Induction of (6-4) photolyase gene transcription by blue light in Xenopus A6 cells.

(6-4) photolyase repairs pyrimidine-pyrimidone (6-4) photoproducts generated in DNA upon UV light exposure. We studied the effects of blue light on the expression of this gene in Xenopus A6 cells. Exposure of the cells to blue light, but not red light, for 12h resulted in more than 20-fold increase of the (6-4) photolyase mRNA. By contrast, levels of the other two photolyase mRNAs, i.e., those for CPD photolyase and cryptochrome DASH, did not change significantly. Oxygen radicals presumably generated within the cells upon exposure to blue light were not the cause of the induction, since addition of neither hydrogen peroxide nor a photosensitizer, phenol red, in the culture medium increased the (6-4) photolyase mRNA level. These results support the possibility that the expression of (6-4) photolyase may be regulated by a mechanism involving an as yet ill-defined blue light photoreceptor in the peripheral tissues of Xenopus.

Role of distal upstream sequence in vitamin D-induced expression of human CYP24 gene.

The level of CYP24 mRNA in cultured human fibroblasts increases up to 20,000-fold in response to 1,25-dihydroxyvitamin D(3). Two vitamin D-responsive elements (VDREs) located immediately upstream of the CYP24 gene are primarily responsible for the induction. We studied roles of other regions in the 5'-flanking sequence of this gene. A series of deletion constructs between nucleotides -1918 and +209 of the gene were examined for their promoter activities employing the luciferase reporter assay. We found that the VDREs were not sufficient to account for the extent of induction. The sequence between nucleotides -548 and -294, which is located immediately upstream of the VDREs and includes three potential Sp1 sites, acted synergistically with the VDREs for the induction. Further upstream sequence and the 5'-untranslated region did not appear to play a major role in the vitamin D response.

Purification, cDNA cloning, and expression profiles of the cyclobutane pyrimidine dimer photolyase of Xenopus laevis.

Photolyase is a light-dependent enzyme that repairs pyrimidine dimers in DNA. Two types of photolyases have been found in frog Xenopus laevis, one for repairing cyclobutane pyrimidine dimers (CPD photolyase) and the other for pyrimidine-pyrimidone (6-4)photoproduct [(6-4)photolyase]. However, little is known about the former type of the Xenopus photolyases. To characterize this enzyme and its expression profiles, we isolated the entire coding region of a putative CPD photolyase cDNA by extending an EST (expressed sequence tag) sequence obtained from the Xenopus database. Nucleotide sequence analysis of the cDNA revealed a protein of 557 amino acids with close similarity to CPD photolyase of rat kangaroo. The identity of this cDNA was further established by the molecular mass (65 kDa) and the partial amino acid sequences of the major CPD photolyase that we purified from Xenopus ovaries. The gene of this enzyme is expressed in various tissues of Xenopus. Even internal organs like heart express relatively high levels of mRNA. A much smaller amount was found in skin, although UV damage is thought to occur most frequently in this tissue. Such expression profiles suggest that CPD photolyase may have roles in addition to the photorepair function.

Characterization of vitamin D-mediated induction of the CYP 24 transcription.

Transcription of the CYP24 (25-hydroxyvitamin D(3)-24-hydroxylase) gene is known to be induced by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). We studied the induction kinetics in detail in human skin-derived fibroblasts. While the basal transcription of this gene was very low, addition of 1,25(OH)2D3 increased the mRNA level by 50-fold within 1h. The induction reached as high as 20000-fold after 12h. DNA microarray analysis also indicated that the induction ratio of the CYP24 gene is exceptionally high among 3800 human genes examined. The increase of mRNA was caused by stimulation of the transcription, but not by stabilization of mRNA. 24(R),25-dihydroxyvitamin D3 (24,25(OH)2D3), a compound metabolically related to 1,25(OH)2D3, also stimulated the CYP24 gene transcription, though at much higher concentrations. However, this stimulation was significantly augmented by synergistic actions of 24,25(OH)2D3 and 1,25(OH)2D3, suggesting that 24,25(OH)2D3 or its metabolites might be playing some roles in the regulation of CYP24 gene transcription.

Rapid and preferential induction of ATF3 transcription in response to low doses of UVA light.

Long-wavelength UV light (UVA) is known to induce transcription of various genes in the cell and to cause a variety of pathological or protective responses in the skin. To find additional UVA-responsive genes, human skin-derived fibroblasts were exposed to UVA under non- or partially lethal conditions, and the effects of UVA on the transcriptional profile were examined by using DNA microarray and RT-PCR. Transcription of several genes including those already known to be UVA-responsive was induced to a significant extent under 50% lethal conditions of exposure. Among those, ATF3 was the most sensitive and its transcription was increased 10-fold within 1h. Even at a non-lethal dose of UVA (8J/cm(2)), it was increased 8-fold, if cells were cultured for 3h post-exposure. Typical immediate-early genes such as c-fos and c-jun were not affected at this dose. We thus suggest that ATF3 could be a key regulator for a variety of cellular responses in the skin, particularly to low doses of UVA.