Biomedical subjects

John H White

Publications and source records attributed to John H White.

At least 19 recordsLinked to original sources

PubMed2006

Genome-wide approaches for identification of nuclear receptor target genes.

Large-scale genomics analyses have grown by leaps and bounds with the rapid advances in high throughput DNA sequencing and synthesis techniques. Nuclear receptor signaling is ideally suited to genomics studies because receptors function as ligand-regulated gene switches. This review will survey the strengths and limitations of three major classes of high throughput techniques widely used in the nuclear receptor field to characterize ligand-dependent gene regulation: expression profiling studies (microarrays, SAGE and related techniques), chromatin immunoprecipitation followed by microarray (ChIP-on-chip), and genome-wide in silico hormone response element screens. We will discuss each technique, and how each has contributed to our understanding of nuclear receptor signaling.

Journal Article↗

PubMed2006

p19INK4D and cell death.

INK4 proteins are members of a family of cyclin-dependent kinase (CDK) inhibitors that function in G(1) to block the activity of CDKs 4 and 6. While they share clear structural similarities, numerous studies have shown that INK4 proteins differ in their expression patterns during development and in the adult, and have differing roles in tumor suppression. A recent study from our laboratory showed that expression of the gene encoding p19(INK4D) is induced by the hormonal form of vitamin D(3) and by retinoids, both of which signal through related nuclear receptor transcription factors. Although vitamin D(3) and retinoids have distinct developmental and physiological functions, both regulate the cell cycle and have been shown to have chemopreventive effects in a range of studies. Induction of p19(INK4D) expression contributed to cell cycle arrest by both ligands. However, knockdown of p19(INK4D) rendered cells sensitive to autophagic cell death, a remarkable phenotype given the hyperproliferative responses to loss of other INK4 proteins. We discuss the relevance of our studies and recent findings of others to the cell death observed in p19(INK4D)-deficient animals and to a possible role for p19(INK4D) induction in chemoprevention.

Animals↗

PubMed2006

Convergence of vitamin D and retinoic acid signalling at a common hormone response element.

Although 1,25-dihydroxyvitamin D3 (1,25D3) and retinoic acid (RA) have distinct developmental and physiological roles, both regulate the cell cycle. We provide molecular and genomic evidence that their cognate nuclear receptors regulate common genes through everted repeat TGA(C/T)TPyN8PuG(G/T)TCA (ER8) response elements. ER8 motifs were found in the promoters of several target genes of 1,25D3 and/or RA. Notably, an element was characterized in the cyclin-dependent kinase (CDK) inhibitor p19ink4d gene, and 1,25D3- or RA-induced p19INK4D) expression. P19ink4d knockdown together with depletion of p27kip1, another CDK inhibitor regulated by 1,25D3 and RA, rendered cells resistant to ligand-induced growth arrest. Remarkably, p19INK4D-deficient cells showed increased autophagic cell death, which was markedly enhanced by 1,25D3, but not RA, and attenuated by loss of p27KIP1. These results show a limited crosstalk between 1,25D3 and RA signalling by means of overlapping nuclear receptor DNA binding specificities, and uncover a role for p19INK4D in control of cell survival.

Autophagy↗

PubMed2005

Opposite effects of histone deacetylase inhibitors on glucocorticoid and estrogen signaling in human endometrial Ishikawa cells.

Histone deacetylase inhibitors (HDACi), which have emerged as a new class of anticancer agents, act by modulating expression of genes controlling apoptosis or cell proliferation. Here, we compared the effect of HDACi on transcriptional activation by estrogen or glucocorticoid receptors (ER and GR, respectively), two members of the steroid receptor family with cell growth regulatory properties. Like other transcription factors, steroid receptors modulate histone acetylation on target promoters. Using episomal reporter vectors containing minimal promoters to avoid promoter-specific effects, we observed that long-term (24-h) incubation with HDACi strongly stimulated GR-dependent but markedly repressed ER-dependent signaling in ER+/GR+ human endometrial carcinoma Ishikawa cells. These effects were reproduced on endogenous target genes and required incubation periods with HDACi substantially longer than necessary to increase global histone acetylation. Repression of estrogen signaling was due to direct inhibition of transcription from multiple ERalpha promoters and correlated with decreased histone acetylation of these promoters. In contrast, the strong HDACi stimulation of GR-dependent gene regulation was not accounted for by increased GR expression, but it was mimicked by overexpression of the histone acetyltransferase complex component transcriptional intermediary factor 2. Together, our results demonstrate striking and opposite effects of HDACi on ER and GR signaling that involve regulatory events independent of histone hyperacetylation on receptor target promoters.

