Shigeaki Kato

Activation of the Estrogen Receptor Through Phosphorylation by Mitogen-Activated Protein Kinase

The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos

Retinoic acid (RA) is a physiological agent that has a wide range of biological activity and appears to regulate developmental programs of vertebrates. However, little is known about the molecular basis of its metabolism. Here we have identified a novel cytochrome P450 (P450RA) that specifically metabolizes RA. In vitro, P450RA converts all‐trans RA into 5,8‐epoxy all‐trans RA. P450RA metabolizes other biologically active RAs such as 9‐cis RA and 13‐cis RA, but fails to metabolize their precursors, retinol and retinal. Overexpression of P450RA in cell culture renders the cells hyposensitive to all‐trans RA. These functional tests in vitro and in vivo indicate that P450RA inactivates RA. The P450RA gene is not expressed uniformly but in a stage‐ and region‐specific fashion during mouse development. The major expression domains in developing embryos include the posterior neural plate and neural crest cells for cranial ganglia. The expression of P450RA, however, is not necessarily inducible by excess RA. These results suggest that P450RA regulates the intracellular level of RA and may be involved in setting up the uneven distribution of active RA in mammalian embryos.

A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5′-TGACC-3′ motifs acting synergistically

We have identified an estrogen-responsive enhancer element (DH3 ERE) in the estrogen-induced DNAase I-hypersensitive region III of the chicken ovalbumin gene, which is located approximately 3.3 kb upstream from the mRNA start site and does not contain palindromic ERE. Four TGACC half-palindromic motifs, separated from each other by more than 100 bp, are responsible for conferring estrogen inducibility either to the proximal ovalbumin gene promoter or to heterologous promoters. Thus, widely spaced half-palindromic ERE motifs can act synergistically. Each half-palindromic motif was shown to bind the estrogen receptor (ER) with a low efficiency in vitro. However, two widely spaced half-palindromic motifs bound the ER cooperatively, much more efficiently than expected from binding to isolated half-ERE motifs. The ovalbumin promoter half-palindromic ERE motif located close to the TATA box was required for the activity of the distal DH3 ERE, but could be replaced by the binding sites of other transactivators.

Selective Interaction of Vitamin D Receptor with Transcriptional Coactivators by a Vitamin D Analog

ABSTRACT The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor. A family of cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and activates the transactivation function of nuclear receptors in a ligand-dependent way. We examined interaction of VDR with these coactivators that was induced by several vitamin D analogs, since they exert differential subsets of the biological action of vitamin D through unknown mechanisms. Unlike other vitamin D analogs tested, OCT (22-oxa-1α,25-dihydroxyvitamin D3) induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1. Consistent with these interactions, only TIF2 was able to potentiate the transactivation function of VDR bound to OCT. Thus, the present findings suggest that the structure of VDR is altered in a vitamin D analog-specific way, resulting in selective interactions of VDR with coactivators. Such selective interaction of coactivators with VDR may specify the array of biological actions of a vitamin D analog like OCT, possibly through activating a particular set of target gene promoters.

Widely spaced, directly repeated PuGGTCA elements act as promiscuous enhancers for different classes of nuclear receptors.

We describe here a novel class of cis-acting response elements for retinoid, vitamin D, and estrogen receptors which are widely spaced (10 to 200 bp) direct repeats (DRs) of the canonical 5-AGGTCA half-site recognition motif (DR10 to DR200). In contrast to the specificity previously observed with shortly spaced DRs (DR1 to DR5), the different receptors bind promiscuously to these novel elements to activate transcription in the presence of retinoic acid (RA), vitamin D, or estrogen. The greatest RA-dependent transactivation, seen with DR15, was similar to that observed with the canonical DR5. Both RA receptors and retinoid X receptors contribute to transactivation through widely spaced DR elements. With the estrogen receptor, DR15 was one-third as efficient as the classical palindromic response element. A further increase of spacer lengths progressively decreased the efficiency of transactivation. No transactivation was seen with widely spaced DRs when the thyroid and retinoid X receptors were coexpressed in the presence of their ligands. The progesterone receptor was also unable to transactivate through a DR10 element composed of its cognate binding motifs. These results considerably extend the response element repertoire of nuclear receptors and suggest the existence of promiscuous transcriptional regulation through common response elements, as well as the possibility of receptor cross-talk.

