The two subtypes of retinoid Z receptor (RZR alpha and beta) and the three splicing variants of retinoid orphan receptor (ROR alpha 1, alpha 2, and alpha 3) form a subfamily within the superfamily of nuclear hormone receptors. Very recently we found that the pineal gland hormone melatonin is a natural ligand of RZR alpha and RZR beta. Ligand-induced transcriptional control is therefore proposed to mediate physiological functions of melatonin in the brain where RZR beta is expressed, but also in peripheral tissues, where RZR alpha was found. However, no natural RZR responding genes have been identified yet. Here, we report that a response element in the promoter of 5-lipoxygenase binds specifically RZR alpha and ROR alpha 1, but not ROR alpha 2 and alpha 3. 5-Lipoxygenase is a key enzyme in the biosynthesis of leukotrienes, which are known to be allergic and inflammatory mediators. We could show that the activity of the whole 5-lipoxygenase promoter as well as of the RZR response element fused to the heterologous thymidine kinase promoter could be repressed by melatonin. The hormone down-regulated the expression of 5-lipoxygenase about 5-fold in B lymphocytes, which express RZR alpha. In contrast, 5-lipoxygenase mRNA levels were not affected in differentiated monocytic and granulocytic cell lines, which do not express RZR alpha. This indicates that 5-lipoxygenase is the first natural RZR alpha responding gene. Furthermore, our results open up a new perspective in understanding the involvement of melatonin in inflammatory and immunological reactions.

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/ROR␣ in the mouse mutation staggerer and the antiproliferative action of melatonin, respectively.Many important physiological functions are controlled at the level of transcriptional regulation by members of the nuclear receptor superfamily. This family of structurally related transcription factors contains the nuclear receptors for steroid hormones (aldosterone, cortisone, estrogen, progesterone, and testosterone), 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), 1 thyroid hormone, and all-trans-retinoic acid (RA), but also Ͼ100 orphan nuclear receptors, for which no ligand was known at the time of their discovery (1).Nuclear receptors activate transcription through DNA sequences in the promoter region of target genes, referred to as response elements. Since the DNA-binding domain is highly conserved in all nuclear receptors (2), they bind in a vast majority to the hexameric consensus motif AGGTCA (3). However, most of them do not have sufficient affinity to bind to DNA as a monomer, so they have to form homodimers or heterodimers with another member of the superfamily (4). Therefore, most natural response elements are formed by two hexameric motifs in a directly repeated or inverted palindromic orientation. Specificity in the recognition of these response elements is mainly obtained by the number of spacing nucleotides (5, 6). In contrast, nuclear receptors that are able to bind as a monomer increase the specificity of their interaction with DNA through the recognition of two to four nucleotides 5Ј-flanking the hexameric consensus motif (7).RZR/ROR belongs to those nuclear receptors that bind DNA as a monomer (8, 9). To date, three RZR/ROR subtypes are known: RZR/ROR␣ is rather ubiquitously expressed (10), whereas RZR/ROR␤ is brai...

Pleiotropic actions of the biologically active form of vitamin D 3 , 1a,25-dihydroxyvitamin D 3 (VD), include antiproliferative effects in both normal human melanocytes and malignant melanoma cell lines. In this study the actions of VD and its low calcemic analogues EB1089 and CB1093, have been examined in two human melanoma cell lines MeWo and WM1341. Both cell lines express similar amounts of vitamin D receptor mRNA and show functional gene regulatory effects in response to VD and its analogues. VD, EB1089 and CB1093 induced apoptosis only in WM1341 cells and not in MeWo cells, even though both cell lines responded well to etoposide, a strong inducer of apoptosis. Additionally, these results were confirmed by analysis of cell morphology. Interestingly in WM1341 cells, CB1093 was found to be more potent in inducing apoptosis than EB1089 and the natural hormone. Moreover, CB1093 appeared to induce apoptosis at a relatively low concentration of 0.1 nM, whereas greater than tenfold higher concentrations of VD and EB1089 were needed to obtain comparable effects. These observations highlight CB1093 as a promising drug for a future treatment against specific types of melanoma. -23yne-24a,26a, 27a-trihomo-1a,25-dihydroxyvitamin D 3 ; CAT, chloramphenicol acetyl transferase; DR3, direct repeat spaced by 3 nucleotides; EB1089, 22,24-diene-24a,26a,27a-trihomo-1a,25-dihydroxyvitamin D 3 ; IP9, inverted palindrome spaced by 9 nucleotides; TNFa, tumor necrosis factor alpha; VD, 1a,25-dihydroxyvitamin D 3 ; VDR, 1a,25-dihydroxyvitamin D 3 receptor; VDRE; VD response element

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.

The aim of this study was to longitudinally characterize the distribution of cells actively expressing the progenitor transcription factor islet-1 (Isl1+) during the embryonic life, the postnatal period, and adulthood. In this study, we have used direct immunohistochemical staining toward the protein Isl1 in a longitudinal rat model. Cells actively expressing Isl1 were traced in embryos from gestational day (GD) 11 until adulthood. In early cardiac development (GD 11), the Isl1+ progenitors were located in a greater abundance in the paracardiac regions, areas suggested to be the second heart field. To a lesser extent, Isl1+ cells were present within the bulbotruncal region and the truncus arteriosus. During the following days until GD 15, the Isl1+ cells were mainly observed at the proximal outflow tract (OFT) and at the inflow area of the right atrium. No Isl1+ cells were detected in the left ventricle. Compared with GD 11, more Isl1+ cells seemed to co-express cardiomyocyte markers and a minority of the Isl1+ cells was undifferentiated. Unexpectedly, only few undifferentiated Isl1+ cells were Ki67+ while a lot of TnT+ cardiomyocytes were proliferating in the ventricles. After birth, immature Isl1+ cells were still present in the OFT where they resided until adulthood. Our data suggest that during embryogenesis, Isl1+ cells migrate from extracardiac regions into the proximal part of the heart, proliferating and giving rise to cardioblasts. Unexpectedly, only a minority of the Isl1+ cells while a majority of ventricular cardiomyocytes were proliferating. The Isl1+ cell pool persists into adulthood, which might open up new strategies to repair damaged myocardium.