Detection of melanogenic proteins in cultured chick-embryo melanocytes

Differentiation

Differentiation (1985) 30:4&46

'I'

Springer-Verlag 1985

Detection of melanogenic proteins in cultured chick-embryo melanocytes William Oetting, Karen Langner*, and John A. Brumbaugh** Section of Genetics, Cellular and Molecular Biology, School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NB 68588-0118, USA

Abstract. The phorbol ester, 12-O-tetradecanoylphorbol-l3acetate (TPA), was used as a reversible inhibitor of melanogenesis. Chick-melanocyte cultures of the black genotype, EIE, were grown in conditioned medium plus TPA. After growth in TPA and after its removal, the cells were pulse labeled with 3H-leucine. The membrane fraction, which included all tyrosinase activity as well as both mature and immature melanosomes, was solubilized with Triton X-100. The proteins were separated using two-dimensional electrophoresis and visualized by fluorography. One defined melanogenic protein, tyrosinase, was isolated, and its location was determined in the two-dimensional protein pattern. The protein patterns for both the TPA-inhibited cells and the cells in which the TPA effects were reversed after removal were compared. in addition to tyrosinase, at least nine TPA-sensitive proteins were found. These were designated as being putative melanogenic proteins which, along with tyrosinase, may be responsible for melanin-granule synthesis.

Introduction The melanosome, or pigment granule, is an organelle produced by the melanocyte and is responsible for its color. The melanosome is formed from the products of two cellular pathways [6, 71. One pathway is responsible for the polymerization of tyrosine to melainin; this reaction is initially catalyzed by the enzyme, tyrosinase, and is further regulated by other controlling factors [2, 321. The second pathway is responsible for the production of the membranebound matrix of the pigment granule, i.e., the premelanosome. Tyrosinase is activated when tyrosinase-containing vesicles fuse with premelanosomes. The enzyme oxidizes tyrosine to dopaquinone. which is then polymerized to melanin and deposited on the premelanosomal matrix [28]. Melanin deposition in normal melanocytes proceeds until a solid ellipsoid granule has been produced. There have been many attempts to determine the number and characteristics of the proteins involved in melanogenesis. There is a great deal of available information about the melanin-producing enzyme, tyrosinase [16, 23, 29, 371 but previous reports describing the proteins of the premelanosome present conflicting results [S, 13,2&22,41]. Significant differences in the molecular weights of the pre-

* **

Present address: Department

of Genetics, lowa State Universi-

ty, Ames, lowa 50010. USA To whom offprint requests should be sent

melanosomal proteins have been reported. In these studies the number of proteins reported to be involved in melanogenesis ranges from 3 to 20, with some being considered premelanosomal matrix proteins and the rest being regarded as membrane-associated proteins. We believe the major reason for these conflicting results is that mature melanosomes were used as the source of premelanosomal proteins. The mature melanosome is a subcellular particle which is very resistant to degradation. The melanin polymer is attached to the amino or sulfhydroxyl groups of the protein matrix via quinone linkages [4,341. Therefore, it is very unlikely that undegraded proteins can be removed from the mature melanosome. This could account for the conflicting reports with regard to both the number and size of the premelanosomal proteins. It would seem advantageous to examine premelanosomal proteins before melanization has occurred. Several methods for culturing chick melanocytes using the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), have been described [18, 24, 331. TPA can act as a mitogen [36], an inhibitor of differentiated cellular functions, or both. This has been demonstrated in chondrogenesis [27] and myogenesis [8, 10-121. The inhibition of melanogenesis in avian melanocytes has been reported by a number of investigators [18, 24, 33, 351. This inhibition of pigmentation due to the presence of TPA is also reversible if the TPA is removed from the growth medium [24, 331. Inhibition of the differentiated function(s) is thought to occur via interference with protein synthesis [lo, 12, 241. Utilizing this reversible inhibitory effect of TPA, melanocytes were grown in the presence of TPA, and their proteins were compared to those of cells aft,er the removal of TPA. Melanocytes which were forming premelanosomes after TPA removal were pulse labeled. The cells were unpigmented or lightly pigmented, and thus omit the premelanosoma1 proteins were susceptible to solubilization in the absence of interference by the melanin polymer. Protein patterns were compared using denaturing two-dimensional electrophoresis. Any proteins sensitive to TPA were considered to be possible melanogenic proteins. The known melanogenic protein, tyrosinase, was used as a marker for the melanogenic response to TPA.