Acetylation↗

PubMed2005

Large-scale in silico and microarray-based identification of direct 1,25-dihydroxyvitamin D3 target genes.

1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] regulates calcium homeostasis and controls cellular differentiation and proliferation. The vitamin D receptor (VDR) is a ligand-regulated transcription factor that recognizes cognate vitamin D response elements (VDREs) formed by direct or everted repeats of PuG(G/T)TCA motifs separated by 3 or 6 bp (DR3 or ER6). Here, we have identified direct 1,25(OH)2D3 target genes by combining 35,000+ gene microarrays and genome-wide screens for consensus DR3 and ER6 elements, and DR3 elements containing single nucleotide substitutions. We find that the effect of a nucleotide substitution on VDR binding in vitro does not predict VDRE function in vivo, because substitutions that disrupted binding in vitro were found in several functional elements. Hu133A microarray analyses, performed with RNA from human SCC25 cells treated with 1,25(OH)2D3 and protein synthesis inhibitor cycloheximide, identified more than 900 regulated genes. VDREs lying within -10 to +5 kb of 5'-ends were assigned to 65% of these genes, and VDR binding was confirmed to several elements in vivo. A screen of the mouse genome identified more than 3000 conserved VDREs, and 158 human genes containing conserved elements were 1,25(OH2)D3-regulated on Hu133A microarrays. These experiments also revealed 16 VDREs in 11 of 12 genes induced more than 10-fold in our previous microarray study, five elements in the human gene encoding the epithelial calcium channel TRPV6, as well as novel 1,25(OH2)D3 target genes implicated in regulation of cell cycle progression. The combined approaches used here thus provide numerous insights into the direct target genes underlying the broad physiological actions of 1,25(OH)2D3.

Animals↗

PubMed2005

Analysis of the domain properties of the novel cytochrome P450 RhF.

The properties of the heme, flavin mononucleotide (FMN) and FeS domains of P450 RhF, from Rhodococcus sp. NCIMB 9784, expressed separately and in combination are analysed. The nucleotide preference, imidazole binding and reduction potentials of the heme and FMN domains are unaltered by their separation. The intact enzyme is monomeric and the flavin is confirmed to be FMN. The two one-electron reduction potentials of the FMN are -240 and -270 mV. The spectroscopic and thermodynamic properties of the FeS domain, masked in the intact enzyme, are revealed for the first time, confirming it as a 2Fe-2S ferredoxin with a reduction potential of -214 mV.

Base Sequence↗

PubMed2005

Histological observations on the microenvironment of osteolytic bone metastasis by breast carcinoma cell line.

