Stem cell factor combined with matrix proteins regulates the attachment and migration of melanocyte precursors of human hair follicles in vitro.

In conjunction with matrix proteins, stem cell factor (SCF) plays an important role in the migration of melanocyte precursors (MPs) derived from the mouse embryo. However, no studies have demonstrated an effect of SCF on human follicular MPs migration in vitro. In this report, first we demonstrate the immature state of the follicular MPs. Then cell attachment rate was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Standard 48-well chemotaxis chambers were used for a transfilter migration assay. F-actin was labeled by rhodamine-conjugated phalloidin, and then organization of the actin cytoskeleton was observed by confocal microscope. In the results, we directly show that MPs adhere more strongly to fibronectin (FN), laminin (LN) and type IV collagen (CIV) than to the negative control. SCF decreased the adhesion of MPs to FN and CIV. A chemotaxis analysis showed that FN and CIV have chemotactic effects on MPs. FN showed an obvious increase in chemotactic effects on MPs with SCF treatment comparing with the control group, but there were no significant changes in the levels of chemotaxis with CIV and LN when the cells were treated with SCF. SCF was chemotactic to MPs, and the presence of FN caused a statistically significant increase in MPs migration at various concentrations of SCF. Furthermore, we showed that SCF, in combination with FN, could induce an apparent increase in actin stress fiber formation in MPs. Our results indicate that SCF, in combination with matrix proteins and in particular with FN, regulates the movement of MPs by both altering cell attachment and increasing cell chemotaxis.

Endothelin-1 combined with extracellular matrix proteins promotes the adhesion and chemotaxis of amelanotic melanocytes from human hair follicles in vitro.

During the repigmentation of vitiliginous skin, amelanotic melanocytes (AMMCs) migrate from the outer root sheath (ORS) of the hair follicles into depigmented skin. It has been shown that endothelin-1 (ET-1) is an important cytokine in the migration of epidermal melanocytes, produced by keratinocytes particularly after irradiation with ultraviolet B (UVB). To further examine the role of ET-1 on the migration of AMMCs, we investigated the effects of ET-1 to the adhesion and chemotaxis of human AMMCs combined with extracellular matrix proteins (ECMP) and observed the effects on the actin and tubulin cytoskeleton of AMMC by ET-1. Human AMMCs were treated with different concentrations of ET-1 (0.1-100 nM) to observe adhesion on culture dishes coated with fibronectin (FN), laminin (LN) and collagen IV (CIV). In addition, chemotaxis on FN, LN and CIV coated micropore filters, with various concentrations of ET-1 as attractants, was investigated using a Boyden chemotaxis chamber. Cellular microfilaments and microtubules were immunostained with Rhodamine labeled actin and FITC labeled beta-tubulin. The effects of ET-1 on cytoskeleton were observed with laser confocal microscopy. The study demonstrated that ET-1 increases human AMMCs adhesion on FN in a dose-dependent manner, but minor increases are found on the coated surface with LN and CIV. ET-1 also induces chemotaxis of AMMCs on CIV, LN and FN in a dose-dependent manner. The greatest effect was seen with CIV. Minor chemotactic effects were observed with non-coated surfaces. A concentration of >or=10nM ET-1 induced an apparent increase in stress fibers underneath the cell membrane, but no effects were found on tubulin. ET-1 has various effects on the adhesion and chemotaxis of AMMCs on various ECMP, which could be partly due to a modulation and reorganization of the actin cytoskeleton.

A modified method for purifying amelanotic melanocytes from human hair follicles.

We describe a modified method for establishing long-term pure cultures of amelanotic melanocytes (AMMC) derived from human hair follicles. Normal human corpse scalp (just after death, 1 h) was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. Hair follicle cell suspensions were prepared by 0.50% trypsin treatment for 30 min and cultured in an optimized melanoblast proliferation nature mitogen medium. Cells attached to the substratum were mostly amelanotic melanocytic in character with small, bipolar shapes in the early stage; only a few keratinocytes and rare fibroblasts were observed. Keratinocytes were easily removed by differential trypsinization. After the third passage, the proliferating cells were all amelanotic melanocytes as confirmed by immunostaining with polyclonal antibodies to alphaPEP7h, which recognized the tyrosinase protein located on melanosomes and NKI/beteb, which is a pre-melanosomal antigen against synthetic peptides corresponding to the carboxyl termini of human melanosomal protein GP100. Cultured AMMC were highly positive to L-dopa reactivity after the addition of IBMX to the culture medium for 7 days. Many stage I and II melanosomes and occasional stage III melanosomes without stage IV melanosomes were found in the cytoplasm by transmission electron microscope. This modified technique is potentially more suitable for cultivating amelanotic melanocytes. The availability of pure cultures of hair-follicle amelanotic melanocytes will facilitate investigations of the roles of those cells in migration and differentiation during treatment of vitiligo.

A gene locus responsible for reticulate pigmented anomaly of the flexures maps to chromosome 17p13.3.

Reticulate pigmented anomaly of the flexures (RPAF), also called Dowling-Degos disease, is a rare autosomal-dominant cutaneous disorder characterized by spotted and reticulate pigmentation of the flexures. The gene, or even the chromosomal location, for RPAF has not yet been identified. In this study, one Chinese family with RPAF was identified and subjected to a genomewide screen for linkage analysis. We identified a locus at chromosome 17p13.3 with a maximum two-point limit of detection score of 3.61 at markers D17S831and D17S1866 (at recombination fraction theta=0.00). Haplotype analyses indicated that the disease gene is located within the 6.8 cM region distal to D17S1798. It is the first locus identified for RPAF. This study provides a map location for isolation of a disease gene causing RPAF.

A new arginine substitution mutation of DSRAD gene in a Chinese family with dyschromatosis symmetrica hereditaria.

BACKGROUND: Dyschromatosis symmetrica hereditaria (DSH) is a pigmentary genodermatosis of autosomal dominant inheritance characterized by a mixture of hyperpigmented and hypopigmented macules distributed on the dorsal aspects of the hands and feet. To date, only three articles testified that DSH is caused by the mutations of DSRAD gene (also called ADAR1) encoding for RNA-specific adenosine deaminase. OBJECTIVE: To identify mutations of DSRAD as the disease-causing gene and recognize different mutations giving a clue to insight into the mechanism of DSH. METHODS: We collected a Chinese DSH family consisting of a total of 11 individuals including five DSH patients (three males and two females). The whole coding region of DSRAD was amplified by polymerase chain reaction and products analyzed by direct sequencing. RESULTS: We detected a transition, 3463 C>T, leading to a missense mutation (R1155W) in genomic DNAs of five patients, and the point mutation was not found in normal individuals in this DSH family and in 100 unrelated, population-match control individuals. CONCLUSION: Our data suggests that R1155W missense mutation is a new mutation in exon 15 of DSRAD gene and further testify that DSRAD gene is the pathogenic gene of DSH.