Methods Genetic stocks Embryos and feather material were obtained from inbred genetic stocks of domestic fowls (Gallus gallus) maintained

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at the School of Biological Sciences in the University of Nebraska-Lincoln. The birds used were of the extendedblack genotype (E/E).Adults and chicks have black plumage and dark eyes. Cell culture The method used for melanocyte cell culture has previously been described [17, 241. In brief, caudal somites from embryos at stages 16-18 [19] were isolated and trypsinized. Cells were plated out as monolayer cultures at a density of 2.5 x 10’ cells per 25-cm2 flask and fed with a medium that was preconditioned with BRL-3A cells originally isolated by Coon [9] from Buffalo-rat liver. The media were conditioned for 2 days, centrifuged and filtered to remove BRL-3A cells, and mixed (3: 1) with fresh F-12 containing 10% calf serum. On day 5 of the culture, TPA (Sigma, St. Louis, Mo) was added at a final concentration of lo-’ M. The TPA was removed on day 30 of culture. The cell purity was monitored in situ by counting the number of melanocytes and nonmelanocytes in 12 randomly selected 0.25-mm2 areas of each flask and then multiplying by 833 to estimate the total number of cells in each 25-cmZflask. Cell labeling Replicate cultures were radioactively labeled on culture day 9 (while the cells were in TPA) and on culture day 13 (72 h after TPA removal). After washing with Hank’s balanced salt solution (HBSS), the cells were labeled for 60 min with 2.0 ml minimum essential medium without leucine (GIBCO, Grand Island, NY) containing 75 pCi/ml ~44-5)’H-leucine ( 1 3 M 50 Ci/mM; Amersham, Arlington Heights, Ill.) with (9-day cultures) or without (13-day cultures) lo-’ M TPA. The cells were scraped from the flasks using a rubber policeman and were homogenized in an Eberbach microhomogenizer. The homogenate was then centrifuged at 100,000g for 60 min, and the resulting pellet was used in the electrophoretic studies. Biochemical determinations showed that the pelleted fraction from cells at the pigmenting stage contained over 98% of the total cellular tyrosinase activity. Both immature premelanosomes and mature melanosomes were seen at the electron-microscope level (unpublished observations). The pellet was solubilized with 1.0% (w/v) Triton X-100, and the samples were subsequently denatured with 9 M urea and 5% (v/v) beta-mercaptoethanol. Each experiment was performed in triplicate.

Isolation of tyrosinase Tyrosinase was isolated from E / E regenerating feathers [40]. The living material from the base of each feather was squeezed out of the feather sheath and placed in a 0.25 M sucrose solution. The material was washed and then resuspended in 5 vol. (v/w) 50 mM sodium phosphate (pH 7.4), 0.25 M sucrose, 25 mM KCl, and 4 mM MgC12, and homogenized using a Brinkman-Polytron homogenizer (4 x 15 s; setting 6). The homogenate was filtered through gauze and centrifuged at 700 g. The supernatant was then centrifuged at 100,000g for 60 min in order to pellet the membrane fraction. The pellet was resuspended at a final protein concentration of 4 mg/ml in 0.5% (w/v) cholic acid and was allowed to solubilize for 60 min with gentle stirring at 4” C. The solution was again centrifuged at 100,000 g (to remove particulate matter), and the supernatant was precipitated with 30% and then with 42.5% saturated (final concentration) ammonium sulfate. The 42.5% precipitate was resuspended in 10 m M phosphate buffer (pH 7.4) containing 0.1 % Triton X-100 and then dialyzed overnight. The dialysate was applied to a column (1 x 5 cm)of DEAEcellulose preequilibrated with 10 mM phosphate buffer (pH 7.4) and 0.1% Triton X-100. The proteins were eluted using a linear gradient of KCl (0.001-0.5 M), and 0.5-ml fractions were collected. The fractions containing tyrosinase activity were then loaded onto preparative nondenaturing IEF tube gels. After the completion of the run, one gel was stained for tyrosinase activity using 1 mg/ml dopa in 0.1 M phosphate buffer, pH 6.8. The tyrosinase-containing bands were then cut out of the unstained gels. This gel fragment was then subjected to two-dimensional electrophoresis as already described. In nondenaturing gels, the enzyme was stained for activity using dopa or was visualized immunologically after transfer to nitrocellulose paper as described elsewhere [30]. The proteins in denaturing gels were visualized using a silver stain [15]. Protein-pattern determination Protein patterns in TPA-treated cells and cells grown after the removal of TPA were compared by direct visualization. Several obvious proteins or groups of proteins which were absent in TPA-treated cells, but which appeared after TPA removal, were studied. Not all protein-pattern changes were examined. Those proteins which were present in TPAtreated cells but which decreased or disappeared after TPA removal were not investigated.