Bone tissue, with its dynamic microenvironment featuring osteoclastic bone resorption, angiogenesis and matrix degradation, appears to facilitate proliferation of tumor cells after the onset of bone metastasis. In this study, we examined metastatic lesions in the femora of BALB/c nu/nu mice two weeks after intracardiac injection with human breast carcinoma MDA-231 cells. Histopathological observations showed the metastatic lesions close to the chondro-osseous junction, and revealed MDA-231 cells loosely intermingled with different cell types such as osteoblasts, fibroblastic stromal cells, osteoclasts and endothelial cells. In the metastatic nest, many tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts accumulated in direct contact with or were close to alkaline phosphatase (ALPase)- or receptor activator of NF-kappaB ligand (RANKL)-positive osteoblastic cells. It seems likely that osteoclastogenesis is mediated through cell-to-cell contacts with ALPase- and RANKL-expressing osteoblastic cells. Formation of many capillaries lacking complete basal membranes and pericytes ratified the results of in situ hybridization, which revealed intense expression of VEGF in tumor nests, and therefore, indicated ongoing tumor-induced angiogenesis. The tumor cells possessed matrix metallo-proteinases (MMPs)-1 and -9, and frequently extended their stout cytoplasmic processes into fragmented fibrillar components of the growth plate cartilage, implicating degradation of cartilaginous matrix. Thus, osteolytic bone metastasis has demonstrated pathological features as tumor-induced angiogenesis and degradation of extracellular matrix, in addition to osteoclastogenesis. This complex interplay between tumor cells and host tissues may enable and nourish the establishment of a microenvironment that facilitates tumor progression.

Animals↗

PubMed2004

Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.

The hormonal form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is an immune system modulator and induces expression of the TLR coreceptor CD14. 1,25(OH)(2)D(3) signals through the vitamin D receptor, a ligand-stimulated transcription factor that recognizes specific DNA sequences called vitamin D response elements. In this study, we show that 1,25(OH)(2)D(3) is a direct regulator of antimicrobial innate immune responses. The promoters of the human cathelicidin antimicrobial peptide (camp) and defensin beta2 (defB2) genes contain consensus vitamin D response elements that mediate 1,25(OH)(2)D(3)-dependent gene expression. 1,25(OH)(2)D(3) induces antimicrobial peptide gene expression in isolated human keratinocytes, monocytes and neutrophils, and human cell lines, and 1,25(OH)(2)D(3) along with LPS synergistically induce camp expression in neutrophils. Moreover, 1,25(OH)(2)D(3) induces corresponding increases in antimicrobial proteins and secretion of antimicrobial activity against pathogens including Pseudomonas aeruginosa. 1,25(OH)(2)D(3) thus directly regulates antimicrobial peptide gene expression, revealing the potential of its analogues in treatment of opportunistic infections.

Adjuvants, Immunologic↗

PubMed2004

Genome-wide identification of high-affinity estrogen response elements in human and mouse.

Although estrogen receptors (ERs) recognize 15-bp palindromic estrogen response elements (EREs) with maximal affinity in vitro, few near-consensus sequences have been characterized in estrogen target genes. Here we report the design of a genome-wide screen for high-affinity EREs and the identification of approximately 70000 motifs in the human and mouse genomes. EREs are enriched in regions proximal to the transcriptional start sites, and approximately 1% of elements appear conserved in the flanking regions (-10 kb to +5 kb) of orthologous human and mouse genes. Conserved and nonconserved elements were also found, often in multiple occurrences, in more than 230 estrogen-stimulated human genes previously identified from expression studies. In genes containing known EREs, we also identified additional distal elements, sometimes with higher in vitro binding affinity and/or better conservation between the species considered. Chromatin immunoprecipitation experiments in breast cancer cell lines indicate that most novel elements present in responsive genes bind ERalpha in vivo, including some EREs located up to approximately 10 kb from transcriptional start sites. Our results demonstrate that near-consensus EREs occur frequently in both genomes and that whereas chromatin structure likely modulates access to binding sites, far upstream elements can be evolutionarily conserved and bind ERs in vivo.

Algorithms↗

PubMed2004

The pleiotropic actions of vitamin D.

General knowledge of the role of vitamin D3 in human physiology has been shaped by its discovery as a preventive agent of nutritional rickets, a defect in bone development due to inadequate uptake of dietary calcium. Studies on the function of the hormonal form of vitamin D3, 1alpha,25-dihydroxyvitamin D3, have been greatly accelerated by the molecular cloning and structural analysis of the vitamin D3 receptor, which is a ligand-activated regulator of gene transcription. Molecular genetic techniques including genomics have helped reveal that 1alpha,25-dihydroxyvitamin D3 can control more than calcium homeostasis. It has widespread effects on cellular differentiation and proliferation, and can modulate immune responsiveness, and central nervous system function. Moreover, accumulating epidemiological and molecular evidence suggests that 1alpha,25-dihydroxyvitamin D3 acts as a chemopreventive agent against several malignancies including cancers of the prostate and colon. Here, we survey the most-recent findings and discuss their implications for the potential therapeutic uses of vitamin D analogues.

