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. 2005 Aug 29;170(5):703-8.
doi: 10.1083/jcb.200505059.

Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation

Claudia Wellbrock  1 , Richard Marais
Affiliations

Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation

Claudia Wellbrock et al. J Cell Biol. .

Abstract

The protein kinase B-RAF is a human oncogene that is mutated in approximately 70% of human melanomas and transforms mouse melanocytes. Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear. In this study, we show that MITF expression is suppressed by oncogenic B-RAF in immortalized mouse and primary human melanocytes. However, low levels of MITF persist in human melanoma cells harboring oncogenic B-RAF, suggesting that additional mechanisms regulate its expression. MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation. Furthermore, differentiation-inducing factors that elevate MITF expression in melanoma cells inhibit their proliferation, but when MITF up-regulation is prevented by RNA interference, proliferation is not inhibited. These data suggest that MITF is an anti-proliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.

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Figures

Figure 1.
Figure 1.
MITF expression is lost in B-RAF–transformed melanocytes. (A) Western blot analysis of melan-a cells, a neoR control line, WTB-RAF–expressing clones B2 and B9, and V600EB-RAF–expressing clone VE16 probed for myc-tagged B-RAF, total B-RAF, and ERK2. (B) Bright field image of melan-a cells, neoR controls, clones B2, B9, and VE16, and G12VRAS- or MEKEE-transformed melan-a cells under growing conditions. (C) Western blot analysis of MITF, phosphorylated ERK (ppERK), and ERK2 in melan-a cells, neoR controls, WTB-RAF–expressing clones (B2 and B9), V600EB-RAF–expressing clones (VE11, VE14, and VE16) and G12VRAS- or MEKEE-expressing cells.
Figure 2.
Figure 2.
Constitutive ERK activation triggers MITF down-regulation. (A) Immunofluorescent analysis of recombinant HA-MITF and HA-S73AMITF in melan-a–VE16 cells using anti-MITF antibody C5. Nuclei are counterstained with DAPI. (B) Western blot analysis of transiently expressed HA-MITF, HA-S73AMITF, ppERK, and ERK2 in melan-a–VE16 cells that were either untreated or treated with 10 μM DMSO or U0126 for 2 h. Control cells were transfected with empty vector, and HA-MITF proteins were detected using anti-HA. (C) Western blot analysis of stably expressed HA-MITF or HA-S73AMITF in melan-a–VE16 cells untreated or treated with 30 μM MG132 for 8 h. HA-MITF proteins were detected using anti-MITF (C5). Total ERK2 is used as a loading control. (D) RT-PCR of melanocyte-specific MITF mRNA expression in parental melan-a cells and clones B2, VE11, VE14, and VE16. GAPDH serves as a loading control. (E) RT-PCR analysis of MITF expression in melan-a and melan-a–VE cells treated with 20 μM forskolin (FO) for the indicated times. GAPDH serves as a loading control. (F) RT-PCR analysis of melanocyte-specific MITF mRNA in melan-a–VE cells treated with 20 μM forskolin for the indicated times in the presence of 10 μM U0126 or DMSO (D) for a 10-min pretreatment. GAPDH serves as a loading control. (G) Western blot analysis for phosphorylated CREB, ppERK, and ERK2 in melan-a–VE cells treated with 10 μM forskolin for 30 min, 10 μM U0126 for a 10-min pretreatment, or DMSO.
Figure 3.
Figure 3.
MITF inhibits proliferation of B-RAF–transformed melanocytes. (A) Melan-a–VE clones VE11 and VE16 were transfected with a Hygromycin resistance plasmid plus empty vector or the MITF expression vector. Western blot shows MITF expression 24 h after transfection in comparison with endogenous MITF in melan-a cells. The cells were then selected for Hygromycin resistance and stained for colonies 15 d after transfection. Results are means from triplicate determinations with error bars to represent the SD. A representative stained cell image is shown to the right of the graph. (B) Western blot analysis of stably expressed HA.ER and HA.ER-MITF in melan-a–VE cells untreated or treated with 200 nM 4-hydroxy-tamoxifen (4-OHT) for 24 h. HA.ER and HA.ER-MITF were revealed with the anti-HA antibody. (C) Luciferase assay of melan-a–VE HA.ER or HA.ER-MITF cells transfected with a tyrosinase promoter luciferase reporter. 24 h after transfection, cells were untreated or treated with 200 nM 4-OHT for a further 24 h. Error bars represent SD from the mean. (D) Growth curve of melan-a–VE HA.ER- or HA.ER-MITF–expressing cells in the absence or presence of 200 nM 4-OHT. Error bars represent SD from the mean.
Figure 4.
Figure 4.
V600EB-RAF activates ERK and suppresses MITF expression in human melanocytes. (A) Western blot analysis of ppERK and ERK2 in primary human melanocytes (NHM) that were untreated or treated with 10 μM U0126 for 24 h or with DMSO control (second lane). (B) Thymidine incorporation into NHM treated with 10 μM U0126 for 24 h, DMSO, or no treatment control in the presence of melanocyte growth factor supplement. (C) Western blot analysis of A-RAF, B-RAF, C-RAF, ppERK, and ERK2 in NHM transfected with either scrambled control siRNA (sc) or siRNAs specific for A-RAF, B-RAF, or C-RAF. (D) Thymidine incorporation into NHM in the presence of melanocyte growth factor supplement 72 h after transfection with the indicated siRNAs. Error bars represent SD. (E) Western blot analysis for myc-tagged B-RAF, ppERK, and ERK2 in NHM transiently expressing either myc-V600EB-RAF or myc-WTB-RAF. B-RAF was revealed using the antibody 9E10. (F) Immunofluorescence analysis of transiently expressed myc-V600EB-RAF and myc-WTB-RAF and of endogenous MITF in NHM transfected with myc-V600EB-RAF or myc-WTB-RAF. B-RAF proteins are revealed with anti-myc, and MITF is revealed with C5. Nuclei are counterstained with DAPI. Arrows indicate nuclei of transfected cells expressing either wild-type B-RAF or mutant V600EB-RAF. (G) Quantification of immunofluorescence data. Means of three experiments are shown (100 cells were counted in each experiment). Vector-transfected cells served as a control.
Figure 5.
Figure 5.
Enhanced MITF expression contributes to cAMP-induced growth inhibition of melanoma cells. (A) Western blot analysis of endogenous MITF, ppERK, and ERK2 in melanoma cell lines expressing oncogenic RAS or B-RAF compared with NHM. MITF was revealed using anti-MITF (D5). (B) Western blot of endogenous MITF (using antibody D5) and ERK2 as a loading control in NHM treated with 20 μM forskolin or DMSO for the indicated times in the absence of melanocyte growth factor supplement. (C) Thymidine incorporation into NHM in the absence of melanocyte growth factor supplement after 20 μM forskolin treatment for 24 h or DMSO treatment. (D) Western blot analysis of MITF (D5) and ERK2 in untreated or 20 μM forskolin-treated (16 h) Colo826 or WM266-4 cells in the presence or absence of either MITF-specific siRNA or scrambled control (sc). (E) Thymidine incorporation in parallel samples from D. Thymidine incorporation in DMSO-treated cells was set at 100%. Results are from triplicate assays with error bars to represent SD from the mean.
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