Two-dimensional electrophoresis Two-dimensional electrophoresis was performed using a modification of O’Farrell’s method [31]. Both denaturing and nondenaturing gels were prepared. In the denaturing gels, the first dimension consisted of isoelectric focusing (IEF). The ampholine concentration was 1.6%, pH 5-8 (LKB; Gaithersburg, Md) and 0.4%, pH 3.5-10 (LKB). The second dimension consisted of an 8.0% acrylamide (0.2% bis-acrylamide) gel and 0.5% (w/v) SDS. After electrophoresis, the gels were processed for fluorography using previously described techniques [3, 251. In the nondenaturing two-dimensional electrophoresis the denaturing agents, urea, beta-mercaptoethanol, and SDS were not used. In the IEF gels, the pH range was 3.510 (2.0% ampholines). The gels contained a Triton-X100 concentration of 0.1 %.

Results Cell culture On day 5 of culture, approximately 25% of the E / E cells were producing small amounts of pigment. Cells at this stage were flat and dendritic. The cultures were surprisingly free of contaminating cell types, these being obvious because of their different morphologies. After 4 days in TPA (day 9), cultures had no observable pigment (Fig. 1A); cells at this stage were rounded or spindle shaped. Seventy-two hours after the removal of TPA (day 13), the cells were again flat and dendritic, as in the day-5 cultures, and some cells were producing pigment (7.5%). Analyses of ten sets of cultures showed that the percentage of contaminating cells ranged from 1.9% to 0.06%,with an average of 0.67%

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Fie. 1 A, B. Cultures of the black genotype, E/E. A Staincd day-9 cells that had been growing in TPA. B Unstained day-I7 cells that had been growing without TPAsinceday 10. x 864

Table 1. Percentage of nonmelanocytes in cullures'

Mean Numbcr of Mean number of Mean total experiments nonmelanocytes number of cellsb percentage of nonmelanocytes 10

a

6.4 1 0 3 + 3.2 x lo3

1.5 x lo6 1 0 . 7 7 x lo6

0.67 0.57

Based on morphological and pigment-producing criteria Mean number of cells (kSD) per 25-cm2 flask

(see Table 1). Control experiments showed that approximately 90%-%% of the melanocytes had produced copious quantities of pigment by day 17 (Fig. 1 B). These morphological changes were confirmed at the electron-microscope level. Both dopa-oxidase activity and premelanosomes were absent in TPA cultures, but these reappeared after TPA removal [24]. Location of tyrosinase on two-dimensional gels

The pattern of isolated tyrosinase in nondenaturing twodimensional gels stained with the substrate dopa is shown in Fig. 2. It consisted of a row of spots located in the acidic region of the IEF separation and toward the top of the gel. Figure 3 shows an immunoblot from a nondenaturing gel. the primary antibody being mouse antichicken tyrosinase (dilution, 1 :10,000 [30,40]). The reaction was visualized with horseradish-peroxidase-conjugated goat antimouse IgG [30]. The reactive region was in the same dopa position (Fig. 3, arrow) as the dopa-stained spots seen in Fig. 2.

The pattern of isolated tyrosinase in denaturing twodimensional gels is shown in Fig. 4 as visualized using a silver stain. As expected, it consisted of a row of spots (Fig. 4, arrows). The PI range was 4.M.5, and the molecular mass was estimated to be 72,000 daltons. Since this sample was an aliquot of the same preparation used in Figs. 2 and 3, the silver stain marked the specific location of tyrosinase in denaturing two-dimensional gels. Fluorography

Day-9 cultures growing in TPA and day-I3 cultures after the removal of TPA were labeled with 'H-leucine. Day 13 was chosen because the cultures showed the greatest increase in pigment production between culture days 13 and 14. We considered that, at this time, the greatest quantity of melanogenic proteins would be produced by the melanocytes, while the minimum amount of interfering melanin would be present. Figure 5 shows the protein pattern of day-9 cell cultures growing in the presence of TPA. Figure 6 shows the protein pattern of day-13 cell cultures growing in the absence of TPA. A row of spots (see Fig. 6, arrows with asterix) with a pl of 4 . M. 5 and a molecular mass of 72,000 daltons (identical to that of tyrosinase; see Fig. 4) was observed, that was not found in cells growing in the presence of TPA (see Fig. 5). A row of spots above this row was similarly affected. The large group of proteins indicated by the three vertical arrows above the tyrosinase in Fig. 6 are almost completely absent in Fig. 5. In Fig. 6 seven other spots in the pattern are also TPA sensitive, so these spots are absent in Fig. 5. The PIS of these monitored proteins were, for the most

