Ultraviolet B suppresses vitamin D receptor gene expression in keratinocytes.

Keratinocytes not only produce vitamin D3 in response to ultraviolet B light (UVB) and convert 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D) but also possess the vitamin D receptor (VDR) and respond to 1,25(OH)2D. We characterized the regulation of the expression of the VDR gene in primary human keratinocytes following UVB irradiation. We report a marked dose-dependent down-regulation of the VDR mRNA and protein within a few hours after irradiation. This occurs independently of de novo protein synthesis and is not due to a change in the half-life of the VDR mRNA. Interestingly, treatment of the cells with sodium salicylate, caffeic acid phenethyl ester and tosylphenylchloromethylketone inhibited this down-regulation. Our results strongly suggest the existence of a feedback mechanism in that UVB initiates vitamin D synthesis in keratinocytes and at the same time limits VDR abundance. They also provide a rational explanation for the reported lack of any additive effect between 1,25(OH)2D and UVB phototherapy in the treatment of psoriasis.

Early ultraviolet B-induced G1 arrest and suppression of the malignant phenotype by wild-type p53 in human squamous cell carcinoma cells.

Wild-type p53 (wt-p53) negatively controls cell cycle progression after cellular stress mediating either a temporary growth arrest or apoptosis, depending on the cell type and nature of the cellular stress. The aberrant proliferation which is characteristic of tumor cells may be suppressed by exogenous wt-p53 and appears to depend strongly on the level of reexpression. We performed retroviral-mediated gene transfer of wt-p53 into a human squamous cell carcinoma cell line from the head and neck region (A253 cell line) lacking endogenous p53. This allowed us to study the effect of wt-p53 on the malignant phenotype and on the response to the DNA damaging agent ultraviolet B (UVB). Restoration of wt-p53 in malignant keratinocytes suppressed tumorigenicity in nude mice although p53-reconstituted cells eventually formed small tumors with long latency. Cells derived from these tumors showed reduced expression of wt-p53. Exogenous wt-p53 increased baseline mRNA expression of the small proline rich proteins 1 and 2, consistent with a prodifferentiating effect. After exposure to a biological UVB dose, only p53-positive A253 cells underwent an early and transient G1 arrest. Both p53-positive and -negative A253 cells displayed a late G2 delay/arrest. We conclude that reexpression of wt-p53 in squamous cell carcinoma A253 cells decreases their malignant phenotype and reestablishes a G1 checkpoint after UVB.

Involvement of two Ets binding sites in the transcriptional activation of the MAGE1 gene.

The MAGE1 gene codes for an antigen recognized on melanoma cell line MZ2-MEL by autologous cytolytic T lymphocytes. It is expressed in a number of tumors of different histological origins, but not in normal tissues except in testis. The MAGE1 promoter region was analyzed by performing transient transfections in MZ2-MEL cells with luciferase reporter plasmids. A fragment extending from nucleotide -792 to +118 exhibited high transcriptional activity. By deletional analysis of this fragment, we identified five activating regions designated C, A, B', B, and D. The activity of region A depends on the presence of region B' and vice versa. Two inverted Ets motifs contained in regions B' and B were found to drive 90% of the activity of the MAGE1 promoter in MZ2-MEL cells. Electrophoretic mobility shift assays performed with a nuclear extract from MZ2-MEL cells and with competitor oligonucleotides containing an Ets consensus site showed that nuclear proteins bind to the Ets motif of regions B' and B. Similar experiments suggested that region A binds transcription factors of the Sp1 family. The MAGE1 promoter was found to exert transcriptional activity in tumor cells where the MAGE1 gene is not expressed, suggesting that other mechanisms, such as demethylation, may contribute to the tumor-specific expression of the gene.

Characterization of an alternative promoter in the human growth hormone gene.

