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First published online 18 December 2007
doi: 10.1242/jcs.013912

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Neurofibromin as a regulator of melanocyte development and differentiation

Dermatology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA

View larger version (37K): [in this window] [in a new window]   Fig. 1. (A) Reduction of ventral spot size in KitW/+ and MitfMi-wh/+ mice with Nf1 haploinsufficiency. Nf1+/– mice were intercrossed with KitW/+ mice and with MitfMi-wh/Mi-wh mice. The litters at 6 weeks of age from both crosses were genotyped for Nf1. The belly-spot area in KitW/+ mice, wild-type and heterozygous for Nf1 and MitfMi-wh/+ mice, wild-type and heterozygous for Nf1 was determined and normalized to the gram body weight of each mouse. Error bars in right panel represent standard deviation from the mean. (B) Models for the regulation of Kit-Mitf signaling by neurofibromin. Neurofibromin may regulate Kit and Mitf through their signaling axis, as depicted in the left panel, or independently through both a non-Mitf effector downstream of Kit and a non-Kit inducer upstream of Mitf. (C) Reduction of dorsal spot in KitW-41/+;MitfMi-wh/+ mice with Nf1 haploinsufficiency. Nf1+/–;KitW-41/W-41 mice were crossed with MitfMi-wh/Mi-wh mice to generate KitW-41/+;MitfMi-wh/+ compound heterozygotes Nf1+/+ or Nf1+/–. Mice at 6 weeks of age were genotyped for Nf1 mutational status, separated by genotype and photographed.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Purification of primary melanocytes by FACS sorting from neonatal dermal suspensions of C57BL/6 mice. Primary cells were cultured from dermal suspensions of C57BL/6 neonates for a period of 10 days with TPA, bFGF and dbcAMP as described. Primary cells lacking additives for 72 hours were double labeled with PE-CD117 (anti-Kit) and APC-CD45 and separated by FACS. The CD117+CD45-population was plated and analyzed for melanocyte content using MEL-5 (anti-Tyrp1) immunofluorescence. (A) Anti-CD117 (against Kit) and anti-CD45 flow cytogram of mixed primary cultures from murine neonatal dermal suspensions of wild-type cells. The CD117+CD45– population was generally 1% of the total population of cells. (B) Bright-field photomicrograph of CD117+CD45– population showing prominent pigmentation and dendrites characteristic of melanocytes. (C) MEL-5 (anti-Tyrp1) immunofluorescence of sorted CD117+CD45– cells. (D) DAPI staining of cells shown in C.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Melanogenic gene expression in purified primary wild-type and Nf1+/– melanocytes. FACS-separated Kit+CD45– wild-type and Nf1+/– melanocytes were plated at 1 x104 cells per well in individual wells of a 24-well plate and cultured in the presence of TPA, bFGF and dbcAMP for 10 days. Growth factors were withdrawn 72 hours prior to RNA isolation. The relative gene expression levels of (A) Tyr, (B) Tyrp1, (C) Dct and (D) Mitf were measured by real time RT-PCR. Results represent the normalized mean ± standard deviation of five independent measurements. P<0.05 for Nf1+/– versus wild-type melanocytes by Student's paired t-test.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Melanogenic protein and gene expression and Erk hyperactivation in Nf1+/– primary melanocytes. (A) Melanogenic protein expression in mixed primary culture wild-type and Nf1+/– cells. Lysates were prepared on day 10 of primary culture with TPA, bFGF and dbcAMP withdrawn 72 hours prior to lysis. Western blotting shows relative expression of p-Erk, Tyr, Tyrp1, Mitf and Dct/TRP-2 as indicated. Expression of Erk2 is used as a loading control. (B) Melanogenic gene expression in mixed primary culture wild-type and Nf1+/– cells. Total RNA was prepared on day 10 of primary wild-type and Nf1+/– cultures. Relative expression of Tyrp1, Dct, and Tyr was