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C Carlberg

Publications and source records attributed to C Carlberg.

At least 55 records · Page 3Linked to original sources
PubMed1997

1 alpha,25-dihydroxyvitamin D3 receptor as a mediator of transrepression of retinoid signaling.

The receptors for retinoic acid (RA) and for 1 alpha,25-dihydroxyvitamin D3 (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-alpha type 1 receptor (TNF alpha RI) gene identified a 3' enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein-protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD.

3T3 Cells↗
PubMed1997

Allosteric interaction of the 1alpha,25-dihydroxyvitamin D3 receptor and the retinoid X receptor on DNA.

Genomic actions of the hormone 1alpha,25-dihydroxy-vitamin D3(VD) are mediated by the transcription factor VDR, which is a member of the nuclear receptor superfamily. VDR acts in most cases as a heterodimeric complex with the retinoid X receptor (RXR) from specific DNA sequences in the promoter of VD target genes called VD response elements (VDREs). This study describes a mutation (K45A) of the VDR DNA binding domain that enhances the affinity and ligand responsiveness of VDR-RXR heterodimers on some VDREs. In analogy to a homologous mutation in the glucocorticoid receptor (K461A), this lysine residue appears to function as an allosteric 'lock'. Interestingly, overexpression of RXR was found to reduce the responsiveness and sensitivity of wild type VDR to VD, but enhance the response of VDRK45A. Moreover, the transactivation domains of both VDR and RXR were shown to be essential for obtaining responsiveness of the heterodimers to VD and 9- cis retinoic acid (the RXR ligand). This indicates that RXR is an active rather than silent partner of the VDR on the VDREs tested. Taken together, transactivation by VDR-RXR heterodimers can be triggered individually by all components of the protein-DNA complex, but full potency appears to be reached through allosteric interaction.

Alitretinoin↗
PubMed1997

Functional conformations of the nuclear 1alpha,25-dihydroxyvitamin D3 receptor.

The nuclear hormone 1alpha,25-dihydroxyvitamin D3 (VD) has important cell regulatory functions. Various synthetic VD analogues are under investigation to identify candidates with an improved therapeutic profile against hyperproliferative diseases. VD directly activates the transcription factor VD receptor (VDR), which in turn stimulates the expression of a cascade of primary and secondary VD-responsive genes. The activation of the VDR through binding of its natural and synthetic ligands is linked to a conformational change presenting the interface with co-activator proteins, referred to as the (trans)activation function 2 (AF-2) domain. Multiple conformations of the VDR might be the key to understanding a selective action of VD analogues. The method of limited protease digestion was used here to characterize up to three different functional VDR conformations stabilized individually by VD and its analogues. The relative potency of VDR ligands can be quantified in the interaction with these VDR conformations by determination of a functional dissociation constant, where a two-concentration-point comparison has already provided important information. In this way seven amino acid residues in the AF-2 domain have been analysed as potential ligand contact points. Interestingly, residues Phe-422 and Val-418 seem to interact with all tested VDR ligands, whereas VD analogues such as the anti-psoriatic drug MC903 displayed additional contact points within the AF-2 domain. Taken together, limited protease digestion is a powerful method for studying functional VDR conformations and seems to be very appropriate for screening VD analogues.

Cell Nucleus↗
PubMed1997

Sensitive induction of apoptosis in breast cancer cells by a novel 1,25-dihydroxyvitamin D3 analogue shows relation to promoter selectivity.

The biologically active form of vitamin D3, the nuclear hormone 1 alpha,25-dihydroxyvitamin D3 (VD), is an important regulator of cellular growth, differentiation, and death. The hormone mediates its action through the activation of the transcription factor VDR, which is a member of the superfamily of nuclear receptors. In most cases the ligand-activated VDR is found in complex with the retinoid X receptor (RXR) and stimulates gene transcription mainly from VD response elements (VDREs) that are formed by two hexameric core binding motifs and are arranged either as a direct repeat spaced by three nucleotides (DR3) or as an inverted palindrome spaced by nine nucleotides (1P9). The two VD analogues CB1093 and EB1089 are both very potent inhibitors of the proliferation of MCF-7 cultured breast cancer cells displaying approximately 100-fold lower IC50 values (0.1 nM) than the natural hormone. In addition, CB1093 is even more potent in vivo than EB1089 in producing regression of experimental mammary tumors. Moreover, both VD analogues induce apoptosis in MCF-7 cells, but CB1093 is effective at concentrations approximately 10-fold lower than EB1089. In accordance, the reduction of Bcl-2 protein expression showed CB1093 to be more potent than EB1089. This suggests that the antiproliferative effect of CB1093 may be related mainly to its apoptosis inducing effect, whereas EB1089 may preferentially have effects on growth arrest. EB1089 is known to result in a selectivity for the activation of IP9-type VDREs, whereas CB1093 shows a preference for the activation of DR3-type VDREs. This promoter selectivity suggests that the effects of VD and its analogues on growth arrest and the induction of apoptosis may be mediated by different primary VD responding genes. In conclusion, CB1093 was found to be a potent inhibitor of rat mammary tumor growth in vivo. CB1093 also displayed a high potency in vitro in the induction of apoptosis, a process that may be linked to a promoter selectivity for DR3-type VDREs.