Animals↗

PubMed2004

Profiling 1,25-dihydroxyvitamin D3-regulated gene expression by microarray analysis.

Vitamin D was discovered as a preventive agent of nutritional rickets, a defect in bone development due to inadequate uptake of dietary calcium. However, a variety of studies over the last several years has revealed that Vitamin D controls much more than calcium homeostasis. In particular, there is widespread evidence that the hormonal form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is an important regulator of cellular differentiation and proliferation. Direct genomic signaling by 1,25(OH)(2)D(3) occurs through the Vitamin D receptor, which is a nuclear receptor and a ligand-activated regulator of gene transcription. 1,25(OH)(2)D(3) can therefore directly regulate patterns of gene expression within a target cell. The development of high throughput genomics technologies have greatly enhanced our capacity to identify the genetic and biochemical changes associated with the physiological actions of 1,25(OH)(2)D(3). Microarray analyses of expression profiles in 1,25(OH)(2)D(3)-treated cells have underlined its widespread effects on cellular differentiation and proliferation. They have provided a molecular basis for the accumulating epidemiological and preclinical evidence indicating that 1,25(OH)(2)D(3) can act as a chemopreventive agent against several malignancies including cancers of the prostate and colon. In addition, they have underlined the therapeutic potential of 1,25(OH)(2)D(3) analogues as modulators of immune system function.

Calcitriol↗

PubMed2004

Corepressor recruitment by agonist-bound nuclear receptors.

Members of the nuclear receptor superfamily are ligand-regulated transcription factors that are composed of a series of conserved domains. These receptors are targets of a wide range of lipophilic signaling molecules that modulate many aspects of physiology and metabolism. Binding of cognate ligands to receptors induces a conformational change in the ligand binding domain (LBD) that creates a pocket for recruitment of coregulatory proteins, which are essential for ligand-dependent regulation of transcription. Several coregulatory proteins that interact with hormone-bound receptors contain characteristic helical LXXLL motifs, known as nuclear receptor (NR) boxes. Generally, ligand binding to receptors is associated with activation of transcription, and most of the NR box-containing proteins characterized to date are coactivators. However, a full understanding of the function of hormone-bound receptors must also incorporate their recruitment of corepressors. The recent identification of ligand-dependent corepressor (LCoR) is a case in point. LCoR contains a single NR box that mediates its hormone-dependent interaction with several nuclear receptors. It functions as a molecular scaffold that recruits several proteins that function in transcriptional repression. Remarkably, although the two proteins share only very limited homology, LCoR and another NR box-containing corepressor RIP140 recruit similar cofactors implicated in transcriptional repression, suggesting many parallels in their mechanisms of action. Corepressors such as LCoR and RIP140 may function in negative feedback loops to attenuate hormone-induced transactivation, act more transiently as part of a cycle of cofactors recruited to target promoters by ligand-bound receptors, or function in hormone-induced target gene repression.

Adaptor Proteins, Signal Transducing↗

PubMed2003

A self-sufficient cytochrome p450 with a primary structural organization that includes a flavin domain and a [2Fe-2S] redox center.

p450 RhF from Rhodococcus sp. NCIMB 9784 is the first example of a new class of cytochrome p450 in which electrons are supplied by a novel, FMN- and Fe/S-containing, reductase partner in a fused arrangement. We have previously cloned the gene encoding the enzyme and shown it to comprise an N-terminal p450 domain fused to a reductase domain that displays similarity to the phthalate family of oxygenase reductase proteins. A reductase of this type had never previously been reported to interact with a cytochrome p450. In this report we describe the purification and partial characterization of p450 RhF. We show that the enzyme is self-sufficient in catalyzing the O-dealkylation of 7-ethoxycoumarin. The p450 RhF catalyzed O-dealkylation of 7-ethoxycoumarin is inhibited by several compounds that are known inhibitors of cytochrome p450. Presteady state kinetic analysis indicates that p450 RhF shows a 500-fold preference for NAPDH over NADH in terms of Kd value (6.6 microm versus 3.7 mm, respectively). Potentiometric studies show reduction potentials of -243 mV for the two-electron reduction of the FMN and -423 mV for the heme (in the absence of substrate).