determined by real time RT-PCR. Data is representative of results from two independent experiments. Error bars represent the standard deviation from the mean of duplicate samples. (C) SCF/Kit-ligand-dependent and SCF/Kit-ligand-independent Erk phosphorylation in wild-type and Nf1+/– cells. Mixed wild-type and Nf1+/– primary cultures were grown for 15 days. TPA, bFGF and dbcAMP were withdrawn 72 hours prior to stimulation with murine SCF for 5 minutes and 15 minutes. Cell lysates were collected and examined for relative expression of phosphorylated Erk and Erk2 by western blotting.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Effects of MEK inhibitor PD98059 upon Erk phosphorylation and melanogenic gene expression in wild-type and Nf1+/– cells. (A) Effect of MEK inhibition on Erk phosphorylation in wild-type and Nf1+/– cells. Lysates were prepared from day 10 mixed wild-type and Nf1+/– primary cultures. TPA, bFGF and dbcAMP were withdrawn 72 hours prior to preparation of cell lysates, and designated Nf1+/– cultures were treated with 50 µM MEK inhibitor PD98059 12 hours prior to cell lysate preparation. One group of PD98059-treated cells was stimulated with SCF (20 ng/ml) for 15 minutes. Cell lysates were probed for phosphorylated Erk and Erk2 by western blotting. (B-D) Effect of MEK inhibition upon melanogenic gene expression in immortalized melanocytes. Melan-a cells grown to confluency on a 10-cm plate were treated with vehicle or PD98059 (50 µM) for 24 or 48 hours prior to RNA isolation. Tyr, Tyrp1 and Dct expression were quantified by real-time RT-PCR. Results represent the average of two independent experiments, each performed with duplicate samples. Error bars indicate the standard deviation from the mean. (E-H) Effect of MEK inhibition upon melanogenic gene expression in FACS-sorted wild-type and Nf1+/– cells. FACS-sorted primary wild-type or Nf1+/– melanocytes were plated at densities of 1.5 x104 cells per well (wild-type cells) or 8 x103 cells per well (Nf1+/– cells) in 24-well plates and cultured for 10 days. TPA, bFGF and dbcAMP were withdrawn 72 hours prior to lysis and cells were treated with vehicle or MEK inhibitor PD98059 overnight prior to RNA isolation. Tyr, Tyrp1, Dct and Mitf expression were measured by quantitative real-time RT-PCR. Results represent mean ± standard deviation of the fold change in expression for five independent measurements of wild-type versus wild-type + PD98059 samples and Nf1+/– versus Nf1+/– + PD98059 samples. **P<0.05 for wild-type and wild-type + PD98059 comparison, and *P<0.05 for Nf1+/– and Nf1+/– + PD98059 comparison by Student's paired t-test.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Effects of reduction of neurofibromin in immortalized murine melanocytes. (A) siRNA-mediated depletion of neurofibromin in melan-a cells. Melan-a cells at a cell density of 2 x105 cells per well in a six-well plate were transfected with three different siRNAs (lanes 1-3) targeting Nf1. Cells were also transfected with a control, scrambled siRNA (lane M) or activated with 40 µM TPA (lane U) for 15 minutes prior to lysis. Cells were lysed 72 hours post-transfection and evaluated for siRNA-mediated knockdown of neurofibromin with western blotting. (B) Melanogenic gene expression in melan-a cells treated with neurofibromin siRNA. Melan-a cells at a cell density of 2 x105 cells per well in a six-well plate were transfected with the same siRNAs targeting Nf1. RNA isolated from cells 48 hours post-transfection was evaluated for Tyr, Tyrp1 and Dct expression by real time RT-PCR. Experiments were performed in triplicate, with error bars representing standard deviation from the mean. The fold increase in gene expression for each siRNA gene pair is relative to the expression level measured for the same gene in cells transfected with the control, scrambled siRNA (Control) described above.