Animals↗
PubMed1997

Functional characterization of a novel type of 1 alpha,25-dihydroxyvitamin D3 response element identified in the mouse c-fos promoter.

The seco-steroid 1 alpha,25-dihydroxyvitamin D3 (VD) is known to inhibit cellular proliferation and to induce differentiation as well as programmed cell death (apoptosis). VD is the ligand of the transcription factor VDR, which is a member of the nuclear receptor superfamily. Primary VD responding genes contain a VD response element (VDRE), on which VDR binds as a dimeric complex. The main heterodimeric partner of VDR is the retinoid X receptor (RXR) and the majority of the known natural VDREs are formed by a direct repeat of hexameric core binding motifs spaced by 3 nucleotides. Most of the genes carrying DR3-type VDREs are associated with the hormone's classical function, which is the regulation of calcium homeostasis. Recently, it has been found that inverted palindromic arrangements spaced by 9 nucleotides also form functional VDREs. This paper reports the identification of a novel IP9-type VDRE in the mouse c-fos promoter. This elements is bound with high affinity by VDR-RXR heterodimers and responds at 10-fold lower concentrations to the potent anti-proliferative VD analogue EB1089 than to VD. This suggests that VD may be directly involved in the transcriptional regulation of the cell cycle via the activation of the c-fos gene.

Animals↗
PubMed1996

The high affinity ligand binding conformation of the nuclear 1,25-dihydroxyvitamin D3 receptor is functionally linked to the transactivation domain 2 (AF-2).

The nuclear receptor for 1,25-dihydroxyvitamin D3 (VD), VDR, is a transcription factor that mediates all genomic actions of the hormone. The activation of VDR by ligand induces a conformational change within its ligand binding domain (LBD). Due to the lack of a crystal structure analysis, biochemical methods have to be applied in order to investigate the details of this receptor-ligand interaction. The limited protease digestion assay can be used as a tool for the determination of a functional dissociation constant (K(df)) of VDR with any potential ligand. This method provided with the natural hormone VD two protease-resistant fragments of the VDR LBD and with the 20-epi conformation of VD, known as MC1288, even an additional fragment of intermediate size. These fragments were interpreted as different receptor conformations and their decreasing size was found to be associated with decreasing ligand binding affinity. A critical amino acid for VDR's high ligand binding conformation has been identified by C-terminal receptor truncations and point mutations as phenylalanine 422. This amino acid appears to directly contact the ligand and belongs to the ligand-inducible activation function-2 (AF-2) domain. Moreover, functional assays supported the observation that high affinity ligand binding is directly linked to transactivation function.

Antigens↗
PubMed1996

Potent gene regulatory and antiproliferative activities of 20-methyl analogues of 1,25 dihydroxyvitamin D3.

The biological active form of vitamin D3, 1,25-dihydroxyvitamin D3 (VD), regulates cellular growth and differentiation. This provides the hormone with an interesting therapeutic potential. However, hypercalcemia is a side effect, which is caused by VD's classical action, the regulation of calcium homeostasis. This made the need for VD analogues with selectively increased cell regulatory properties. Studies with 20-epi analogues pointed out the importance of the carbon-20 position and led to the development of 20-methyl derivatives of VD. In this report the biological properties of the compounds ZK161422 and ZK157202, which are 20-methyl- and 20-methyl-23-eneanalogues, respectively, have been analyzed in comparison with VD. Both compounds show about 2-fold lower affinity to the VD receptor (VDR) than VD. However, compared to VD, their antiproliferative effect is up to 30-fold higher on human peripheral blood mononuclear cells and even up to 300-fold higher on human breast cancer MCF-7 cells. Whereas the hypercalcemic effect for ZK157202 is also increased 10-fold, ZK161422 has the same calcium-mobilizing potency as VD. Moreover, ZK161422, but not ZK157202, showed preference for gene activation from a promoter carrying a VD response element with a palindromic arrangement of two hexameric receptor binding sites spaced by 9 nucleotides (IP9) rather than for activation from a response element formed by a direct repeat spaced by 3 nucleotides (DR3). This observation supports a model, in which promoter selectivity reflects the selectively increased antiproliferative effect of VD analogues.

