Basal and parathyroid hormone induced expression of the human 25-hydroxyvitamin D 1α-hydroxylase gene promoter in kidney AOK-B50 cells: role of Sp1, Ets and CCAAT box protein binding sites

Gao, Xiu Hui; Dwivedi, Prem P.; Choe, S; Alba, Frances; Morris, Howard A.; Omdahl, John L.; May, Brian K.

doi:10.1016/s1357-2725(01)00165-0

Cited by 44 publications

(40 citation statements)

References 31 publications

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“…CYP27B1 5¢-flanking-luciferase constructs and expression plasmids 5k-deletion constructs of the CYP27B1 1501 bp 5k-flanking region fused to the firefly luciferase reporter gene in the pGL3 vector have been previously described and are designated as pCYP27B1(-1501)-Luc, pCYP27B1(-997)-Luc, pCYP27B1(-884)-Luc, pCYP27B1(-531)-Luc and pCYP27B1(-305)-Luc (Gao et al 2002). Further 5k-deletions of the 5k-flanking sequence (together with 44 bp of CYP27B1-untranslated region) were generated by the polymerase chain reaction (PCR) during this study and named pCYP27B1(-1306)-Luc, pCYP27B1(-1200)-Luc and pCYP27B1(-1100)-Luc.…”

Section: Methodsmentioning

confidence: 99%

“…Control elements present in the -305 bp proximal promoter have been previously identified by Gao et al (2002) and are shown in Figure 3. A CCAAT box (5k-ATTGGCT-3k) at -75 to -70 and a GC rich sequence that is an Sp1 binding site (5k-CCAGCCCCG-3k) at -133 to -125, underlie basal expression in transfected kidney cells (Gao et al 2002), with no significant contribution from a putative Ets protein binding site (5k-CTGTTCCTGG-3k), designated as EBS at -120 to -111 on the antisense strand and located between the CCAAT box and GC rich sequence.…”

Section: Characterization Of the Proximal Promotermentioning

confidence: 96%

“…Mutations in the CAAT box, Sp1 binding site and EBS located in the -305 bp region of the CYP27B1 gene have been described earlier (Gao et al 2002). The GFI1 over-expression clone, pcDNA3-Myc-GFI1, has been described previously (Grimes et al 1996 a).…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…Cells were cultured overnight and harvested in 50 ml Passive Lysis Buffer (Promega). Luciferase activity in cell lysates was determined as described previously (Gao et al 2002) using the Dual Luciferase Reporter assay kit (Promega) and measured using a Luminometer model TD 20/20 (Turner Design Instruments, Sunnyvale, CA, USA). All experiments reported here were repeated on at least three separate occasions and the data shown are from one representative experiment.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…A CCAAT box (5k-ATTGGCT-3k) at -75 to -70 and a GC rich sequence that is an Sp1 binding site (5k-CCAGCCCCG-3k) at -133 to -125, underlie basal expression in transfected kidney cells (Gao et al 2002), with no significant contribution from a putative Ets protein binding site (5k-CTGTTCCTGG-3k), designated as EBS at -120 to -111 on the antisense strand and located between the CCAAT box and GC rich sequence. An analysis of promoter mutant constructs in DU145 cells established that the CCAAT box binding site and the GC box are critically important for expression driven by the pCYP27B1(-305)-Luc construct in DU145 cells (Fig.…”

Section: Characterization Of the Proximal Promotermentioning

confidence: 99%

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Identification of growth factor independent-1 (GFI1) as a repressor of 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) gene expression in human prostate cancer cells

Dwivedi

Anderson²,

Omdahl³

et al. 2005

Endocrine Related Cancer

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The hormone 1,25-dihydroxyvitamin D (1,25D) may play a protective role in prostate cancer. 25-hydroxyvitamin D 1-a hydroxylase (CYP27B1) is the enzyme responsible for the regulation of cellular 1,25D levels. CYP27B1 is substantially repressed in prostate cancer cells. We have investigated the molecular basis for this inhibition. First, we identify a repressive region between -997 and -1200 in the human CYP27B1 promoter following transient transfection analysis in the prostate cancer cell lines DU145, PC3 and LNCaP. Next, we demonstrate a role for the transcription factor growth factor independent-1 (GFI1) in the repression of CYP27B1. Electrophoretic mobility assays with nuclear extracts from prostate cancer cell lines established binding of GFI1 to the sequence 5k-TGGTACAATCATAACTCACTGCAG-3k present at -997 to -1200 in the repressive region. Site directed mutagenesis of the core GFI1 binding sequence (5k-AATC-3k) substantially increased while forced expression of GFI1 decreased the expression of the CYP27B1 reporter construct. Importantly, GFI1 repression is dependent on an intact GFI1 binding site in the -997 to -1200 region. GFI1 is an oncoprotein known to form a large protein complex with co-repressors that recruit histone deacetylases. We propose that the formation of such a repressive complex on the inhibitory domain of the CYP27B1 gene in prostate cancer cells could lead to silencing of either the nearby enhancer or proximal promoter domains and lead to cancer progression by reducing local production of 1,25D. These studies provide the basis for a more detailed understanding of CYP27B1 repression in prostate cancer cells and could provide a novel insight in future diagnosis and treatment.