Base Sequence↗

PubMed2003

Ligand-dependent nuclear receptor corepressor LCoR functions by histone deacetylase-dependent and -independent mechanisms.

LCoR (ligand-dependent corepressor) is a transcriptional corepressor widely expressed in fetal and adult tissues that is recruited to agonist-bound nuclear receptors through a single LXXLL motif. LCoR binding to estrogen receptor alpha depends in part on residues in the coactivator binding pocket distinct from those bound by TIF-2. Repression by LCoR is abolished by histone deacetylase inhibitor trichostatin A in a receptor-dependent fashion, indicating HDAC-dependent and -independent modes of action. LCoR binds directly to specific HDACs in vitro and in vivo. Moreover, LCoR functions by recruiting C-terminal binding protein corepressors through two consensus binding motifs and colocalizes with CtBPs in the nucleus. LCoR represents a class of corepressor that attenuates agonist-activated nuclear receptor signaling by multiple mechanisms.

Adult↗

PubMed2003

Inhibition of F-Box protein p45(SKP2) expression and stabilization of cyclin-dependent kinase inhibitor p27(KIP1) in vitamin D analog-treated cancer cells.

Treatment of cancer cells with 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D(3)] or its analogs induces growth arrest and expression of the cyclin-dependent kinase inhibitor p27(KIP1). Although 1,25(OH)(2)D(3) transiently enhances p27(kip1) gene transcription in some cells, its effects on p27(KIP1) protein levels are generally more gradual and sustained. This suggests that 1,25(OH)(2)D(3) treatment may be stabilizing p27(KIP1) protein, which is sensitive to modification by the SCF(SKP2) protein ubiquitin ligase and proteosomal degradation. Here, we show that treatment of AT-84 head and neck squamous carcinoma cells with the 1,25(OH)(2)D(3) analog EB1089 increases p27(KIP1) protein levels without significantly affecting expression of its mRNA. EB1089 treatment repressed expression of mRNAs encoding the F-box protein p45(SKP2), a marker of poor head and neck cancer prognosis, and the cyclin kinase subunit CKS1, which is essential for targeting p45(SKP2) to p27(KIP1). This coincided with a reduction of total p45(SKP2) protein, and p45(SKP2) associated with p27(KIP1). Consistent with these findings, turnover of p27(KIP1) protein was strongly inhibited in the presence of EB1089. A similar reduction in p45(SKP2) expression and stabilization of p27(KIP1) protein was observed in 1,25(OH)(2)D(3)-sensitive UF-1 promyelocytic leukemia cells, which also respond by transiently increasing p27(kip1) gene transcription. Our results reveal that 1,25(OH)(2)D(3) analogs increase levels of p27(KIP1) in different cell types by inhibiting expression of SCF(SKP2) subunits and reducing turnover of p27(KIP1) protein.

Animals↗

PubMed2002

Diversity in the mechanisms of gene regulation by estrogen receptors.

The sequencing of the human genome has opened the way for using bioinformatics to identify sets of genes controlled by specific regulatory signals. Here, we review the unexpected diversity of DNA response elements mediating transcriptional regulation by estrogen receptors (ERs), which control the broad physiological effects of estrogens. Consensus palindromic estrogen response elements are found in only a few known estrogen target genes, whereas most responsive genes contain only low-affinity half palindromes, which may also control regulation by other nuclear receptors. ERs can also regulate gene expression in the absence of direct interaction with DNA, via protein-protein interactions with other transcription factors or by modulating the activity of upstream signaling components, thereby significantly expanding the repertoire of estrogen-responsive genes. These diverse mechanisms of action must be taken into account in screening for potential estrogen-responsive sequences in the genome or in regulatory regions of target genes identified by expression profiling.

