All-trans Retinoic Acid Induces Differentiation and Apoptosis of Murine Melanocyte Precursors with Induction of the Microphthalmia-Associated Transcription Factor

Abstract

The effects of all-trans retinoic acid on the differentiation and proliferation of immature melanocyte precursors were studied. NCC-melb4 cells are an immortal cloned cell line established from mouse neural crest cells using a single-cell cloning method. These cells were positive for tyrosinase-related protein 1, tyrosinase-related protein 2 and KIT, but were negative for tyrosinase and had no dihydroxyphenylalanine reaction. They contained only stage I melanosomes without any melanosomes in more advanced stages. After treatment with all-trans retinoic acid, many of the cells became tyrosinase- and dihydroxyphenylalanine-reaction-positive, changed from polygonal to dendritic in shape, and had stage III to IV melanosomes. These findings indicate that treatment with all-trans retinoic acid induced the differentiation of NCC-melb4 cells. Reverse transcription polymerase chain reaction analysis revealed a marked increase in expression of microphthalmia-associated transcription factor mRNA after all-trans retinoic acid treatment, suggesting that microphthalmia-associated transcription factor may be the key molecule in this event. Enhanced expression of protein kinase Calpha following treatment with all-trans retinoic acid was also demonstrated. The proliferation of NCC-melb4 cells was inhibited by all-trans retinoic acid in a dose-dependent manner. Increased apoptosis after all-trans retinoic acid treatment was observed by electron microscopy, the TUNEL method, DNA fragmentation assay, and flow cytometry. All-trans retinoic acid upregulated caspase-3 and downregulated bcl-2. Electron microscopy showed that apoptotic cells contained melanosomes of advanced stages, suggesting that mature melanocytes may tend to undergo apoptosis after all-trans retinoic acid treatment. This study provides important clues towards understanding the roles and working mechanisms of retinoic acids in melanocyte development and melanogenesis.

Mitf expression is sufficient to direct differentiation of medaka blastula derived stem cells to melanocytes

“…In other studies on melanocyte differentiation, treatment with retinoic acid (Watabe et al, 2002), or seeding ES cells, melanocyte precursors or neural crest cells on a cell layer (Yamane et al, 1999) have been used as the inducing stimulus. These experiments led to melanocytes, but the mechanisms that triggered the differentiation process could not be elucidated.…”

“…Some melanocytes were obtained after seeding undifferentiated ES cells on a bone marrow-derived stromal cell line and treating the cells with dexamethasone (Yamane et al, 1999). Mature melanocytes were obtained from a cell line of immature melanocytes treating the cells with retinoic acid (Watabe et al, 2002). An increase of expression of microphtalmia transcription factor (Mitf) was observed indicating an important role in this process.…”

Peroxisome proliferator-activated receptors-γ activator, ciglitazone, inhibits human melanocyte growth through induction of apoptosis

“…Of interest, our observation is similar to that observed in the ligands of other nuclear hormone receptors including retinoic acid receptor in melanocytes. Retinoic acid has inhibitory effects on the growth of melanocytes, which was accompanied by the induction of apoptosis in melanocytes [21]. Watabe et al [21] suggested that mature differentiated melanocytes are removed through apoptosis, which may be clinically beneficial, as topical application of retinoic acid reportedly improved hyperpigmented skin lesions like melasma.…”

Is apoptosis involved in the development of balloon cell nevus? Suggestion from a case report

“…Melanogenesis may be related to the apoptosis. 8,9 Expression of apoptosis regulatory molecules (ie, Bcl down-regulation and Bax up-regulation) are closely involved in melanogenesis. 8 In addition, it was reported that mature melanocytes may tend to undergo apoptosis after alltrans retinoic acid treatment.…”

“…8 In addition, it was reported that mature melanocytes may tend to undergo apoptosis after alltrans retinoic acid treatment. 9 Further study is needed for more precise understanding of the ballooning changes in association with apoptotic activity.…”