Biomedical subjects

Christian Frank

Publications and source records attributed to Christian Frank.

11 recordsLinked to original sources

PubMed2005

Influence of amphiphilic block copolymers on lyotropic liquid crystals in water-oil-surfactant systems.

In ternary water-oil-nonionic alkyl polyglycol ether (C(i)E(j)) microemulsions, an increase in efficiency is always accompanied by the formation of a lamellar (L(alpha)) phase. The addition of an amphiphilic block copolymer to the ternary base system increases the efficiency of the microemulsion drastically while suppressing--at least partly--the formation of the L(alpha) phase. However, amphiphilic block copolymers can be used not only to suppress the formation of lyotropic liquid crystals but also for the opposite effect, namely, to induce their formation. To understand to what extent the increase in efficiency is accompanied by the formation of lyotropic liquid crystals, we studied phase diagrams of water-n-alkane-n-alkyl polyglycol ethers (C(i)E(j))-PEPX-PEOY at a constant volume fraction of oil in the water/oil mixture. Using polymers of the poly(ethylene propylene)-copoly(ethylene oxide) type, with M(PEP) = X kg mol(-1) and M(PEO) = Y kg mol(-1), we determined phase diagrams as a function of the polymer concentration, size, and symmetry. Moreover, the influence of a particular polymer mixture was studied, which turned out to be the best system if both a high efficiency and a low tendency to form an L(alpha) phase are needed.

Journal Article↗

PubMed2005

Spatio-temporal activation of chromatin on the human CYP24 gene promoter in the presence of 1alpha,25-Dihydroxyvitamin D3.

The vitamin D3 24-hydroxylase gene (CYP24) is one of the most strongly induced genes known. Despite this, its induction by the hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3) has been characterized only partially. Therefore, we monitored the spatio-temporal, 1alpha,25OH2D3-dependent chromatin acetylation status of the human CYP24 promoter by performing chromatin immunoprecipitation (ChIP) assays with antibodies against acetylated histone 4. This was achieved by performing PCR on 25 contiguous genomic regions spanning the first 7.7 kb of the promoter. ChIP assays using antibodies against the 1alpha,25OH2D3 receptor (VDR) revealed that, in addition to the proximal promoter, three novel regions further upstream associated with VDR. Combined in silico/in vitro screening identified in three of the four promoter regions sequences resembling known VDREs and reporter gene assays confirmed the inducibility of these regions by 1alpha,25OH2D3)=. In contrast, the fourth VDR-associated promoter region did not contain any recognizable classical VDRE that could account for the presence of the protein on this region. However, re-ChIP assays monitored on all four promoter regions simultaneous association of VDR with retinoid X receptor, coactivator, mediator and RNA polymerase II proteins. These proteins showed a promoter region-specific association pattern demonstrating the complex choreography of the CYP24 gene promoter activation over 300 minutes. Thus, this study reveals new information concerning the regulation of the CYP24 gene by 1alpha,25OH2D3, and is a demonstration of the simultaneous participation of multiple, structurally diverse response elements in promoter activation in a living cell.

Acetylation↗

PubMed2005

The human peroxisome proliferator-activated receptor delta gene is a primary target of 1alpha,25-dihydroxyvitamin D3 and its nuclear receptor.

Peroxisome proliferator-activated receptor (PPAR) delta is the most widely expressed member of the PPAR family of nuclear receptor fatty acid sensors. Real-time PCR analysis of breast and prostate cancer cell lines demonstrated that PPARdelta expression was increased 1.5 to 3.2-fold after three hours stimulation with the natural vitamin D receptor (VDR) agonist, 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). In silico analysis of the 20 kb of the human PPARdelta promoter revealed a DR3-type 1alpha,25(OH)2D3 response element approximately 350 bp upstream of the transcription start site, which was able to bind VDR-retinoid X receptor (RXR) heterodimers and mediate a 1alpha,25(OH)2D3-dependent upregulation of reporter gene activity. Chromatin immuno-precipitation assays demonstrated that a number of proteins representative for 1alpha,25(OH)2D3-mediated gene activation, such as VDR, RXR and RNA polymerase II, displayed a 1alpha,25(OH)2D3-dependent association with a region of the proximal PPARdelta promoter that contained the putative DR3-type VDRE. This was also true for other proteins that are involved in or are the subject of chromatin modification, such as the histone acetyltransferase CBP and histone 4, which displayed ligand-dependent association and acetylation, respectively. Finally, real-time PCR analysis demonstrated that 1alpha,25(OH)2D3 and the synthetic PPARdelta ligand L783483 show a cell and time-dependent interference in each other's effects on VDR mRNA expression, so that their combined application shows complex effects on the induction of VDR target genes, such as CYP24. Taken together, we conclude that PPARdelta is a primary 1alpha,25(OH)2D3-responding gene and that VDR and PPARdelta signaling pathways are interconnected at the level of cross-regulation of their respective transcription factor mRNA levels.

