Immunohistochemical survey of the distribution of epidermal melanoblasts and melanocytes during the development of UVB-induced pigmented spots.

BACKGROUND: Repeated exposures to ultraviolet B radiation (UVB) induce pigmented spots on dorsal skin of (HR-1 x HR/De) F(1) hairless mouse. We showed previously that this mouse is suitable for studies of melanocyte function. OBJECTIVE: To clarify the mechanism of development of pigmented spots induced by chronic UVB exposure. METHODS: We used light and fluorescence microscopy to quantify changes in the numbers of differentiated melanocytes containing melanin pigments (MM) and melanoblasts/melanocytes immunohistochemically positive for tyrosinase-related protein (TRP)-1, TRP-2 (dopachrome tautomerase), and c-kit in epidermis during the development of pigmented spots in hairless mice chronically exposed to UVB (99 mJ/cm(2), 3 times/week, 8 weeks). RESULTS: The change in the number of TRP-1-positive cells during chronic UVB exposure was similar to that of MM: both increased dramatically during the stage of acute pigmentation, then decreased sharply after cessation of UVB, concomitantly with depigmentation; subsequently they increased gradually with the development of pigmented spots. In contrast, after two UVB exposures, no c-kit-positive cells were detected, then the number gradually increased during UVB irradiation, and continued to increase after cessation of irradiation; TRP-2-positive cells showed a rather similar pattern, except that they did not disappear initially. CONCLUSION: Our results indicate that chronic UVB irradiation induces differentiation and proliferation of melanoblasts, followed by an increase of differentiated melanocytes, leading to the development of pigmented spots. The sequence of expression of markers appeared to be c-kit, TRP-2, TRP-1, and finally melanin, as it is during normal melanocyte differentiation.

MIP-1beta, a novel biomarker for in vitro sensitization test using human monocytic cell line.

In order to seek a novel biomarker for predicting skin sensitization, changes in the gene expression profile of THP-1 cells on exposure to 2,4-dinitrochlorobenzene (DNCB), p-phenylenediamine (pPD) and nickel sulfate (Ni) were assessed using oligo-DNA microarrays. While the change in gene expression varied depending on the sensitizers, up-regulation of MIP-1 beta mRNA expression was detected in both DNCB-treated and Ni-treated THP-1 cells. This finding was validated by RT-PCR and confirmed at the protein level by ELISA. Secretion of MIP-1 beta from THP-1 was detected after 24-h treatment with sensitizers such as DNCB, Ni, 2-mercaptobenzothiazole (2-MBT) and cobalt sulfate (Co), while pPD and non-sensitizers such as sodium dodecyl sulfate (SDS) and benzalkonium chloride (BC) had no effect. The use of both MIP-1 beta production and CD86 expression as criteria reduced the number of false-negatives, and the results were in good agreement with those of in vivo assays. MIP-1 beta may be useful as a novel biomarker in in vitro sensitization assay using THP-1 cells, either alone or in combination with known markers.

Expression profiling and cellular localization of genes associated with the hair cycle induced by wax depilation.

The hair cycle is a highly regulated process controlled by multiple factors. Systematic analysis of gene expression patterns in each stage of the hair cycle would provide information useful for understanding this complicated process. To identify genes associated with the hair cycle, we used DNA microarray hybridization to analyze sequential gene expression patterns in mouse skin following hair cycle synchronization by wax depilation. Messenger RNA levels in mouse skin at various times after depilation were compared with those prior to depilation (resting phase). According to their expression patterns, upregulated genes were categorized into four groups: early anagen, middle anagen, late anagen/early catagen, and middle/late catagen, and processes that take place in each stage were evaluated. We identified 12 new components that are specifically expressed in the hair follicle, 11 genes in anagen including carbonic anhydrase 6, cytokeratin 12, and matrix metalloproteinase-11 in catagen that were confirmed using in situ hybridization. The strategy used here allowed us to identify unknown genes or process previously not suspected to have a role in hair biology. These analyses will contribute to elucidating the mechanisms of hair cycle regulation and should lead to the identification of novel molecular targets for hair growth and/or depilation agents.

Upregulation of the IFN-gamma-stimulated genes in the development of delayed pigmented spots on the dorsal skin of F1 mice of HR-1 x HR/De.

DNA microarray hybridization was used to measure the changes of mRNA levels over time during the development of delayed pigmented spots on the dorsal skin of F1 mice of HR-1 x HR/De. Upregulation of a number of interferon (IFN)-gamma-stimulated genes was detected in delayed pigmented lesions, suggesting that IFN-gamma may play a pivotal role in the development of delayed pigmented spots in this model. Upregulation of these genes was further supported by the increased protein expression level of IFN-gamma in the lesions. Epidermal infiltration of CD8(+) T lymphocytes and mast cell accumulation in the dermis were observed in delayed pigmented spots. Genes encoding chemokines such as monocyte chemoattractant protein-2 (MCP-2), IFN-inducible protein 10 (IP-10), and monokine induced by IFN-gamma (MIG) were among those upregulated by IFN-gamma. We hypothesize that chemokines produced in the epidermis induce migration of inflammatory cells, such as T lymphocytes, mast cells, and macrophages, to the vicinity of melanocytes. Keratinocytes, T lymphocytes, mast cells, and macrophages would become involved in an interactive network, providing a suitable local environment for melanocyte activation. In this environment, melanocytes are exposed to an extensive array of secreted mediators. Reciprocal activation among these cells to maintain this interactive network results in constitutive melanocyte activation and chronic melanin synthesis in delayed pigmented lesions.

Involvement of microphthalmia-associated transcription factor (MITF) in expression of human melanocortin-1 receptor (MC1R).

Analysis of the nucleotide sequence of human melanocortin-1 receptor (MC1R) promoter indicated that an E-box (CANNTG) is present immediately upstream of the transcriptional initiation site. The presence of the CATGTG motif suggests that MC1R gene expression may be regulated by a basic helix-loop-helix-leucine-zipper (bHLH-LZ) type transcription factor. The microphthalmia-associated transcription factor (MITF), which belongs to the family of bHLH-LZ type transcription factors, regulates the transcription of melanogenesis-related enzyme genes such as the tyrosinase and TRP-1 genes. We investigated whether MITF regulates human MC1R gene expression through the same transcriptional mechanism as tyrosinase and TRP-1 genes in melanocytes. For this purpose, the effect of co-expression of cDNA encoding MITF on MC1R promoter activity in NIH/3T3 cells was studied. MC1R promoter activity was induced to the extent of approximately 5-fold in the presence of MITF. In addition, electrophoretic mobility shift assay indicated that nuclear extracts of human SK-Mel-2 cells contain a protein that binds specifically to the MC1R promoter region containing the CATGTG motif. These results suggested that MITF regulates not only the expression of enzymes involved in melanin synthesis, but also the expression of a receptor which plays an essential role in melanocyte functions.