Transcription of the human growth hormone (hGH) gene depends on cis-acting elements contained within 300 base pairs of its 5'-flanking sequence. An earlier in vitro study of the transcriptional activity of this 5'-flanking region suggested that transcription can start upstream from position +1. We have investigated this phenomenon by cell-free transcription and transient transfection of chimeric constructs in cultured pituitary cells and in HeLa cells and by analysis of RNA from human pituitary glands and HeLa cells. Transcription initiation sites were identified at positions -54 and -197 by cell-free transcription assays and by RNAse mapping of human pituitary RNA. In transfection assays, the hGH gene 5'-flanking sequence upstream from position -197 displayed transcriptional activity, which critically depended on the upstream stimulatory factor-binding site located between positions -253 and -266. Transcripts initiated upstream from position +1 were detected in human pituitary RNA by polymerase chain reaction amplification and Northern blotting. These transcripts were longer than the mRNA encoding hGH. They might control initiation at position +1 or code for a novel peptide.

Sp1 can displace GHF-1 from its distal binding site and stimulate transcription from the growth hormone gene promoter.

DNase I footprinting experiments showed that binding activities of Sp1 and of GHF-1 to its distal site on the human growth hormone gene promoter are mutually exclusive. The kinetics of GHF-1 binding were indicative of positive cooperativity. The Sp1 site did not affect promoter activity in cell-free transcription. Still, Sp1 could compensate partially for the decreased stimulation of transcription seen at low GHF-1 concentrations.

Nuclear factor-I and activator protein-2 bind in a mutually exclusive way to overlapping promoter sequences and trans-activate the human growth hormone gene.

Transcription of the human growth hormone (hGH) gene and its regulation are controlled by trans-acting factors that bind to hGH gene promoter sequences. Several DNase I footprints have been described within 500 bp of this promoter, one of which (-289 to -267) has not yet been ascribed to a defined factor. By DNase I footprinting, gel mobility shift, and methylation interference assays with extracts from HeLa cells and GH-producing pituitary tumor (GC) cells, we show that this factor belongs to the NF-I family. When NF-I was competed out of the cell extracts, the trans-acting factor AP-2 bound to the same site as NF-I. AP-2 was present not only in HeLa cells, but also in GC cells albeit at a much lower concentration. Consistent with the mutually exclusive binding of NF-I and AP-2, their methylation interference patterns included four guanine residues that were crucial for binding of both NF-I and AP-2. Cell-free transcription from the hGH gene promoter showed that these two factors can transactivate this gene.

Evidence that the upstream stimulatory factor and the Sp1 transcription factor bind in vitro to the promoter of the human-growth-hormone gene.

Expression of the human-growth-hormone gene is restricted to pituitary somatotrophs. Two protein-DNA complexes that are specific to the pituitary, and two that are not, had been demonstrated in vitro on the promoter of this gene. The two pituitary-specific footprints had been ascribed to a single protein called growth hormone factor 1. We have now characterized the factors responsible for the two other footprints by means of deoxyribonuclease-I protection and gel-retardation experiments. The first footprint, located between -257 and -290 relative to the transcription initiation site, involves at least two factors present in pituitary cells. One of these factors binds between nucleotides -257 and -267, and is indistinguishable from the upstream stimulatory factor, also called major late transcription factor or upstream element factor, initially described in HeLa cells. Earlier work by others had shown that the activator protein 2 purified from HeLa cells can bind to nucleotides -263 and -290. Our experiments suggest that a factor different from activator protein 2 is involved in the protection of this region against deoxyribonuclease I. The second footprint, located between nucleotides -116 and -140, involves only one factor. This factor, present in pituitary cells, recognizes a GC box and is indistinguishable from transcription factor Sp1, previously described in HeLa cells. The human-growth-hormone gene is therefore a candidate for regulation by these factors in vivo.

Analysis of cis- and trans-acting elements in the hormone-sensitive human somatotropin gene promoter.

The expression of the human growth hormone (hGH) gene is regulated by several transcription factors. Basal level transcription factors include TATA box-binding proteins, Sp1, USF and CTF/NF-1. The hGH gene is expressed only in pituitary somatotrophs, and the pituitary-specific GHF-1/Pit-1 protein is a potent transcriptional stimulator. Glucocorticoid and thyroid hormones, insulin, and GHRH which acts via cAMP, also control hGH gene transcription via trans-acting factors some of which are the hormone receptors themselves. Three transcription initiation sites were detected when hGH gene promoter activity was studied in a cell-free system. This system enabled us to delineate the respective role of some transcription factors and to propose a model that accounts for the basal, pituitary-specific, and hormonal control of hGH gene expression.