Cell Line, Tumor↗

PubMed2005

Identification of pregnane X receptor binding sites in the regulatory regions of genes involved in bile acid homeostasis.

The nuclear receptor pregnane X receptor (PXR) acts as a sensor for a broad variety of natural and synthetic lipophilic compounds, such as bile acids and rifampicin, and regulates the expression of proteins that are involved in the metabolism and transport of these compounds. PXR binds as a heterodimer with the retinoid X receptor (RXR) to specific DNA sites, called response elements (REs), within the promoter regions of genes it activates transcriptionally. In this study we created a position weight matrix (PWM) for PXR-RXR heterodimers that took the relative in vitro binding strength and not only the sequence of natural and synthetic PXR binding sites (PXREs) into account. We further extended the discriminatory power of the matrix by including the variation of the dinucleotides 5'-flanking the hexameric binding motifs, which we show to have a significant effect on PXR binding ability. To test this PWM, it was used to screen the promoter regions of the human organic anion transport protein 2 (OATP2) and small heterodimer partner 1 (SHP1) genes. This resulted in the identification of 17 potential PXREs, of which seven bound PXR-RXR heterodimers in vitro. Furthermore, in HepG2 human hepatoma cells, PXR and RXR occupied chromatin regions that contained four of these REs. Induction of OATP2 and SHP1 mRNA expression by rifampicin confirmed that both genes are primary human PXR responding genes. This observation increases the understanding of the physiological role of PXR in the homeostasis of bile acids in humans.

Bile Acids and Salts↗

PubMed2004

Agonist-dependent and agonist-independent transactivations of the human constitutive androstane receptor are modulated by specific amino acid pairs.

The constitutive androstane receptor (CAR) is an interesting member of the nuclear receptor superfamily because of its exceptionally high constitutive activity due to ligand-independent interaction of the ligand-binding domain with co-activator proteins. This study compares the agonist-dependent and agonist-independent activities of human CAR with those of mouse CAR and the vitamin D receptor and demonstrates that the constitutive activity of CAR is mediated by at least three contacts between the amino acids of helix 12, partner amino acids in helices 4 and 11, and a charge clamp between helices 12 and 3. The stabilization of helix 12 by a contact between its C terminus and the lysine of helix 4 has the same impact in human and mouse CARs. In addition, the charge clamp between the glutamate in helix 12 and the lysine in helix 3 is also important for the constitutive activity of both receptor orthologs but less critical for the agonist-dependent stabilization of their respective helices 12. Interestingly, Cys-357 in mouse CAR has significantly more impact on the stabilization of helix 12 than does the orthologous position Cys-347 in human CAR. This deficit appears to be compensated by a more dominant role of Ile-330 in human CAR over Leu-340 in mouse CAR because it is more efficient than Cys-347 in controlling the flexibility of helix 12 in the presence of an agonist. The constitutive activity of other members of the nuclear receptor superfamily could be explained by a homologous hydrophobic interaction between large, non-polar amino acids of helices 11 and 12.

Amino Acid Sequence↗

PubMed2004

Using chromatin immunoprecipitation to monitor 1alpha,25-dihydroxyvitamin D3-dependent chromatin activity on the human CYP24 promoter.

We applied the chromatin immunoprecipitation (ChIP) method for the analysis of 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3))-dependent chromatin activity on the human 24-hydroxylase (CYP24) promoter in MCF-7 human breast cancer cells. In this pilot study we concentrated on the proximal promoter (+22 to -424) of the CYP24 gene, which includes the known 1,25-D(3) response element (VDRE) cluster. A constitutively active region of the human histone 4a gene (-40 to +285) served for normalization. Chromatin activity snapshots were taken 0, 30, 60, 120, 180, 240 and 300 min after the onset of stimulation with 1,25-D(3) and anti-acetylated histone 4 antibodies were used for ChIP. Our results suggest that ChIP is suitable for monitoring 1,25-D(3)-dependent changes of chromatin organization and can be used to reveal information about chromatin activity in living cells.

Base Sequence↗

PubMed2004

The genes of the coactivator TIF2 and the corepressor SMRT are primary 1alpha,25(OH)2D3 targets.

The complex of the receptor for the hormone 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), Vitamin D(3) receptor (VDR), the retinoid X receptor (RXR) and a 1alpha,25(OH)(2)D(3) response element (VDRE) is considered to be the molecular switch for nuclear 1alpha,25(OH)(2)D(3) signaling. In the presence of ligand the VDR-RXR complex interacts with coactivator (CoA) proteins that in turn contact components of the basal transcriptional machinery resulting in an enhanced transcription of 1alpha,25(OH)(2)D(3) target genes. In the absence of ligand the VDR remains bound to the DNA and interacts with corepressor (CoR) proteins that are involved in gene silencing activity. We treated MCF-7 breast cancer cells with 1alpha,25(OH)(2)D(3) for increasing amounts of time, extracted mRNA and screened by real-time PCR the members of the p160 CoA and NCoR CoR families. We find that of the p160 coactivators, only TIF2 was responsive to 1alpha,25(OH)(2)D(3). Similarly SMRT but not NCoR1 gene transcription was sensitive to 1alpha,25(OH)(2)D(3) treatment. In silico analysis revealed that both TIF2 and SMRT promoters have substantial numbers of VDREs compared to the promoters of the other family members. These VDREs are formed by direct repeats of the core binding motif RGKTCA with a three nucleotide spacing (DR3). We suggest that some or all of these DR3-type VDREs are responsible for the observed responsiveness of TIF2 and SMRT to 1alpha,25(OH)(2)D(3).

Calcitriol↗

PubMed2003

Characterization of DNA complexes formed by the nuclear receptor constitutive androstane receptor.

The nuclear receptor constitutive androstane receptor (CAR) acts as a xenobiotic sensor and regulates the expression of enzymes, such as several cytochromes P450s and the UDP-glucuronosyltransferase (UGT) type 1A1. CAR binds as a heterodimer with the retinoid X receptor (RXR) to specific DNA sites, called response elements (REs). Clusters of CAR REs, referred to as phenobarbital response enhancer modules (PBREMs), have been identified in several CAR target genes. In this study we confirm that REs formed by direct repeats of two AGTTCA hexamers with 4 spacing nucleotides are optimal for the binding of CAR-RXR heterodimers. In addition, we found that the heterodimers also form complexes on everted repeat-type arrangements with 8 spacing nucleotides. We also observed that CAR is able to bind DNA as a monomer and to interact in this form with different coregulators even in the presence of RXR. Systematic variation of the nucleotides 5'-flanking to both AGTTCA hexamers showed that the dinucleotide sequence modulates the DNA complex formation of CAR monomers and CAR-RXR heterodimer by a factor of up to 20. The highest preference was found for the sequence AG and lowest for CC. The increased DNA affinity of CAR is mediated by the positively charged arginines 90 and 91 located in the carboxyl-terminal extension of the DNA-binding domain of the receptor. Furthermore, we show that one of the three CAR REs of the human UGT1A1 PBREM is exclusively bound by CAR monomers and this is regulated by ligands that bind to this nuclear receptor. This points to a physiological role for CAR monomers. Therefore, both CAR-RXR heterodimers and CAR monomers can contribute to the gene activating function of PBREMs in CAR target genes.

Amino Acid Motifs↗

PubMed2002

Differential nuclear receptor signalling from DR4-type response elements.

Nuclear receptors form a large family of highly related transcription factors that transform an incoming signal in the form of a lipophilic hormone into an activation of the basal transcriptional machinery. The specific recognition of nuclear receptor DNA binding sites, referred to as response elements (REs), determines the genes that can be regulated by nuclear hormones. In this study, it was shown that the complexes of the retinoid X receptor (RXR) with either the vitamin D3 receptor (VDR), the thyroid hormone receptor (T3R) or the liver X receptor (LXR) have comparable functionality on a RE of the rat pit-1 gene that is formed by a direct repeat of two hexameric binding motifs spaced by 4 nucleotides (DR4). The sequence of two nucleotides 5'-flanking the downstream binding motif of this DR4-type RE and, interestingly, also those flanking the upstream motif were shown to have in part rather drastic and receptor-specific effects on heterodimer complex formation on DNA. In particular, a downstream substitution into GA reduced the complex formation for LXR specifically, while upstream substitutions into AA or TA increase complex formation for LXR and, to a lesser extent, T3R. The preference of this in vitro complex formation was shown to correlate well with the functional activity of the nuclear receptors in living cells. The results of this study allow (i) a more detailed understanding of known REs, (ii) a more straightforward search for putative REs in newly identified promoter sequences, for example, of the whole human genome, and (iii) a more precise prediction of the hormone responsiveness of the respective genes.

Animals↗

PubMed2002

Toxicity assessment of cyanobacterial toxin mixtures.

A method is presented for the toxicity assessment of mixtures of cyanobacterial toxins with hepatotoxic, neurotoxic, and carcinogenic effects. The method is based on the derivation of toxicity equivalent factors obtained from toxicological data presented for cyanobacterial toxins in the literature and on the use of toxicity equivalents in analogy with the method used for polychlorinated dibenzo[p]dioxins and dibenzofurans. Because the method easily can include all toxicological data for cyanobacterial toxins at any time, the quality of the toxicity assessment of these toxins can be increased continuously depending on data availability. Generally, all toxicologically relevant cyanobacterial toxins for which data are available should be included in this assessment. The method is considered useful in the toxicity assessment of cyanobacterial toxin mixtures in surface water bodies and in drinking water. When using the total sum of the toxicity equivalents, the approach proposed here seems to lead to a more realistic toxicity assessment of cyanobacterial toxin mixtures than does the worst-case approach. However, the availability of data about acute and, especially, chronic toxicity must be increased significantly in the future in order to establish toxicologically validated exposure limit values for cyanobacterial toxins, especially those with hepatotoxic, neurotoxic, and carcinogenic effects.

Bacterial Toxins↗

PubMed2002

Modulation of mouse and human phenobarbital-responsive enhancer module by nuclear receptors.

The constitutive androstane receptor (CAR) regulates mouse and human CYP2B genes through binding to the direct repeat-4 (DR4) motifs present in the phenobarbital-responsive enhancer module (PBREM). The preference of PBREM elements for nuclear receptors and the extent of cross-talk between CAR and other nuclear receptors are currently unknown. Our transient transfection and DNA binding experiments indicate that binding to DR4 motifs does not correlate with the activation response and that mouse and human PBREM are efficiently 'insulated' from the effects of other nuclear receptors despite their substantial affinity for DR4 motifs. Certain nuclear receptors that do not bind to DR4 motifs, such as peroxisome proliferator-activated receptor-alpha and farnesoid X receptor, can suppress PBREM function via a coactivator-dependent process that may have relevance in vivo. In competition experiments, mouse PBREM is clearly more selective for CAR than human PBREM. Pregnane X, vitamin D, and thyroid hormone receptors can potentially compete with human CAR on human PBREM. In contrast to the selective nature of PBREM, CYP3A enhancers are highly and comparably responsive to CAR, pregnane X receptor, and vitamin D receptor. In addition, the ligand specificities of human and mouse CAR were defined by mammalian cotransfection and yeast two-hybrid techniques. Our results provide new mechanistic explanations to several previously unresolved aspects of CYP2B and CYP3A gene regulation.

Animals↗