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Abstract
The microphthalmia-associated transcription factor (MITF), has been described as the master regulator of the basic helix-loop-helix leucine zipper family, involves melanogenesis in melanocytes. MITF consists of at least six isoforms, called MITF-M, MITF-A, MITF-B, MITF-C, MITF-H, and MITF-J. Previously, we found that not only MITF-M is expressed in the human hair follicle, but also MITF-A, MITF-C, MITF-H, and MITF-J isoforms are expressed in the skin. The aim of this study was to conform the MITF isoforms expressed in human skin, and investigate novel role of MITF isoforms in the melanocytes. Expression of MITF-M and MITF-A was found in primary melanoctyes and the melanoma cell lines. Interestingly, when MITF-M and MITF-A were overexpressed in the SK-MEL-24 melanoma cells by adenoviral transfection, length of the dendrites, serves as the principal conduit for melanosomes transfer, was significantly increased in the MITF-M overexpressed cells compared with the control group, and number of the dendtrites was significantly increased in the MITF-A overexpressed cells. A signal molecule involve in actin polymerization during dendrite formation, Rac1, was increased in the SK-MEL-24 melanoma cells treated with adenoviral MITF-M and MITF-A vectors. These results suggest that MITF-M and MITF-A induce dendrite formation via Rac1 signaling in the melanocytes.
References
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Fig. 1.
Gene expression of MITF isoforms in various cells. (A) MITF isoforms expression in normal cells, DP cells (dermal papilla cells of hair follicle), ORS cells (outer root sheath cells of hair follicle, melanocytes, keratinocytes, and fibroblast. (B) MITF isoforms expression in melanoma cell lines, SK-MEL-24, SK-MEL-28, HM3KO, and MNT-1 cells.
Fig. 2.
The change of morphology of the MITF-overexpressed SK-MEL-24 melanoma cells. Number of the dendrites, mainly long (red arrows), are increased in the MITF-M-overexpressed SK-MEL-24 melanoma cells (B and E) compared to the Ad/Lac-Z control (A and D). Number of the dendrites, mainly short (white arrows), are increased in the MITF-A-overexpressed SK-MEL-24 melanoma cells (C and F) compared with the Ad/Lac-Z control (A and D). E and F, Magnified photographs of the rectangular region in the Fig. B and Fig. C, respectively. Original magnification, A-C, × 100; D-F, × 400.
Fig. 3.
The graphs for the morphomentric analysis of Fig. 2 data. (A) Differential cell number in the control (Ad/Lac-Z), MITF-M-overex-prssed cells (Ad/MITF-M), and MITF-A-overexpressed cells. (B) Dendritic length (% control) in the control, MITF-M-overexprssed cells, and MITF-A-overexpressed cells. (C) % cell with >3 dendrites in the control, MITF-M-overexprssed cells, and MITF-A-overexpressed cells. Results are expressed as the mean±SD of independent experiments (n=3). ∗ Significantly different (p<0.05) from the control.
Fig. 4.
Expression of total-Rac1 protein and phospho-Rac-1 protein, active form of Rac1, are increased in the MITF-M-over-expressed cells (Ad/MITF-M), and in the MITF-A-overexpressed cells (Ad/MITF-A) compared with the control (Ad/Lac-Z). The band of Flag shows that adenoviral vectors including MITF-M and MITF-A respectively are transfected well in each cells.
Table 1.
List of primer sequences used in RT-PCR
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