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

Wellbrock C, Marais R - J. Cell Biol. (2005)

Bottom Line: Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear.MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation.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.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Team, Cancer Research UK Centre of Cell and Molecular Biology, The Institute of Cancer Research, London SW3 6JB, England, UK.

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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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.
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fig2: 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.

Mentions: Previous studies have shown that ERK phosphorylates S73 of MITF, targeting it for degradation (Wu et al., 2000; Xu et al., 2000), so we analyzed whether this mechanism underlies MITF loss in our cell lines. Transiently expressed HA-tagged MITF localizes to the nucleus of melan-a–VE cells (Fig. 2 A). On SDS gels, it migrates as a single band whose mobility is increased when the cells are treated with the MEK inhibitor U0126 (Fig. 2 B); these effects were previously attributed to ERK-dependent phosphorylation on S73 (Hemesath et al., 1998). Accordingly, MITF in which S73 is mutated to alanine (S73AMITF) comigrates with MITF in U0126-treated cells (Fig. 2 B). In melan-a–VE lines, ectopic MITF is expressed at low levels, but these increase when the cells are treated with the proteasome inhibitor MG132 (Fig. 2 C). This suggests that MITF is degraded by the ubiquitin pathway after S73 phosphorylation by ERK. However, when S73AMITF is expressed, it also fails to accumulate unless the cells are treated with MG132 (Fig. 2 C). This effect does not appear to be caused by mislocalization because S73AMITF also resides in the nucleus (Fig. 2 A). Although our data directly implicate the ubiquitin-mediated proteasome pathway in MITF stability in melan-a–VE cells, MG132 did not induce the accumulation of endogenous MITF in these cells (Fig. 2 C, control), suggesting that additional mechanisms regulate MITF expression. RT-PCR analysis revealed that MITF mRNA levels are significantly lower in melan-a–VE cells than in parental or melan-a–B-RAF cells (Fig. 2 D), but the cAMP-elevating agent forskolin still induces MITF expression (Fig. 2 E). Importantly, forskolin-induced MITF expression (Fig. 2 F) and CREB phosphorylation (Fig. 2 G) are not inhibited by U0126, demonstrating that MEK–ERK signaling is not required for MITF regulation by cAMP in these cells.


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

Wellbrock C, Marais R - J. Cell Biol. (2005)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: Previous studies have shown that ERK phosphorylates S73 of MITF, targeting it for degradation (Wu et al., 2000; Xu et al., 2000), so we analyzed whether this mechanism underlies MITF loss in our cell lines. Transiently expressed HA-tagged MITF localizes to the nucleus of melan-a–VE cells (Fig. 2 A). On SDS gels, it migrates as a single band whose mobility is increased when the cells are treated with the MEK inhibitor U0126 (Fig. 2 B); these effects were previously attributed to ERK-dependent phosphorylation on S73 (Hemesath et al., 1998). Accordingly, MITF in which S73 is mutated to alanine (S73AMITF) comigrates with MITF in U0126-treated cells (Fig. 2 B). In melan-a–VE lines, ectopic MITF is expressed at low levels, but these increase when the cells are treated with the proteasome inhibitor MG132 (Fig. 2 C). This suggests that MITF is degraded by the ubiquitin pathway after S73 phosphorylation by ERK. However, when S73AMITF is expressed, it also fails to accumulate unless the cells are treated with MG132 (Fig. 2 C). This effect does not appear to be caused by mislocalization because S73AMITF also resides in the nucleus (Fig. 2 A). Although our data directly implicate the ubiquitin-mediated proteasome pathway in MITF stability in melan-a–VE cells, MG132 did not induce the accumulation of endogenous MITF in these cells (Fig. 2 C, control), suggesting that additional mechanisms regulate MITF expression. RT-PCR analysis revealed that MITF mRNA levels are significantly lower in melan-a–VE cells than in parental or melan-a–B-RAF cells (Fig. 2 D), but the cAMP-elevating agent forskolin still induces MITF expression (Fig. 2 E). Importantly, forskolin-induced MITF expression (Fig. 2 F) and CREB phosphorylation (Fig. 2 G) are not inhibited by U0126, demonstrating that MEK–ERK signaling is not required for MITF regulation by cAMP in these cells.

Bottom Line: Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear.MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation.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.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Team, Cancer Research UK Centre of Cell and Molecular Biology, The Institute of Cancer Research, London SW3 6JB, England, UK.

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.

Show MeSH
Related in: MedlinePlus