Breast Neoplasms↗
PubMed1996

The potent anti-proliferative effect of 20-epi analogues of 1,25 dihydroxyvitamin D3 in human breast-cancer MCF-7 cells is related to promoter selectivity.

The hormone 1,25-dihydroxyvitamin D3 (VD) has a potential use as an anti-tumor agent, but its clinical applications are restricted by its strong calcemic activity. This has led to the development of VD analogues with selectively increased growth-inhibitory activity. One of the most potent analogues is KH1060, which is a 20-epi-22-oxa-derivative of VD. In human breast cancer MCF-7 cells, we studied the growth-inhibitory activities of a set of 8 analogues that cover conservative structural changes from 20-epi-VD (MC1288) to KH1060. In the same cellular system, we analyzed the potential of these 8 analogues to stimulate reporter gene activity driven by a recently discovered novel-type VD response element. We found that this VD response element is more appropriate than classical VD response elements to correlate anti-proliferative effects of VD analogues with their gene-regulatory properties.

Amino Acid Sequence↗
PubMed1996

High-affinity nuclear receptor binding of 20-epi analogues of 1,25-dihydroxyvitamin D3 correlates well with gene activation.

The hormone 1,25-dihydroxyvitamin D3 (VD) has the potential for clinical use in several diseases, such as cancer, osteoporosis, and psoriasis. The action of VD is mediated by primary responding genes that contain in their promoter region a binding site for the transcription factor VDR. Most of the known VD response elements are formed by a direct repeat of two hexameric core binding motifs spaced by three nucleotides (DR3) bound by a heterodimer of VDR and the retinoid X receptor (RXR). Various VD analogues have been developed in order to optimize the therapeutic profile of VD. This report presents a novel experimental system that may help in the understanding of the structural basis for the high potency of a VD analogue like KH1060, which is a 20-epi-22-oxa-derivative of VD. In human breast cancer cells, MCF-7, the half-maximal gene activation values for KH1060 and seven of its structural precursors were determined on a DR3-type VD response element. These eight analogues cover conservative structural changes from 20-epi-VD (MC1288) to KH1060. With a modified version of the limited protease digestion assay the functional affinity of the analogues to VDR was measured. The functional receptor affinity of the eight analogues was found to be directly proportional to their potency in VDR-RXR-mediated gene activity.

Binding Sites↗
PubMed1996

Identification of natural monomeric response elements of the nuclear receptor RZR/ROR. They also bind COUP-TF homodimers.

The receptor RZR/ROR is an important member of the nuclear receptor superfamily and has recently been shown to be the nuclear receptor for the pineal gland hormone melatonin. RZR/ROR binds as a monomer to DNA, and the human 5-lipoxygenase gene has been identified as the first RZR/ROR/melatonin-responding gene. Another prominent nuclear receptor is COUP-TF, which binds as a dimer to DNA. In this study, the sequences of known promoter regions of genes that may be involved in the physiological action of melatonin have been screened for putative monomeric RZR/ROR response elements. The binding of RZR/ROR and COUP-TF was compared and quantified on a set of 12 putative response elements. Interestingly, COUP-TF homodimers were found to bind with high affinity to some of the monomeric RZR/ROR response elements. Four RZR/ROR response elements, found in the genes of the mouse bifunctional enzyme, the rat bone sialoprotein, mouse Purkinje cell protein 2, and human p21(WAF1/CIP1), were shown to be inducible by melatonin under conditions of low constitutive activity. Surprisingly, the constitutive activity of COUP-TF was also stimulated by an unknown serum compound. The novel Purkinje cell protein 2 and p21(WAF1/CIP1) RZR/ROR/melatonin-responding genes may be the key for understanding the role of RZR/RORalpha in the mouse mutation staggerer and the antiproliferative action of melatonin, respectively.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

Thiazolidine diones, specific ligands of the nuclear receptor retinoid Z receptor/retinoid acid receptor-related orphan receptor alpha with potent antiarthritic activity.