43

part, acidic while the molecular masses ranged from 42,000 to 100,000 daltons. Not all of the proteins that were affected by the presence or absence of TPA were investigated; only major and obvious changes in TPA-sensitive proteins were documented. Proteins which disappeared or whose levels were obviously reduced after TPA removal were not studied. A comparison of the TPA-insensitive spots in the two patterns (with TPA and without TPA) revealed a high degree of homology both with regard to the location of spots and the relative intensity of their appearance on the fluorographs. Discussion TPA was found to be effective as a mitogen, thus making it possible to recover large numbers of melanocytes. The use of BRL-3A conditioned medium without TPA produced a maximum of 2 x lo5 differentiated melanocytes per stage-16 to -18 embryo [ l l . The same medium plus TPA, as described in the present study, produced a maximum of 4 x lo6 differentiated melanocytes per embryo.This is consistent with the results of Glimelius and Weston I181 and Langner et al. [24], but is contradictory to the results of Payette et al. [33], who did not find mitotic stimulation of melanocytes in the presence of TPA. Two major factors could account for this difference. First, the culture systems were not identical; the culture system used by Langner et al. [24] and in the present study empolyed the stimulatory effects of BRL-conditioned medium in conjunction with TPA, while the other culture systems did not. Second, the pigment genotypes used by Payette et al. [33] were very different from those of the wild quail used by Glimelius and Weston [18] and the inbred black chick line used in the present study. We have found that genotypes resembling those used by Payette et al. [33] do not respond mitogenically to TPA (unpublished observations). On the other hand, the reversal of the inhibitory effect of TPA on pigment synthesis was definitive, like that reported by Payette et al. [33]. Cultures normally produced pigment by day 5, but cells ceased producing pigment in the presence of TPA. On the removal of TPA, the cultures began producing pigment again within 3 days. Thus, we suggest that TPA is an effective switch which can turn pigmentation off (see [ti]). The interpretation of the changing protein patterns that occur in response to TPA depends, to a large degree, on the purity of the cultures used. If the cultures contain vir-

Fig. 2. The upper-right quadrant of a nondendturing two-dimensional gel of isolated tyrosinase. The gel was stained with dopa. The small arrows indicate a diagonal row of spots exhibiting tyrosinase activity

Fig. 3. The upper right quadrant of an immunoblot from a nondenaturing two-dimensional gel of tyrosinase. The arrow indicates the location of the reaction to antityrosinase mouse serum, this being found in the same position as the tyrosinase reaction product seen in Fig. 2 Fig. 4. Two-dimensional gel of isolated, denatured tyrosinase; the gel was silver stained. The small arrows point to a diagonal row of spots indicating the location of tyrosinase. The pZ range is 4 . M . 5 , and the molecular mass is 72,000 daltons

Fig. 5. Two-dimcnsional fluorograph of 3H-lcucinc-labcled proteins of day-9 cultures growing in the presence of TPA. The small arrows indicate vacant areas which are occupied by spots in Fig. 6. The row of arrows with asterix indicates location of tyrosinase. Fig. 6. Two-dimensional fluorograph of 3H-leucine-labeled proteins of day-1 3 cultures during the pigmenting phase after the removal of TPA. The small arrows indicate spots which appeared after the removal of TPA. The row of arrows with asterix indicates location of tyrosinase.

