosomes. Immunoblotting analysis under nonreducing conditions suggested the presence of some other polypeptides interacting with the 115 kDa matrix protein by disulfide bonding (data not shown). The mechanism in melanosome construction would be better understood if the structures of these melanosomal components were analyzed particularly by the in vitro reconstruction experiments of melanosome, using these molecules. As expected, MC/1 can serve as a reagent to specify the differentiated phenotype of pigmented cells in vivo and in vitro (Figs. 1, 2). In past studies of transdifferentiation of PEC into lens in the culture system of Itoh and Eguchi (1986a, b), specificities of PEC have been identified in both

melanin contents and tyrosinase activities. Thus, it remained to be elucidated, using markers other than these two, whether bipotent dePECs produced PEC-specific proteins or not. The absence of lens-specific molecules, 6-crystallin, in dePEC has already been reported (Itoh and Eguchi, 1986b). Immunofluorescent and immunoblotting examinations were performed, and the 115 kDa melanosomal matrix protein was not detected in the dePEC (Fig. 7). Thus, a dedifferentiated property of dePEC was confirmed not only by the conventional markers thus far utilized but also by the absence of this matrix protein. Although dePECs do not synthesize 8-crystallin, they contain the transcripts of 8-crystallin gene (Agata and Eguchi, 1984). Analysis with DNA probe of the gene coding for the present melanosomal matrix protein is under investigation in respect to the transdifferentiation system (Itoh and Eguchi, 1986b). MC/1 antibody not only recognized PECs but also melanocytes in the skin that are derived from the neural crest and are morphologically different from PECs. This indicates that these two species of pigment cells share a common melanosomal matrix antigen. Unfortunately, the molecular weight of the antigen protein in the dermal melanocytes is not known due to the difficulty in isolating a sufficient quantity of melanocytes from the chicken skin for the blotting analysis. However, MC/1 can serve as a useful reagent for following the differentiation and migration processes of melanocytes and their precursors from the neural crest, particularly, since non-pigmented precursors of melanocyte can be identified by MC/1 antibody.

Acknowledgments We thank Professor T.S. Okada, Director-General, National Institute for Basic Biology for his critical reading of the manuscript and Ms. Toyoko Tsuge for her kind assistance in preparing the manuscript. This study was supported in part by Grants-in-Aid for General Research and for Basic Cancer Research from the Ministry of Education to G.E.

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Itoh, Y. and G. Eguchi: Enhancement of expression of lens phenotype in cultures of pigmented epithelial cells by hyaluronidase in the presence of phenylthiourea. Cell Differ. 18, 173-182 (1986a). Itoh, Y. and G. Eguchi: In vitro analysis of cellular metaplasia from pigmented epithelial cells to lens phenotypes: A unique model system for studying cellular and molecular mechanisms of "transdifferentiation". Dev. Biol. 115, 352-362 (1986b). Jimbow, K., M. Jimbow and M. Chiba: Characterization of structural properties for morphological differentiation of melanosomes: II. Electron microscopic and SDS-PAGE comparison of melanosomal matrix proteins in BI6 and Harding Passey melanomas. J. Invest. Dermatol. 78, 75-81 (1982). KtShler, G. and C. Milstein: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497 (1975). Laemmli, U.K.: Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227, 680-685 (1970). Seiji, M., K. Shimano, S.C. Birbeck and T.B. Fitzpatrick: Subcellular localization of melanin biosynthesis. Ann. N.Y. Acad. Sci. 100, 497-533 (1963). Towbin, H., T. Staehelin and J. Gordon: Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354 (1975). Yasuda, K., T.S. Okada, G. Eguchi and M. Hayashi: A demonstration of a switch of cell type in human fetal eye tissue in vitro: Pigmented cells of the iris and the retina can transdifferentiate into lens. Exp. Eye Res. 26, 591-595 (1978). Yelton, D.E., B.A. Diamond, S.P. Kwan and M.D. Scharef: Fusion of mouse myeloma and spleen cells. Curr. Top. Microbiol. Immunol. 81, 1-7 (1978). Zimmerman, J.: Four new proteins of the eumelanosome matrix of the chicken pigment epithelium. J. Exp. Zool. 219, 1-6 (1982).