PPAR gamma regulates MITF and beta-catenin expression and promotes a differentiated phenotype in mouse melanoma S91

Abstract

Melanoma represents one of the most rapidly metastasizing, hence deadly tumors due to its high proliferation rate and invasiveness, characteristics of undifferentiated embryonic tissues. Given the absence of effective therapy for metastatic melanoma, understanding more fully the molecular mechanisms underlying melanocyte differentiation may provide opportunities for novel therapeutic intervention. Here we show that in mouse melanoma S91 cells activation of the peroxisome proliferator activated receptor (PPAR) gamma induces events resembling differentiation, such as growth arrest accompanied by apoptosis, spindle morphology and enhanced tyrosinase expression. These events are preceded by an initial transient increase in expression from the Microphthalmia-associated transcription factor gene, (MITF) promoter, whereas exposure to a PPAR gamma ligand- ciglitazone that exceeds 8 h, causes a gradual decrease of MITF, until by 48 h MITF expression is substantially reduced. Beta-catenin, an MITF transcriptional activator, shows a similar pattern of decline during ciglitazone treatment, consistent with previous reports that activated PPAR gamma inhibits the Wnt/beta-catenin pathway through induction of beta-catenin proteasomal degradation. We suggest that the PPAR gamma-mediated beta-catenin down-regulation is likely to be responsible for changes in MITF levels. The data suggest that PPAR gamma, besides its well-established role in mesenchymal cell differentiation towards adipocytes, might regulate differentiation in the melanocytic lineage.

Figure 1

Mouse melanoma cells S91 and B16F10 express transcriptionally active PPAR γ but inactive PPAR α. (A) PPARs protein levels checked by western blot. BAT – brown adipose tissue, positive control for PPAR γ, Liver – positive control for PPAR α. GRB2 - loading control. (B) Transcriptional activity of PPAR α and PPAR γ, determined by luciferase assay. Plot: mean ± SEM, *P < 0.05. Symbols: Ctrl – control, DMSO treated cells, F – Fenofibrate, C – Ciglitazone, 9cRA – 9–cis-retinoic acid.

Figure 2

PPAR γ activators induce growth arrest in S91 cells. (A) Growth kinetics. Plot: mean ± SD. Symbols: Ctrl – control, DMSO treated cells, C – Ciglitazone, 9cRA – 9-cis-retinoic acid. (B) Cell cycle analysis for S91 cells treated with ciglitazone together with 9-cis-retinoic acid for 24, 48 and 72 h. Time point 0 represents control cells.

Figure 3

PPAR γ activators induce morphological changes. Phase contrast microphotographs from S91 cell culture after 72 h incubation in the indicated conditions. Scale bar: 100 µm. Symbols: Ctrl – control DMSO treated cells, C – Ciglitazone, 9cRA – 9–cisretinoic acid, Forsk – forskolin, NM – normal human melanocytes.

Figure 4

PPAR γ activation affects expression of genes involved in melanocyte differentiation. (A) PPAR γ activation increases DOPA-oxidase activity of tyrosinase. The enzyme activity is expressed as: [product mol/l* min⁻¹* mg of protein⁻¹]*10⁻¹⁰. Positive control – forskolin treated cells. Plot: mean ± SEM, *P < 0.05. Symbols: Ctrl – control DMSO treated cells, C – Ciglitazone, 9cRA – 9–cis-retinoic acid, Forsk – forskolin. (B) Tyrosinase mRNA levels (Tyr) checked by RT PCR, β-actin as an internal control, Neg – PCR purity control. (C) Effects of ciglitazone on tyrosinase, MITF and β-catenin protein levels (detected by western blot) are reversed by a specific PPAR γ inhibitor, GW 9662. Forskolin-treated cells are a positive control, β-actin – a loading control. In tyrosinase blot, the arrows indicate the high molecular weight, glycosylated form and the lower molecular weight, unglycosylated form. (D) MITF and (E) Tyrosinase promoter activities determined by luciferase assay. Plots: mean ± SEM, *P < 0.05. (F) Time-dependent changes in MITF and β-catenin protein levels (detected by western blot) during the incubation with ciglitazone; β-actin – a loading control. (G) Immunofluorescent detection of β-catenin in S91 cells, the nuclei stained with DAPI. LiCl (30 mM), which blocks β-catenin degradation, is used as a positive control. Symbols: Ctrl – control DMSO treated cells, C – Ciglitazone, 9cRA – 9-cis-retinoic acid, GW – GW 9662, Forsk – forskolin.