AbstractExpression of the platelet-derived growth factor-A subunit (PDGF-A) is regulated to a significant degree by DNA elements located in the 5′-distal region of the gene. A potent basal enhancer (ACE66) located approximately 7 kb upstream of the transcription start site contains a number of half-sites for nuclear receptor binding, two of which are arranged in the form of direct repeat-3 motif that corresponds to a consensus vitamin D response element (VDRE). Electrophoretic mobility shift assays confirmed that the ACE66 sequence was recognized as a high affinity target for binding of heterodimers of recombinant vitamin D receptor (VDR) and its partner, retinoid-X receptor-α (RXRα). VDRE activity was localized by transient transfection analysis to a direct repeat-3 motif within the 5′-portion of the ACE66 element. Moreover, 1,25-(OH)2D3 was validated as a regulator of the endogenous PDGF-A gene by the vitamin D-stimulated upregulation of PDGF-A mRNA levels in a VDR-expressing clone of JEG-3 cells. Thus, PDGF-A represents a novel mitogenic target of 1,25-(OH)2D3 whose expression is induced via binding of hormone-activated VDR to a response element located far upstream of the transcription start site.KeywordsPDGFVitamin D receptorTranscriptionDNA elements1,25-(OH)D, 1,25-dihydroxyvitamin DACE, A-chain cell-specific enhancerDMEM, Dulbecco's modified Eagle's mediumEMSA, electrophoretic mobility shift assayOC, osteocalcinOP, osteopontinPDGF, platelet-derived growth factorPTH, parathryoid hormoneRPA, ribonuclease protection assayRXR, retinoid-X receptorVDR, vitamin D receptorVDRE, vitamin D response element
25-Hydroxyvitamin D3 (25OHD3) is used as a clinical biomarker for assessment of vitamin D status. Blood levels of 25OHD3 represent a balance between its formation rate and clearance by several oxidative and conjugative processes. In the present study, the identity of human uridine 5'-diphosphoglucuronyltransferases (UGTs) capable of catalyzing the 25OHD3 glucuronidation reaction was investigated. Two isozymes, UGT1A4 and UGT1A3, were identified as the principal catalysts of 25OHD3 glucuronidation in human liver. Three 25OHD3 monoglucuronides (25OHD3-25-glucuronide, 25OHD3-3-glucuronide, and 5,6-trans-25OHD3-25-glucuronide) were generated by recombinant UGT1A4/UGT1A3, human liver microsomes, and human hepatocytes. The kinetics of 25OHD3 glucuronide formation in all systems tested conformed to the Michaelis-Menten model. An association between the UGT1A4*3 (Leu48Val) gene polymorphism with the rates of glucuronide formation was also investigated using human liver microsomes isolated from 80 genotyped livers. A variant allele dose effect was observed: the homozygous UGT1A4*3 livers (GG) had the highest glucuronidation activity, whereas the wild type (TT) had the lowest activity. Induction of UGT1A4 and UGT1A3 gene expression was also determined in human hepatocytes treated with pregnane X receptor/constitutive androstane receptor agonists, such as rifampin, carbamazepine, and phenobarbital. Although UGT mRNA levels were increased significantly by all of the known pregnane X receptor/constitutive androstane receptor agonists tested, rifampin, the most potent of the inducers, significantly induced total 25OHD3 glucuronide formation activity in human hepatocytes measured after 2, but not 4 and 24 hours, of incubation. Finally, the presence of 25OHD3-3-glucuronide in both human plasma and bile was confirmed, suggesting that the glucuronidation pathway might be physiologically relevant and contribute to vitamin D homeostasis in humans.
The identification of a highly conserved specificity protein 1 (Sp1) DNA element in mammalian PTH promoters was recently reported. However, the presence of a novel DNA-binding complex was subsequently observed exclusively with the human PTH (hPTH) Sp1 element in mobility shift studies. Point mutations in the hPTH Sp1 element revealed the factor recognized a CAAT-like sequence resulting from a single nucleotide difference unique to the human sequence relative to other mammalian promoters. A consensus nuclear factor Y (NF-Y) element was able to specifically compete for formation of the novel complex, whereas antiserum directed against the B-subunit of NF-Y supershifted the complex without disturbing binding by the Sp3/Sp1 proteins. Moreover, immunocytochemistry confirmed the nuclear localization of NF-Y in parathyroid gland cells. Transient expression of a dominant negative form of NF-Y impaired basal hPTH promoter activity in opossum kidney cells. Studies in Drosophila SL2 cells revealed that an intact NF-Y complex was required to strongly activate transcription from the hPTH promoter, and mutational analysis confirmed the identity of the NF-Y and Sp1 DNA elements. Finally, coexpression studies in SL2 cells indicated that NF-Y and Sp1 competed for binding to their adjoining sites in the hPTH promoter. In summary, an NF-Y enhancer DNA element has been identified that is uniquely positioned in the hPTH promoter and partially overlaps with the species-conserved Sp1 element. Binding appears to be mutually exclusive by the two transcription factors to this site and suggests that separate signaling pathways may be using this DNA locus to enhance transcription of the hPTH gene.
Abstract Vitamin D response elements (VDREs) that are required for negative regulation of rat parathyroid hormone (rPTH) gene expression have been characterized. Gel mobility shift assays using DNA restriction enzyme fragments and recombinant proteins for vitamin D and retinoic acid X receptors (VDR/RXR) revealed a sequence between −793 and −779 that bound a VDR/RXR heterodimer with high affinity (VDRE1). Furthermore, a lower affinity site (VDRE2) was detected that acted in combination with VDRE1 to bind a second VDR/RXR complex. As determined by ethylation interference analysis, the nucleotide sequence of VDRE1 consisted of GGTTCA GTG AGGTAC, which is remarkably similar to the sequence of the negative VDRE found in the chicken PTH (cPTH) gene. Using the same technique, VDRE2 was identified between positions −760 and −746 and contained the sequence AGGCTA GCC AGTTCA. Functional analysis was determined by transfection studies with plasmid constructs that expressed the gene for chloramphenicol acetyl transferase (CAT). The ability of the VDREs to regulate gene expression was tested in their native context with the rPTH promoter as well as when positioned immediately upstream from the cPTH promoter. With either plasmid construct, exposure to 10−8 M 1,25(OH)2D3 resulted in a 60–70% decrease in CAT gene expression when both VDRE1 and VDRE2 were present. Examination of the individual VDREs showed that inhibition by 10−8 M 1,25(OH)2D3 was only 35–40% when just VDRE1 was present. By itself, VDRE2 was even less effective, as significant inhibition of CAT activity (20%) was observed only in the presence of higher concentrations of 1,25(OH)2D3 (10−7 M) or when a plasmid vector that overexpressed the VDR protein was cotransfected. In conclusion, the rPTH gene contains two negative VDREs that act in concert to bind two RXR/VDR heterodimer complexes and that both VDREs are required for maximal inhibition by 1,25(OH)2D3.