Rat adjuvant arthritis is a chronic T cell-dependent autoimmune disease with many similarities to rheumatoid arthritis. We have identified a class of thiazolidine diones with high potency in suppressing chronic inflammation and joint destruction in this experimental model. The lead compound CGP 52608 (1-(3-allyl-4-oxothiazolidine-2-ylidene)-4-methylthiosemicarbazone) exhibits antiarthritic activity at daily oral doses between 0.01 and 1 mg/kg and was shown to specifically activate the retinoid Z receptor/retinoid acid receptor-related orphan receptor alpha (RZR/RORalpha) in low nanomolar concentrations. This receptor is a novel member of the superfamily of ligand-inducible transcription factors, and we have recently identified the pineal gland hormone melatonin as a natural ligand. Structure-activity relationship studies with 13 closely related analogues of CGP 52608 revealed a striking correlation between RZR/RORalpha activation and antiarthritic activity. We therefore suggest that nuclear signaling via RZR/RORalpha is a key mechanism in mediating the antiarthritic effects of these thiazolidine diones and may open a novel therapeutic approach for the treatment of rheumatoid arthritis and other autoimmune diseases. The existence of a nuclear melatonin receptor may lead to a better understanding of the immunomodulatory actions of melatonin.

Acetamides↗
PubMed1996

Identification of a vitamin D3 response element in the fibronectin gene that is bound by a vitamin D3 receptor homodimer.

Fibronectin (FN) is an important adhesive noncollagenous glycoprotein involved in maintenance of the extracellular matrix and cell adhesiveness, loss of which has been implicated in the metastatic potential of cells. Regulation of FN occurs at the transcriptional level by the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Transient transfection of homologous and heterologous promoter reporter constructs into ROS 17/2.8 (rat osteosarcoma), NIH 3T3 (mouse fibroblast), and MCF-7 (human mammary carcinoma) cell lines showed a consistent two- to threefold induction of transcription when stimulated with 1,25-(OH)2D3. These heterologous promoter transfection studies with gel shift analysis locate a third, natural DR6-type vitamin D responsive element (VDRE) at nucleotide positions -171 to -154 in the murine FN promoter. Interestingly, this VDRE is also present in rat and human FN promoters. This study shows that 1,25-(OH)2D3 induces FN transcription from an existing elevated basal transcriptional activity by acting through two putative hexameric core binding motifs which bind VDR homodimers. Furthermore, the FN VDRE is the first homodimer-type VDRE that is not overlaid by a DR3-type structure.

3T3 Cells↗
PubMed1996

Functional characterization of a 1,25-dihydroxyvitamin D3 receptor binding site found in the rat atrial natriuretic factor promoter.

The classical action of the hormone 1,25-dihydroxyvitamin D3 (VD) is the regulation of calcium metabolism. In contrast, the peptide hormone atrial natriuretic factor (ANF) is one of the few known nonclassical VD responding genes. We screened the promoter of the rat ANF gene and identified a typical VD receptor (VDR) binding site formed by a direct repeat of two hexameric core binding motifs spaced by three nucleotides, between positions -907 and -891. Like most of the DR3-type VD response elements this sequence is bound with high affinity (Kd = 0.53 nM) by a heterodimer formed by VDR and retinoid X receptor. In a heterologous promoter context one copy of this sequence mediated an about fourfold gene activation by VD and a half-maximal activation (EC50) value of 0.48 nM VD. This characterizes the identified sequence as one of the most potent VD response elements.

Animals↗
PubMed1996

Diagnostic sensitivity of immunodominant epitopes of glutamic acid decarboxylase (GAD65) autoantibodies in childhood IDDM.