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Section: Methodsmentioning

confidence: 99%

Section: Characterization Of the Proximal Promotermentioning

confidence: 96%

Section: Site-directed Mutagenesismentioning

confidence: 99%

Section: Site-directed Mutagenesismentioning

confidence: 99%

Section: Characterization Of the Proximal Promotermentioning

confidence: 99%

See 3 more Smart Citations

Identification of growth factor independent-1 (GFI1) as a repressor of 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) gene expression in human prostate cancer cells

Dwivedi

Anderson²,

Omdahl³

et al. 2005

Endocrine Related Cancer

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PTH and Vitamin D

Khundmiri

Murray

Lederer

2016

Comprehensive Physiology

224

158

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PTH and Vitamin D are two major regulators of mineral metabolism. They play critical roles in the maintenance of calcium and phosphate homeostasis as well as the development and maintenance of bone health. PTH and Vitamin D form a tightly controlled feedback cycle, PTH being a major stimulator of vitamin D synthesis in the kidney while vitamin D exerts negative feedback on PTH secretion. The major function of PTH and major physiologic regulator is circulating ionized calcium. The effects of PTH on gut, kidney, and bone serve to maintain serum calcium within a tight range. PTH has a reciprocal effect on phosphate metabolism. In contrast, vitamin D has a stimulatory effect on both calcium and phosphate homeostasis, playing a key role in providing adequate mineral for normal bone formation. Both hormones act in concert with the more recently discovered FGF23 and klotho, hormones involved predominantly in phosphate metabolism, which also participate in this closely knit feedback circuit. Of great interest are recent studies demonstrating effects of both PTH and vitamin D on the cardiovascular system. Hyperparathyroidism and vitamin D deficiency have been implicated in a variety of cardiovascular disorders including hypertension, atherosclerosis, vascular calcification, and kidney failure. Both hormones have direct effects on the endothelium, heart, and other vascular structures. How these effects of PTH and vitamin D interface with the regulation of bone formation are the subject of intense investigation.

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Expression, structure‐function, and molecular modeling of vitamin D P450s

Omdahl

Bobrovnikova

Annalora

et al. 2003

J of Cellular Biochemistry

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Although vitamin D(3) is a natural product of a sunlight-mediated process in the skin, the secosteroid's biological function is dependent upon specific cytochrome P450 enzymes that mediate the parent vitamin's bioactivation and inactivation. Cytochrome P450C1 (CYP27B1) is the regulatory rate-limiting enzyme that directs the bioactivation process through introduction of a C-1alpha hydroxyl group. The resultant 1,25-dihydroxyvitamin D(3) (1,25D) is the biologically active secosteroid hormone that directs the multitude of vitamin D-dependent actions involved with calcium homeostasis, cellular differentiation and growth, and the immune response. The circulating and cellular level of 1,25D is regulated through a coordinated process involving the hormone's synthesis and degradation. Central to the degradation and turnover of 1,25D is the regulatory multi-catalytic cytochrome P450C24 (CYP24) enzyme that directs the introduction of C-24R groups onto targeted 25-hydroxy substrates. Discussed in this article is the action of the rat CYP24 to catalyze the side-chain oxidation and cleavage of 25-hydroxylated vitamin D metabolites. Expression and characterization of purified recombinant rat CYP24 is discussed in light of mutations directed at the enzyme's active site.

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Basal and parathyroid hormone induced expression of the human 25-hydroxyvitamin D 1α-hydroxylase gene promoter in kidney AOK-B50 cells: role of Sp1, Ets and CCAAT box protein binding sites

Cited by 44 publications

References 31 publications

Identification of growth factor independent-1 (GFI1) as a repressor of 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) gene expression in human prostate cancer cells

Identification of growth factor independent-1 (GFI1) as a repressor of 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) gene expression in human prostate cancer cells

PTH and Vitamin D

Expression, structure‐function, and molecular modeling of vitamin D P450s

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