Amino Acid Sequence↗

PubMed2002

Association between AAAG repeat polymorphism in the P3 promoter of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene and adult height, urinary pyridinoline excretion, and promoter activity.

The PTH/PTHrP receptor (PTHR1) plays an essential role in skeletal development and mediates many other functions of PTH and PTHrP. Human PTHR1 gene transcription is controlled by three promoters, P1-P3. The most proximal promoter, P3, is active in bone and osteoblast-like cell lines and accounts for the majority of renal transcripts in adults. We have identified a tetranucleotide repeat (AAAG)n polymorphism in the P3 promoter. In 214 unrelated Japanese, the repeat number (n) ranged from 3-8, with the AAAG5 allele being the most frequent (59%). In 55 unrelated Caucasians, n ranged from 5-7, and the frequency of the AAAG5 allele was 78%. The most frequent genotypes in a cohort of 85 young (18-20 yr) female Japanese were 5/5, 5/6, and 6/6. The 6/6 genotype was associated with greater height (5/5 vs. 6/6; P < 0.02) and lower urinary deoxypyridinoline and pyridinoline (P < 0.02), which are markers of bone resorption. The height of an additional 71 healthy female Japanese subjects, aged 14-17 yr, having genotype 5/5, 5/6, or 6/6 was also in the order of genotype 5/5 < 5/6 < 6/6 (5/5 vs. 6/6, P < 0.05). There was no significant difference in lumbar and femoral bone mineral density between genotypes. Likewise, there was no difference in circulating intact PTH levels between groups. The activity of P3 promoter-luciferase reporter constructs in transcription assays in 2 human osteoblast-like cell-lines varied according to repeat number, with AAAG6 being the least active. In conclusion, the P3 promoter (AAAG)n polymorphism is frequent in both Japanese and Caucasians and has potential as a linkage marker for the PTHR1 locus. In addition, it may influence the expression of the receptor in target tissues and have functional consequences on the developing skeleton.

Adult↗

PubMed2002

Expression profiling in squamous carcinoma cells reveals pleiotropic effects of vitamin D3 analog EB1089 signaling on cell proliferation, differentiation, and immune system regulation.

The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is key mediator of calcium homeostasis and is a component of the complex homeostatic system of the skin. 1,25-(OH)2D3 regulates cellular differentiation and proliferation and has broad potential as an anticancer agent. Oligonucleotide microarrays were used to assess profiles of target gene regulation at several points over a 48 h period by the low calcemic 1,25-(OH)2D3 analog EB1089 in human SCC25 head and neck squamous carcinoma cells. One hundred fifty-two targets were identified, composed of 89 up- and 63 down-regulated genes distributed in multiple profiles of regulation. Results are consistent with EB1089 driving SCC25 cells toward a less malignant phenotype, similar to that of basal keratinocytes. Targets identified control inter- and intra-cellular signaling, G protein-coupled receptor function, intracellular redox balance, cell adhesion, and extracellular matrix composition, cell cycle progression, steroid metabolism, and more than 20 genes modulating immune system function. The data indicate that EB1089 performs three key functions of a cancer chemoprevention agent; it is antiproliferative, it induces cellular differentiation, and has potential genoprotective effects. While no evidence was found for gene-specific differences in efficacy of 1,25-(OH)2D3 and EB1089, gene regulation by 1,25-(OH)2D3 was generally more transient. Treatment of cells with 1,25-(OH)2D3 and the cytochrome P450 inhibitor ketoconazole produced profiles of regulation essentially identical to those observed with EB1089 alone, indicating that the more sustained regulation by EB1089 was due to its resistance to inactivation by induced 24-hydroxylase activity. This suggests that differences in action of the two compounds arise more from their relative sensitivities to metabolism than from differing effects on VDR function.

Biomarkers, Tumor↗