MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells

Abstract

As a survival factor for melanocytes lineage cells, MiTF plays multiple roles in development and melanomagenesis. What role MiTF plays in the DNA damage response is currently unknown. In this report we observed that MiTF was phosphorylated at serine 73 after UVC radiation, which was followed by proteasome-mediated degradation. Unlike after c-Kit stimulation, inhibiting p90RSK-1 did not abolish the band shift of MiTF protein, nor did it abolish the UVC-mediated MiTF degradation, suggesting that phosphorylation on serine 73 by Erk1/2 is a key event after UVC. Furthermore, the MiTF-S73A mutant (Serine 73 changed to Alanine via site-directed mutagenesis) was unable to degrade and was continuously expressed after UVC exposure. Compared to A375 melanoma cells expressing wild-type MiTF (MiTF-WT), cells expressing MiTF-S73A mutant showed less p21WAF1/CIP1 accumulation and a delayed p21WAF1/CIP1 recovery after UVC. Consequently, cells expressing MiTF-WT showed a temporary G1 arrest after UVC, but cells expressing MiTF-S73A mutant or lack of MiTF expression did not. Finally, cell lines with high levels of MiTF expression showed higher resistance to UVC-induced cell death than those with low-level MiTF. These data suggest that MiTF mediates a survival signal linking Erk1/2 activation and p21WAF1/CIP1 regulation via phosphorylation on serine 73, which facilitates cell cycle arrest. In addition, our data also showed that exposure to different wavelengths of UV light elicited different signal pathways involving MiTF.

NADPH Oxidase 1 Overexpression Enhances Invasion via Matrix Metalloproteinase-2 and Epithelial–Mesenchymal Transition in Melanoma Cells

“…Cells were lysed in RIPA 250 buffer by sonication (without centrifugation) (Liu et al, 2010). A 2 Â urea buffer (25 mM Tris, pH 6.8, 12 M urea) was added to cell lysate at 1:1 (V:V) ratio, and placed on ice for 10 minutes.…”

“…For both PCR and quantitative real-time RT-PCR, b-actin was used as internal control. The relative mRNA level was calculated by DDCt method as described before (Liu et al, 2010). The chromatin immunoprecipitation protocol followed one described previously (Liu and Lee, 2006).…”

UVB-Stimulated TNFα Release from Human Melanocyte and Melanoma Cells Is Mediated by p38 MAPK

“…Some of these molecular interactions can give rise to genetic alteration, activation/suppression of cell signaling pathways, resulting in either the upregulation or downregulation of cytokine release. The molecular interactions of the B-Raf/extracellular-signal regulated kinase (ERK) pathway in melanoma cells have been widely investigated [1–3]. Unlike ERK, the other mitogen-activated protein kinase (MAPK); p38 MAPK and c-jun terminal kinase (JNK), and nuclear factor-κB (NFκB) pathways have not been frequently linked to melanoma incidences [1].…”

“…These pathways have been suggested to also play a role in malignant transformation of melanocytes, although their anti-tumorigenic activities have also been reported [8–12]. Therefore, it is highly likely that besides the B-Raf/ERK pathway, the other MAPK pathways may on their own or in conjunction with ERK play a role in the development and progression of melanoma [3]. …”

Continuing to illuminate the mechanisms underlying UV-mediated melanomagenesis

“…UV-A and UV-B treatment occurred every Tuesday and Friday during the treatment period. UV-B radiation was performed as previously described (63). Briefly, Cells were grown to about 70% confluence and media was removed completely for UV-B radiation.…”