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tually only melanocytes, then the change in protein patterns must be largely due to changes in melanogenesis in these cells. If they are contaminated with other cell types, some of the protein changes could be due to the nonmelanocytes. As can be seen in Table 1 each culture flask contained approximately 6,400 nonmelanocytes out of a total of approximately 1.5 x lo6 cells. The worst contamination encountered was just under 2% of the cell population. Thus, it can be concluded that the vast majority of proteins in the samples were derived from melanocytes. This high level of melanocyte purity is not attained when unmigrated neural crest is excised from embryos [18, 351. The method used by Giss et al. [17] and Langner etal. [24] as well as in the present study involves selection both for migrating melanoblasts and against the other cell types in somites. A great deal of cell selection occurs during the first 3 days of culture, as previously reported [42]. The selection is virtually complete by the 5th culture day when the TPA is added. The reason for this selection is unknown. Presumably, cellular contact is necessary for other cell types to survive. Contaminating cells were frequently seen as clumps of cells that had not been completely separated by the original trypsinization. It is possible to hypothesize that, unlike the mixed population present in unmigrated neural crest, neural-crest cells migrating through the somites are determined melanoblasts. Tyrosinase was isolated, and its position in a denaturing two-dimensional gel was determined. Its molecular mass was found to be about 72,000 daltons, in agreement with the results of Doezema [14]. Within the two-dimensional pattern, tyrosinase produced a series of spots that differed with regard to their PI. Although tyrosinase is a glycoprotein [40], these isotypes were not merely the product of differing numbers of sialic-acid moieties. The addition of neuraminidase does not alter the number of isotypes, but does change their position in IEF gels [40]. Culture day 13 was used for cell labeling, because the cells were actively synthesizingmelanosomes, but pigmentation was still light to moderate. Cytochemical studies using transmission-electron-microscopy have shown that, 24 h after the removal of TPA, tyrosinase activity is present and premelanosomes begin to reappear [24]. Thus, melanogenic proteins are being synthesized on day 13, but these are presumably not tightly bound to melanin within the melanosome. Therefore, premelanosomal proteins should be easy to solubilize with detergents. A subset of new proteins appeared after the removal of TPA, although the majority of the proteins remained unchanged. Tyrosinase was one of the nine proteins which were monitored for their response to TPA. Each of the nine proteins were TPA sensitive. The molecular masses of these proteins ranged from 42,000 to 100,000daltons. The molecular masses of these proteins were in the same range as those reported by some investigators for premelanosomal proteins [20,21], but they differed from the findings of others [5, 13, 411 who have reported ranges of from 12,700 to 14,500 daltons and up to 180,000 daltons. Using B16/C3 melanoma cells, Laskin et al. [26] have also found a number of TPA-sensitive proteins in the molecular-mass range of 33,000-72,000 daltons. The most probable interpretation of these findings is that proteins found in both cultures are proteins responsible for functions necessary for the maintenance of the cell. The proteins that are sensitive to TPA could be considered as

being putative melanogenic proteins (see also [26]). Tyrosinase is absent in cells grown in the presence of TPA, indicating that at least one melanogenic protein is sensitive to TPA. The fact that premelanosomes are absent in TPAtreated cells and appear after its removal strongly suggests that at least one other melanogenic protein is also TPA sensitive (see also [33]). Studies in our laboratory using cycloheximide have shown that TPA exerts its effect at least at the translational level [24]. This is in agreement with the results of Croop et al. [12] and Cossu et al. [lo]. It would be reasonable - in view of the results of studies of the effects of TPA on chick myogenesis and chondrogenesis [ l l , 12, 271 - to suggest that a subset of proteins involved in melanogenic functions and morphology are TPA sensitive. Striated myofibrils in myotubes were affected by TPA, but other constituitively produced fibrils were not. Similar results were seen in chondroblasts. Two chondroblast-specific molecules were inhibited by TPA but not the more generalized type-111 sulfated proteoglycan. TPA seems to preferentially affect molecules associated with differentiated cellular functions [l 11. Using classical and heterokaryon crosses involving six loci, a theoretical hierarchy of gene control for the melanosome has been proposed [l, 38, 391. Two basic complementation groups were found to exist, one group being involved in the formation of the enzyme tyrosinase and the second being involved in the formation of the premelanosome. It is not known whether these mutations affect the structural genes themselves or affect melanogenic regulatory genes. The aim of the present investigation was to develop a system in which the molecular basis of the different pigment mutants could be studied. In future studies we will investigate pigment mutants, with particular emphasis on TPA-sensitive proteins. Preliminary observations indicate that tyrosinase and tyrosinaselike molecules are TPA sensitive in three mutant types ([30]; unpublished observations). The clarification of differences between TPA-sensitive proteins (other than tyrosinase) in these and other mutants lead to an understanding of the genetic basis of melanogenesis. Acknowledgements. This work was supported by a grant (GM 18969) from the National Institutes of Health and an NIH Biomedical Research Support Grant (RR 07055). The authors wish to thank Tom Bargar, Dana Scheele, and Roxanne Martin for their assistance in the preparation of this manuscript.

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