Stimulation of Osteoclast Formation by 1,25-Dihydroxyvitamin D Requires Its Binding to Vitamin D Receptor (VDR) in Osteoblastic Cells: Studies Using VDR Knockout Mice

Previous studies have shown that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays important roles in the formation of osteoclasts through its actions on osteoblastic cells. We have generated mice lacking vitamin D receptor (VDR) by gene targeting (VDR-/-). These mice had tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and exhibited similar levels of parameters for bone resorption to those in wild type mice. The present studies were undertaken to clarify whether effects of 1,25(OH)2D on osteoclast formation require VDR in osteoblasts, and to examine mechanisms of the formation of osteoclasts without VDR-mediated actions using VDR-/- mice. When wild-type calvarial osteoblasts and spleen cells were co-cultured with 1,25(OH)2D, TRAP-positive osteoclasts were formed regardless of the genotypes of spleen cells. In contrast, when osteoblasts from VDR-/- mice were co-cultured, no osteoclasts could be formed even with wild-type spleen cells. Parathyroid hormone and interleukin-1alpha stimulated osteoclast formation by co-cultures from VDR-/- mice, and the generated osteoclasts showed resorbing activity. These results demonstrate that VDR-mediated actions of 1,25(OH)2D in osteoblasts are essential for osteoclast formation by 1,25(OH)2D, and that functionally intact osteoclasts can be formed without 1,25(OH)2D actions under stimulations by other agents. It is suggested that osteoclastic bone resorption can be maintained without 1,25(OH)2D actions by other stimulatory agents.

Intron retention generates a novel isoform of the murine vitamin D receptor that acts in a dominant negative way on the vitamin D signaling pathway.

We identified and characterized a novel rat vitamin D receptor isoform (rVDR1), which retains intron 8 of the canonical VDR (rVDR0) during alternative splicing. In this isoform protein directed by the stop codon in this newly identified exon, a part of the ligand binding domain (86 amino acids) is truncated at the C-terminal end but contains 19 extra amino acids. The rVDR1 transcript was expressed at a level 1/15 to 1/20 of that of rVDR0 in the kidney and intestine in adult rats but not in embryos. The recombinant rVDR1 protein showed no ligand binding activity. Homo- and heterodimers of the recombinant rVDR0 and rVDR1 proteins bound to a consensus vitamin D response element (VDRE) but not to consensus response elements for thyroid hormone and retinoic acid. However, unlike rVDR0, rVDR1 did not form a heterodimeric complex with RXR on the VDRE. A transient expression assay showed that this isoform acted as a dominant negative receptor against rVDR0 transactivation. Interestingly, the dominant negative activities of rVDR1 differed among VDREs. Thus, the present study indicates that this new VDR isoform negatively modulates the vitamin D signaling pathway, through a particular set of target genes.

CLONING AND SEQUENCING OF THE GENE ENCODING THE MOUSE VITAMIN D RECEPTOR

The mouse vitamin D receptor (VDR)-encoding cDNA was cloned and the coding regions were sequenced. Comparison of the amino-acid sequence to that of humans and rats revealed high homology in the DNA- and ligand-binding domains. Divergence appeared in the internal region between the two domains.

Retinoic acid up‐regulates erythropoietin production in hepatoma cells and in vitamin A‐depleted rats

Retinoic acid (RA) stimulated the production of erythropoietin (Epo) in a human hepatoma cell line, HepG2 cells. The stimulation was due to the accumulation of Epo mRNA. The Epo production in HepG2 cells was also dependent on O2 tension for cell culture but the enhancement of Epo production by RA was independent of O2 tension, indicating that RA exerts its effect through a pathway different from O2. Oral administration of RA to the vitamin A‐depleted rats elevated the concentration of Epo in serum. These results suggest that RA up‐regulates EPO production in vivo as well as in vitro.

THE ROLE OF VITAMIN D METABOLITES IN THE TREATMENT OF OSTEOPOROSIS

The Second Symposium on Osteoporosis was organized in Tokyo, Japan on November 6, 1994, with the purpose of summarizing the current views on the biology of vitamin D, and the applications of vitamin D metabolites in osteoporosis. The Symposium was sponsored by Chugai Pharmaceuticals, Co., and Teijin Ltd., Tokyo, Japan. Dr. Etsuro Ogata (Cancer Research Institute, Tokyo, Japan) introduced the symposium observing that although vitamin D metabolites and analogs are currently being used in Japan and other countries for the treatment of osteoporosis, there is still controversy on their therapeutic role, especially in North America and Europe. Because the molecular aspects of vitamin D action on bone cells constitute the basis for understanding how vitamin D affects bone remodeling, and for the design of analogs with selective actions on bone, the first part of the symposium focused on the biologic action of vitamin D at the cellular level. The second part reviewed the role of vitamin D in the pathogenesis of osteoporosis, and in the last session the most important and controversial issues on the clinical application of active vitamin D analogs in osteoporosis were discussed.