Cell-Density-Dependent Regulation of Expression and Glycosylation of Dopachrome Tautomerase/Tyrosinase-Related Protein-2  Thomas J. Hornyak, Daniel J. Hayes  Journal of Investigative Dermatology  Volume 115, Issue 1, Pages 106-112 (July 2000) DOI: 10.1046/j.1523-1747.2000.00030.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cell-density-induced expression of Dct. (a) Photomicrograph of a colony of stably transfected cells following selection in G418. Colonies were identified and stained for β-galactosidase expression 18 d after selection was begun. (b) Basal transcriptional activity of Dct promoter. Melan-a cells were plated at 1 × 106 cells per 10 cm plate and transfected 16 h later with 5 μg of either pPDct/luc, RSV/luc, or pGL2-Basic with cotransfection of 5 μg CMV/lacZ as internal control with total plasmid DNA brought to 20 μg by addition of pcDNA3. Transfected cells were then incubated for 53 h after removal of calcium phosphate precipitate prior to collection of cell lysate and assay for luciferase and β-galactosidase activities. Data represent the mean corrected ratios of luciferase to β-galactosidase activities from four replicates, with error bars signifying mean ± SD. (c) Autoradiograms of northern blots. Total RNA was isolated from melan-a cells at confluence (*) and on days 1–7 after plating at low density. Total RNA (6.7 μg) from melan-a cells as described was loaded onto a denaturing formaldehyde gel and transferred to nitrocellulose membrane following electrophoresis. Autoradiograms show results after exposure following hybridization with cDNA probes from Dct (Jackson et al. 1992), gapdh, and β-actin. (d) Quantification of autoradiography signals from northern blot. A scanning laser densitometer was used to quantify the signals from the Dct probes and the gapdh probes. The ratio of Dct/gapdh = 1 was assigned to cells at confluence (*) and used to normalize all other ratios. The ratio of Dct/gapdh is designated as relative Dct RNA expression. Journal of Investigative Dermatology 2000 115, 106-112DOI: (10.1046/j.1523-1747.2000.00030.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Cell-density-induced production of TRP-2 and TRP-1. Confluent melan-a cells were trypsinized, plated at 5 × 105 cells per 10 cm plate, and grown for 7 d. Media and TPA were changed and replenished daily. Total protein was obtained from cells at confluence (*) and on days 1–7 after plating at low density. One hundred micrograms of total protein from each set of cells were loaded into individual lanes of an 8% SDS-polyacrylamide gel and electrophoresed. (a) TRP-2 was detected by Western immunoblotting with αPEP8 antisera, horseradish-peroxidase-conjugated antirabbit, and chemiluminescent substrate. (b) TRP-1 was detected by Western immunoblotting with αPEP7 antisera, horseradish-peroxidase-conjugated antirabbit, and chemiluminescent substrate. (c) β-actin was detected by Western immunoblotting with mouse antiactin monoclonal antibody, horseradish-peroxidase-conjugated antimouse, and chemiluminescent substrate. (d) Determination of cell densities. Cells at confluence prior to low density plating (*) and on days 1–7 after low density plating were counted following trypsinization and cell densities at trypsinization calculated. Error bars represent the mean ± SD of four independent cell counts. Journal of Investigative Dermatology 2000 115, 106-112DOI: (10.1046/j.1523-1747.2000.00030.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Endoglycosidase sensitivities of TRP-2 glycoforms. Melan-a cells at confluence (2.0 × 107 cells per 10 cm plate or 2.6 × 105 cells per cm2) were scraped from the plate, counted, suspended in growth media, washed twice with ice-cold PBS, and lysed with RIPA buffer. Total protein concentration determination was performed against BSA standards. Eighty micrograms of total protein were loaded onto individual lanes of an 8% SDS-polyacrylamide gel before (lane 1) and after (lanes 2–5) digestion with N-glycosidase F and endoglycosidase H. TRP-2 glycoforms were detected by Western immunoblotting with αPEP8 antisera, horseradish-peroxidase-conjugated antirabbit, and chemiluminescent substrate. Journal of Investigative Dermatology 2000 115, 106-112DOI: (10.1046/j.1523-1747.2000.00030.