The prevalence and titre of epitope-specific autoantibodies to glutamic acid decarboxylase (GAD65) in 155 insulin-dependent diabetic (IDDM) and 9 GAD65 antibody (Ab)-positive healthy children were determined using four GAD65/67 chimaeric molecules which discriminate among the N-terminal (N), middle (M) and C-terminal (C) epitopes of GAD65. Radioligand binding assays for IgGAb used immunoprecipitation of in vitro translated 35S-GAD. We found autoantibodies to GAD65 in 116 of 155 (75%), to GAD67 in 19 of 155 (12%) (p < 0.0001) and to the GAD65-N-67 chimaera in 25 of 155 (16%) (p < 0.0001) IDDM sera. GAD67Ab were found almost exclusively (17 of 19, 89%) in GAD65Ab-positive sera and the levels of GAD67Ab correlated with those of GAD65Ab (r2 = 0.5913; p = 0.009). GAD65Ab directed to GAD65-M were found in 104 of 155 (67%), to GAD65-C in 104 of 155 (67%) and to GAD65-M + C in 116 of 155 (75%) of IDDM sera, and indicated reactivity to at least two distinct epitopes. Among the nine GAD65Ab-positive healthy children, two (22%) were also positive with GAD67, nine (100%) with GAD65-M + C, seven (78%) with GAD65-M, eight (89%) with GAD65-C and two (22%) with GAD65-N-67. Titres of GAD65Ab (p = 0.007), GAD65-C-Ab (p = 0.002) and GAD65-C + M-Ab (p = 0.003), but not of GAD65-M-Ab (p = 0.101) were significantly higher in IDDM than in healthy children. We conclude that GAD65Ab in IDDM and healthy children are directed to middle and C-terminal epitopes, and propose that levels of antibodies specifically directed to the carboxy-terminal end of GAD65 may distinguish IDDM from healthy children.

Adolescent↗
PubMed1996

The concept of multiple vitamin D signaling pathways.

Nuclear signaling by 1 alpha, 25-dihydroxyvitamin D3 (VD) is mediated by its nuclear receptor (VDR). It is widely accepted that VDR forms heterodimers with retinoid X receptors (RXRs) that bind in 5'-RXR-VDR-3' polarity to response elements formed by direct repeats of hexameric core binding motifs spaced by three nucleotides. This model, however, might be too simple to explain the multiplicity of nuclear signaling by VD. Recently, it was reported that VDR forms homodimers and heterodimers with the nuclear receptors for all-trans retinoic acid and thyroid hormone. All four different VDR complexes bind not only to direct repeats, but also to inverted palindromes; moreover, in heterodimeric complexes VDR can also take the 5' position. Taken together, this discriminates at least seven different types of VDR complexes binding each to two types of response elements. For half of these 14 cases, representative natural VD response elements have already been identified, which demonstrates that nature takes advantage of this concept of diversity. It is our hypothesis that the different physiological functions of VD can be related to different VD signaling pathways. Therefore, the identification of VD analogs that specifically trigger these pathways is the goal of our present and future investigations.

Animals↗
PubMed1996

Vitamin D-retinoid association: molecular basis and clinical applications.

The molecular structure of the biological active form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 (VD), and the vitamin A derivatives all-trans and 9-cis retinoic acid (RA) are not related. The nuclear receptors for VD (VDR) and retinoids (RAR and RXR), however, are members of the same superfamily of ligand-activated transcription factors. We observed stable VDR-RXR and VDR-RAR heterodimers in solution and their transcriptional activity on different types of response elements. Both heterodimeric complexes are activated by VD, but, depending on the relative expression of the nuclear receptors, retinoids can have either co-stimulating or repressing effects. This demonstrates that VD and retinoid signaling are linked at the level of gene regulation and may explain the similar effects of both hormones on cell proliferation and differentiation. This concept may be applied for treating skin diseases, with the hope that a synergism will be observed, allowing better responses with lower doses of each compound. Preliminary observations suggest that psoriasis, cutaneous T-cell lymphomas, and actinic keratoses might be potential targets for VD-retinoid associations.

Calcitriol↗
PubMed1995

The anti-proliferative effect of vitamin D3 analogues is not mediated by inhibition of the AP-1 pathway, but may be related to promoter selectivity.

The hormone 1,25-dihydroxyvitamin D3 (VD) is able to induce cellular differentiation and to inhibit cellular proliferation, which provides it with an interesting therapeutic potential in cancer. However, side effects of VD on homeostasis (eg hypercalcemia) had made the need for the development of VD analogues with low calcemic effect. On the human breast cancer cell line MCF-7 we obtained with the VD analogue EB1089 an about 100-fold higher anti-proliferative effect than with VD. We found that this difference in biological activity is neither related to increased functional affinity to the VD receptor nor to repression of AP-1 activity. The physiologically most prominent complex of the VD receptor is a heterodimer with the retinoid X receptor that binds VD response elements formed two hexameric core binding motifs being arranged either as direct repeats spaced by 3 nucleotides (DR3s) or as inverted palindromes spaced by 9 nucleotides (IP9s). We observed that EB1089 stimulates transcriptional activation from IP9-type elements at clearly lower concentrations than from DR3-type elements. It is possible that IP9-type response elements play an important role in or contribute to the control of cell proliferation, so that promoter-selectivity may explain the high anti-proliferative effect of EB1089.

Antineoplastic Agents↗