ABSTRACT
The rodent chloride channel regulatory proteins mCLCA2 and its porcine and human homologues pCLCA2 and hCLCA2 are expressed in keratinocytes but their localization and significance in the epidermis have remained elusive. hCLCA2 regulates cancer cell migration, invasion and apoptosis, and its loss predicts poor prognosis in many tumors. Here, we studied the influences of epidermal maturation and UV-irradiation (UVR) on rCLCA2 (previous rCLCA5) expression in cultured rat epidermal keratinocytes (REK) and correlated the results with mCLCA2 expression in mouse skin in vivo. Furthermore, we explored the influence of rCLCA2 silencing on UVR-induced apoptosis. rClca2 mRNA was strongly expressed in REK cells, and its level in organotypic cultures remained unchanged during the epidermal maturation process from a single cell layer to fully differentiated, stratified cultures. Immunostaining confirmed its uniform localization throughout the epidermal layers in REK cultures and in rat skin. A single dose of UVR modestly downregulated rClca2 expression in organotypic REK cultures. The immunohistochemical staining showed that CLCA2 localized in basal and spinous layers also in mouse skin, and repeated UVR induced its partial loss. Interestingly, silencing of rCLCA2 reduced the number of apoptotic cells induced by UVR, suggesting that by facilitating apoptosis, CLCA2 may protect keratinocytes against the risk of malignancy posed by UVB-induced corrupt DNA.
Subject(s)
Chloride Channels/biosynthesis , Epidermis/metabolism , Ultraviolet Rays , Animals , Apoptosis , Cells, Cultured , Down-Regulation , Keratinocytes/metabolism , Mice , RatsABSTRACT
BACKGROUND: Hyaluronan is a large, linear glycosaminoglycan present throughout the narrow extracellular space of the vital epidermis. Increased hyaluronan metabolism takes place in epidermal hypertrophy, wound healing and cancer. Hyaluronan is produced by hyaluronan synthases and catabolized by hyaluronidases, reactive oxygen species and KIAA1199. OBJECTIVES: To investigate the changes in hyaluronan metabolism during epidermal stratification and maturation, and the impact of vitamin C on these events. METHODS: Hyaluronan synthesis and expression of the hyaluronan-related genes were analysed during epidermal maturation from a simple epithelium to a fully differentiated epidermis in organotypic cultures of rat epidermal keratinocytes using quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blotting, in the presence and absence of vitamin C. RESULTS: With epidermal stratification, both the production and the degradation of hyaluronan were enhanced, resulting in an increase of hyaluronan fragments of various sizes. While the mRNA levels of Has3 and KIAA1199 remained stable during the maturation, Has1, Has2 and Hyal2 showed a transient upregulation during stratification, Hyal1 transcription remained permanently increased and transcription of the hyaluronan receptor, Cd44, decreased. At maturation, vitamin C downregulated Has2, Hyal2 and Cd44, whereas it increased high-molecular-mass hyaluronan in the epidermis, and reduced small fragments in the medium, suggesting stabilization of epidermal hyaluronan. CONCLUSIONS: Epidermal stratification and maturation is associated with enhanced hyaluronan turnover, and release of large amounts of hyaluronan fragments. The high turnover is suppressed by vitamin C, which is suggested to enhance normal epidermal differentiation in part through its effect on hyaluronan.
Subject(s)
Ascorbic Acid/pharmacology , Epidermis/drug effects , Hyaluronic Acid/metabolism , Keratinocytes/drug effects , Animals , Cell Differentiation/drug effects , Cell Line , Epidermis/chemistry , Epidermis/metabolism , Gene Expression Profiling , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Hyaluronan Synthases/genetics , Hyaluronan Synthases/metabolism , Hyaluronic Acid/analysis , Hyaluronoglucosaminidase/genetics , Hyaluronoglucosaminidase/metabolism , Keratinocytes/chemistry , Keratinocytes/metabolism , RNA, Small Interfering/metabolism , Rats , Up-Regulation/drug effectsABSTRACT
BACKGROUND: Excessive skin exposure to solar radiation damages proteins and DNA, ultimately leading to skin ageing and cancers. OBJECTIVES: To identify new ultraviolet B (UVB) target genes to understand the mechanisms behind the detrimental effects of UVB. METHODS: Organotypic, stratified cultures of rat keratinocytes were exposed to UVB and analysed using a genome-wide expression array, quantitative real-time polymerase chain reaction and histology. The most downregulated gene, rClca2, was further characterized in rat keratinocytes and mouse skin models. RESULTS: A single, 30 mJ cm(-2) dose of broadband UVB proved effective in the organotypic epidermal culture. The expression of 627 genes was changed 24 h postirradiation. In silico analysis of the data indicated activation of DNA repair, metabolism, cell cycle control and amino acid metabolism, but only limited inflammation under these conditions. We selected for further investigation the most downregulated gene, rClca2, previously suggested to regulate keratinocyte differentiation and adhesion, and found that UVB caused a long-lasting downregulation in its expression. Both the rClca2 full-length isoform (expressed in the differentiating cells) and the truncated isoform (expressed in the basal layers) were reduced by UVB. Immunohistochemistry of mouse skin samples with isoform-specific antibodies showed a similar, epidermal differentiation-related pattern. In mouse specimens exposed to chronic ultraviolet radiation (UVR) the staining intensities were reduced and the differentiation-related isoform was disturbed in the hyperplastic and carcinomatous areas induced by UVR. CONCLUSIONS: The data show that rClca2 is a novel UVB target gene and suggest that it might play a role in epidermal differentiation and UV-dependent skin malignancies.