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Time and TPA independence of cell-density-induced TRP-2 production. Confluent melan-a cells (at 2.2 × 105 cells per cm2) were trypsinized and plated at increasing densities of 5 × 105 cells per 10 cm plate (0.6 × 104 cells per cm2), 1 × 106 cells per 10 cm plate (1.3 × 104 cells per cm2), 2 × 106 cells per 10 cm plate (2.5 × 104 cells per cm2), and 5 × 106 cells per 10 cm plate (6.4 × 104 cells per cm2). Cells were grown in culture in the presence or absence of 200 nM TPA for 3 d, harvested by scraping, and cell lysate was prepared by the addition of RIPA buffer. Total protein concentrations were determined by comparison with BSA standards. (a) Total protein (7.5 μg) from cells grown in the presence (lanes 1–4) and in the absence (lanes 5–8) of TPA was loaded into individual lanes of an 8% SDS-polyacrylamide gel and electrophoresed. TRP-2 glycoforms were detected by Western immunoblotting with αPEP8 antisera, horseradish-peroxidase-conjugated antirabbit, and chemiluminescent substrate. (b) Determination of final cell densities. Cells from samples initially plated at increasing densities were counted following removal from the plate by scraping and final cell densities were calculated. Error bars represent mean ± SD of four or six independent cell counts. Journal of Investigative Dermatology 2000 115, 106-112DOI: (10.1046/j.1523-1747.2000.00030.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Lack of dependence of cell-density-induced TRP-2 production on soluble factors. (a) Confluent melan-a cells (at 2.2 × 105 cells per cm2) were trypsinized and plated at densities of 5 × 106 cells per 10 cm plate (6.4 × 104 cells per cm2) (set A) or 5 × 105 cells per 10 cm plate (0.6 × 104 cells per cm2) (sets B, C, and D). Thirty-two hours after plating, conditioned media from sets A (high density plates) and C (low density plates) was transferred to sets B (low density plates) and D (low density plates), respectively, from which media had previously been removed. Fresh growth media and TPA were subsequently added to sets A and C. Cells were harvested by scraping 75–78 h after initial plating, counted, washed twice with ice-cold PBS, and lysed with RIPA buffer after centrifugation. Total protein concentrations were determined by comparison with a set of BSA standards, and 25 mg of total protein from each set of cells A-D were loaded into individual lanes of an 8% SDS-polyacrylamide gel and electrophoresed. Relative amounts of TRP-2 were detected by Western immunoblotting with αPEP8 antisera, horseradish-peroxidase-conjugated antirabbit, and chemiluminescent substrate. (b) Determination of cell densities. Cells were counted after scraping from the plates and final cell densities were calculated. Error bars represent mean ± SD of six independent cell counts. Journal of Investigative Dermatology 2000 115, 106-112DOI: (10.1046/j.1523-1747.2000.00030.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Cell-density-induced upregulation of tyrosinase-related protein genes. (a) Confluent melan-a cells (at 1.9 × 105 cells per cm2) were trypsinized and plated at 5 × 105 cells per 10 cm plate (0.6 × 104 cells per cm2). Total RNA was isolated from confluent cells initially (*) and from cells on days 3, 5, 7, 8, and 10 after plating. Total RNA (9.4 μg) from each sample was loaded onto individual lanes of a denaturing 1% formaldehyde agarose gel and separated by electrophoresis. RNA was transferred to nitrocellulose by northern blotting. Blot was hybridized with cDNA probes from the 3′ end of Dct (nucleotides 1658–2182;Jackson et al. 1992), from the 3′ end of Tyrp1 (nucleotides 2150–2479;Shibahara et al. 1986), and from gapdh (Clontech). Hybridization of the blot with both probes simultaneously (data not shown) showed two distinct bands, slightly different in size, as evidence that the probes did not cross-hybridize with each other's mRNA. (b) Determination of cell densities. Cells were counted after trypsinization and cell densities were calculated. Error bars represent mean ± SD of six or eight independent cell counts. Journal of Investigative Dermatology 2000 115, 106-112DOI: (10.1046/j.1523-1747.2000.00030.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions