The organic osmolyte betaine induces keratin 2 expression in rat epidermal keratinocytes - A genome-wide study in UVB irradiated organotypic 3D cultures.

The moisturizing and potentially protective properties of the organic osmolyte betaine (trimethylglycine) have made it an attractive component for skin care products. Its wide use despite the lack of comprehensive studies addressing its specific effects in skin led us to characterize the molecular targets of betaine in keratinocytes and to explore, whether it modifies the effects of acute UVB exposure. Genome-wide expression analysis was performed on organotypic cultures of rat epidermal keratinocytes, treated either with betaine (10mM), UVB (30 mJ/cm(2)) or their combination. Results were verified with qRT-PCR, western blotting and immunohistochemistry. Additionally, cell proliferation and differentiation were analyzed. Among the 89 genes influenced by betaine, the differentiation marker keratin 2 showed the highest upregulation, which was also confirmed at protein level. Expression of Egr1, a transcription factor, and Purkinje cell protein 4, a regulator of Ca(2+)/calmodulin metabolism, also increased, while downregulated genes included several ion-channel components, such as Fxyd2. Bioinformatics analyses suggest that genes modulated by betaine are involved in DNA replication, might counteract UV-induced processes, and include many targets of transcription factors associated with cell proliferation and differentiation. Our results indicate that betaine controls unique gene expression pathways in keratinocytes, including some involved in differentiation.

Wild blueberries (Vaccinium myrtillus) alleviate inflammation and hypertension associated with developing obesity in mice fed with a high-fat diet.

BACKGROUND: Low-grade metabolic inflammation and hypertension are primary mechanisms involved in obesity-associated adverse health effects. Berries, especially Nordic wild blueberries (hereafter referred to as bilberries), represent an important source of dietary anthocyanins, a group of polyphenols with potential beneficial effects to combat obesity-associated metabolic disturbances. METHODS: The effects of 5% or 10% (w/w) of whole bilberries (BB) were studied on the development of obesity and its metabolic disturbances in C57BL mice fed with a high-fat diet (HFD) for three months. Cytokines, inflammatory cells, systolic blood pressure, glucose tolerance, insulin sensitivity, weight gain, body fat, food consumption and energy metabolism were assessed. RESULTS: Bilberries ameliorated type 1 pro-inflammatory responsiveness induced by HFD. This was indicated by the altered cytokine profile and the reduced prevalence of interferon gamma -producing T-cells, in particular T helper type 1 cells. Bilberries also prevented the progression of obesity associated long term increase in systolic blood pressure in mice. CONCLUSIONS: Bilberries reduce the development of systemic inflammation and prevent the progression of chronic hypertension, thus supporting their potential role in alleviating the adverse health effects associated with developing obesity.

Selective use of multiple vitamin D response elements underlies the 1 alpha,25-dihydroxyvitamin D3-mediated negative regulation of the human CYP27B1 gene.

The human 25-hydroxyvitamin D3 (25(OH)D3) 1alpha-hydroxylase, which is encoded by the CYP27B1 gene, catalyzes the metabolic activation of the 25(OH)D3 into 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), the most biologically potent vitamin D3 metabolite. The most important regulator of CYP27B1 gene activity is 1alpha,25(OH)2D3 itself, which down-regulates the gene. The down-regulation of the CYP27B1 gene has been proposed to involve a negative vitamin D response element (nVDRE) that is located approximately 500 bp upstream from transcription start site (TSS). In this study, we reveal the existence of two new VDR-binding regions in the distal promoter, 2.6 and 3.2 kb upstream from the TSS, that bind vitamin D receptor-retinoid X receptor complexes. Since the down regulation of the CYP27B1 gene is tissue- and cell-type selective, a comparative study was done for the new 1alpha,25(OH)2D3-responsive regions in HEK-293 human embryonic kidney and MCF-7 human breast cancer cells that reflect tissues that, respectively, are permissive and non-permissive to the phenomenon of 1alpha,25(OH)2D3-mediated down-regulation of this gene. We found significant differences in the composition of protein complexes associated with these CYP27B1 promoter regions in the different cell lines, some of which reflect the capability of transcriptional repression of the CYP27B1 gene in these different cells. In addition, chromatin architecture differed with respect to chromatin looping in the two cell lines, as the new distal regions were differentially connected with the proximal promoter. This data explains, in part, why the human CYP27B1 gene is repressed in HEK-293 but not in MCF-7 cells.

Controlling the chromatin organization of vitamin D target genes by multiple vitamin D receptor binding sites.

An essential prerequisite for the direct modulation of transcription by 1alpha,25-dihydroxy vitamin D(3) (1alpha,25(OH)(2)D(3)) is the location of at least one activated vitamin D receptor (VDR) protein close to the transcription start site of the respective primary 1alpha,25(OH)(2)D(3) target gene. This is achieved through the specific binding of VDR to a 1alpha,25(OH)(2)D(3) response element (VDRE). Although these elements are well characterized in vitro, the function of VDREs in living cells in the context of chromatin is still largely unknown. To resolve this issue, approximately 8kB of the promoter regions of the primary 1alpha,25(OH)(2)D(3) target genes CYP24, cyclin C and p21((Waf1/Cip1)) were screened by chromatin immunoprecipitation (ChIP) assays for VDR binding sites using antibodies against VDR and its partner proteins. This approach identified three to four functional VDREs per gene promoter. In parallel, in silico screening of the extended gene areas (i.e. 10kB of promoter, introns, exons and 10kB of the downstream region) of all six members of the insulin-like growth factor binding protein (IGFBP) gene family was performed. Gel shift, reporter gene and ChIP assays identified in total 10 functional VDREs in the genes IGFBP1, IGFBP3 and IGFBP5. Taken together, both screening approaches suggest that a reasonable proportion of all VDR target genes, if not all, are under the control of multiple VDREs.

The insulin-like growth factor-binding protein 1 gene is a primary target of peroxisome proliferator-activated receptors.

Insulin-like growth factor-binding protein 1 (IGFBP-1) is a biomarker for metabolic and hyperproliferative diseases. At the same time, the nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the development of both the metabolic syndrome and various cancers. Here we demonstrate, in human hepatocellular carcinoma cells and in normal mouse liver, that IGFBP-1 mRNA expression is under the primary control of PPAR ligands. We applied an improved in silico screening approach for PPAR response elements (PPREs) and identified five candidate PPREs located within 10 kb of the transcription start site (TSS) of the IGFBP-1 gene. Chromatin immunoprecipitation assays showed that, in living cells, the genomic region containing the most proximal PPRE, at position -1200 (relative to the TSS), preferentially associates with multiple PPAR subtypes and various other components of the transcriptional apparatus, which include their heterodimerizing partner, retinoid X receptor, as well as phosphorylated RNA polymerase II, co-repressor, co-activator, and mediator proteins. Moreover, further chromatin immunoprecipitation assays demonstrated that the TSS regions of the IGFBP-1 gene and those of the related IGFBP-2, -5, and -6, but not of IGFBP-3 and -4 genes, bind PPARs as well. We also show that these additional PPAR binding genes contain a number of candidate PPREs and that their mRNA levels respond quickly to the presence of PPAR ligands, indicating that they are also primary PPAR target genes.

The impact of chromatin organization of vitamin D target genes.

The vitamin D receptor (VDR), the nuclear receptor for 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), controls gene expression by binding discrete DNA sequences in promoter regions of target genes, referred to as 1alpha,25(OH)2D3 response elements (VDREs). Although these elements are well characterized in vitro, the function of VDREs in living cells in the context of chromatin is still largely unknown. To resolve this issue, 7 to 8 kB of the promoter regions of the primary 1alpha,25(OH)2D3 target genes CYP24, cyclin C and p21(Waf1/Cip1) were studied by chromatin immunoprecipitation (ChIP) assays using antibodies against acetylated histone H4 (to assess the global chromatin status) and various other components of VDR-dependent gene activation, such as VDR, retinoid X receptor (RXR), coactivator (CoA) and corepressor proteins. This approach identified three to four functional VDREs per gene promoter. In parallel, the extended analysis of the gene areas, of all six members of the insulin-like growth factor binding protein (IGFBP) family (i.e., 10 kB of promoter, introns, exons and 10 kB of the downstream region) were screened in silico for putative VDR-binding sites. Gel shift, reporter gene and ChIP assays identified, in total, ten functional VDREs in the genes IGFBP1, 3 and 5 and real-time PCR confirmed that these genes are primary VDR targets. Taken together, these results suggest that a reasonable proportion of all VDR target genes, if not all, are under the control of multiple VDREs. These results will have an impact on the development of therapeutic regimes for diseases, such as cancer, that use 1alpha,25(OH)2D3 and its analogs.

An integrated biological approach to nuclear receptor signaling in physiological control and disease.

The nuclear receptors (NRs)--vitamin D receptor (VDR); peroxisome proliferator-activated receptor (PPAR) alpha, delta, gamma; and pregnane X receptor (PXR)--act as sensors for various molecules encountered by the body on a daily basis. The effects of these ligands can be understood by the fact that numerous genes involved in the cellular processes, such as general homeostasis, growth, and defense against microbes, are under the control of these five NRs. The target gene and protein expression patterns of VDR, PPARs, and PXR; the resulting changes in metabolite levels; and their physiological consequences create a network that can be monitored by high-throughput methods and analyzed by multimodal approaches, such as systems biology. We suggest that the fine regulation of this NR network is specific to each human individual and depends, in part, on the constellation of regulatory small nucleotide polymorphisms (SNPs) in his or her genome. When regulatory SNPs affect NRs response elements, lifetime exposure to food components will have different accumulative consequences on the expression of the respective NR target genes. These differences will influence the individual's susceptibility to aging-related diseases, such as type 2 diabetes, atherosclerosis, cancer, and osteoporosis. Furthermore, it is anticipated that systems biology methods will also help to identify the most critical genes, proteins, or metabolites in the NR network that will serve as biomarkers for the early detection of these diseases.

A role of melatonin in neuroectodermal-mesodermal interactions: the hair follicle synthesizes melatonin and expresses functional melatonin receptors.

Since mammalian skin expresses the enzymatic apparatus for melatonin synthesis, it may be an extrapineal site of melatonin synthesis. However, evidence is still lacking that this is really the case in situ. Here, we demonstrate melatonin-like immunoreactivity (IR) in the outer root sheath (ORS) of mouse and human hair follicles (HFs), which corresponds to melatonin, as shown by radioimmunoassay and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The melatonin concentration in organ-cultured mouse skin, mouse vibrissae follicles, and human scalp HFs far exceeds the respective melatonin serum level and is significantly increased ex vivo by stimulation with norepinephrine (NE), the key stimulus for pineal melatonin synthesis. By real-time PCR, transcripts for the melatonin membrane receptor MT2 and for the nuclear mediator of melatonin signaling, retinoid orphan receptor alpha (ROR)alpha, are detectable in murine back skin. Transcript levels for these receptors fluctuate in a hair cycle-dependent manner, and are maximal during apoptosis-driven HF regression (catagen). Melatonin may play a role in hair cycle regulation, since its receptors (MT2 and RORalpha) are expressed in murine skin in a hair cycle-dependent manner, and because it inhibits keratinocyte apoptosis and down-regulates ERalpha expression. Therefore, the HF is both, a prominent extrapineal melatonin source, and an important peripheral melatonin target tissue. Regulated intrafollicular melatonin synthesis and signaling may play a previously unrecognized role in the endogenous controls of hair growth, for example, by modulating keratinocyte apoptosis during catagen and by desensitizing the HF to estrogen signaling. As a prototypic neuroectodermal-mesodermal interaction model, the HF can be exploited for dissecting the obscure role of melatonin in such interactions in peripheral tissues.

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.

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.

The human hyaluronan synthase 2 gene is a primary retinoic acid and epidermal growth factor responding gene.

Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis and subject to large concentration changes associated with keratinocyte proliferation, migration, and differentiation induced by paracrine and endocrine factors like epidermal growth factor (EGF) and all-trans-retinoic acid (RA). We found that in REK cells EGF and all-trans-RA up-regulated hyaluronan synthase 2 (Has2) gene expression within 2 h 4-fold each and in HaCaT human immortal keratinocytes 8- and 33-fold, respectively. The first 10 kb of the human Has2 promoter were scanned in silico and in vitro for potential response elements of signal transducer and activator of transcription (STAT) or RA receptor (RAR) proteins. We identified a STAT-response element in the proximal promoter region and confirmed its functionality in response to EGF by chromatin immunoprecipitation (ChIP) assays. Direct in vitro binding of RARs to four RARE candidates within the Has2 promoter could not be observed at stringent gel shift conditions, but reporter gene assays demonstrated functionality of a complex of two of these RAREs located approximately 1200 bp upstream of the transcription start site. Moreover, ChIP assays using antibodies against nine nuclear proteins monitored all-trans-RA-dependent binding of RAR, retinoid X receptor, mediator protein, and RNA polymerase II and also histone 4 acetylation to a promoter region containing the complex RARE. Taken together, the human Has2 gene is a potent primary EGF and all-trans-RA responding gene with a complex regulation.

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.

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).

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.

The highly conserved region of the co-repressor Sin3A functionally interacts with the co-repressor Alien.

The Sin3 proteins are evolutionarily conserved co-repressors (CoR) that function as mediators of gene repression for a variety of transcriptional silencers. The paired amphipathic helices of Sin3A were identified and studied as protein-protein interacting domains. Previously we have shown the interaction of Sin3A with the CoR Alien in vivo and in vitro. Here, we show that Alien and Sin3A reside together in vivo with the vitamin D3 receptor on the human 24-hydroxylase (CYP24) promoter containing vitamin D3 response elements by chromatin immunoprecipitation. We delineated and characterized the interaction domains of Sin3A with Alien. Interestingly, the highly conserved region (HCR) of Sin3A, which has not yet been functionally characterized, interacts with Alien. The HCR encompasses only 134 amino acids, shares more than 80% identity with Sin3B and binds to the N-terminus of Alien, which harbours a transferable silencing function. Functionally, co-expression of Sin3A enhances Alien-mediated gene repression and overexpression of the HCR alone leads to the inhibition of Alien-mediated repression and to the induction of the endogenous CYP24 promoter. Our results therefore indicate a novel functional role of the Sin3 HCR and give novel insights into Alien-mediated gene repression.

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.