Nuclear Receptor-binding Domain Research Articles

THU412 Role of Glucocorticoid On Bone Metabolism Through AKAP13

Abstract Disclosure: K. Igarashi: None. A. Ishida: None. H. Koide: None. K. Yokote: None. It is not completely clear to what extent glucocorticoids are involved in which stages and sites of bone differentiation, development, and maintenance. we investigated the mechanism of glucocorticoid regulation through the regulation of Rho activation using a regulatory system for the expression of the RhoA activator AKAP13. To examine the effect of glucocorticoids on preosteoblasts, immortalized bone marrow mesenchymal stem cells (imBMSCs) were sown in 6-well plates at a concentration of 2x105/well and treated with various concentrations (0-1000 nM) of dexamethasone (Dex) 24 hr later, and The expression of GILZ, osteoblast and adipocyte-associated markers were evaluated using qRT-PCR. AKAP13 expression vector and AKAP13 siRNA were used to examine the response to Dex. GILZ was induced in a Dex-dependent manner on both day1 and day4. In the differentiation phase, PPARG2 expression was not changed by Dex, but GILZ was induced in a similar Dex-dependent manner. In proliferating imBMSCs, Dex treatment tended to enhance PPARG2 expression in a dose-dependent manner by introducing AKAP13 expression vectors. On the other hand, AKAP13 gene expression was significantly down-regulated in imBMSCs treated with AKAP13 siRNA, and Alp2 mRNA was up-regulated in imBMSCs treated with Dex compared to those not treated with Dex. It has a nuclear receptor binding domain and is known to be involved in osteogenesis. On the other hand, AKAP13 expression was decreased by osteoblast differentiation in an in vitro system, suggesting that AKAP13 may influence the regulation of GC action between the proliferative and differentiation phases of BMSCs. Presentation: Thursday, June 15, 2023

Yeast Two-Hybrid Detection Systems That Are Highly Sensitive to a Certain Kind of Endocrine Disruptors

We tested the effects of several combinations of bait and fish components of the yeast two-hybrid detection system for estrogenic activity. A combination of the full-length human estrogen receptor alpha with the nuclear receptor-binding domain of co-activator steroid receptor co-activator-1 (SRC-1) or transcriptional intermediate factor-2 (TIF-2) was most effective for estrogen-dependent induction of the chromosome-integrated UAS(GAL)-CYC1(p)-lacZ reporter construct among the two-hybrid systems so far tested.

The Interaction of TRβ1-N Terminus with Steroid Receptor Coactivator-1 (SRC-1) Serves a Full Transcriptional Activation Function of SRC-1

Steroid receptor coactivator-1 (SRC-1) plays a crucial role in nuclear receptor-mediated transcription including thyroid hormone receptor (TR)-dependent gene expression. Interaction of the TR-ligand binding domain and SRC-1 through LXXLL motifs is required for this action. However, potential interactions between the TRbeta1-N terminus (N) and SRC-1 have not been explored and thus are examined in this manuscript. Far-Western studies showed that protein construct containing TRbeta1-N + DNA binding domain (DBD) bound to nuclear receptor binding domain (NBD)-1 (amino acid residue, aa 595-780) of SRC-1 without ligand. Mammalian two-hybrid studies showed that NBD-1, as well as SRC-1 (aa 595-1440), bound to TRbeta1-N+DBD in the absence of ligand in CV-1 cells. However, NBD-2 (aa 1237-1440) did not bind to this protein. Glutathione-S-transferase pull-down studies showed that TRbeta1-N (aa 1-105) bound to the broad region of SRC-1-C terminus. Expression vectors encoding a series of truncations and/or point mutations of TRbeta1 were used in transient transfection-based reporter assays in CV-1 cells. N-terminal truncated TRbeta1 (DeltaN-TRbeta1) showed lower activity than that of wild-type in both artificial F2-thyroid hormone response element and native malic enzyme response element. These results suggest that there is the interaction between N terminus of TRbeta1 and SRC-1, which may serve a full activation of SRC-1, together with activation function-2 on TRbeta1-mediated transcription.

Employment of the human estrogen receptor beta ligand-binding domain and co-activator SRC1 nuclear receptor-binding domain for the construction of a yeast two-hybrid detection system for endocrine disrupters.

To screen a wide variety of chemicals for endocrine disrupters, and to develop an effective microbial degradation system for them, a good system is needed for the rapid and accurate evaluation of the endocrine-disrupting activities of suspected chemicals and their degradation products. We constructed two-hybrid systems that co-express the Gal4p DNA binding domain/ligand-binding domain of human estrogen receptor (hER) alpha or beta and the Gal4p transactivation domain/nuclear receptor-binding domain of co-activator SRC1, TIF2, or AIB1 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of Gal4p-binding sites. We found that the combination of the hERbeta ligand-binding domain and SRC1 nuclear receptor-binding domain was most effective for the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by known xenoestrogens and phytoestrogens was found to correlate well with their estrogenic activities as measured by the previous system with rat ERalpha. This system detects estrogenic activity in some chemicals that have not been suspected of being positive. We also applied this assay system to test the microbial degradation products of gamma-hexachlorocyclohexane (gamma-HCH) by Sphingomonas paucimobilis. Among the gamma-HCH metabolites, 2,5-dichlorohydroquinone and chlorohydroquinone had estrogenic activities similar to the original chemical, while hydroquinone, a later stage metabolite, showed no activity, suggesting the necessity of evaluating intermediate metabolites in microbial degradation systems.

HCaRG, a Novel Calcium-regulated Gene Coding for a Nuclear Protein, Is Potentially Involved in the Regulation of Cell Proliferation

Since a negative calcium balance is present in spontaneously hypertensive rats, we searched for the gene(s) involved in this dysregulation. A cDNA library was constructed from the spontaneously hypertensive rat parathyroid gland, which is a key regulator of serum-ionized calcium. From seven overlapping DNA fragments, a 1100-base pair novel cDNA containing an open reading frame of 224 codons was reconstituted. This novel gene, named HCaRG (hypertension-related, calcium-regulated gene), was negatively regulated by extracellular calcium concentration, and its basal mRNA levels were higher in hypertensive animals. The deduced protein showed no transmembrane domain, 67% alpha-helix content, a mutated calcium-binding site (EF-hand motif), four putative "leucine zipper" motifs, and a nuclear receptor-binding domain. At the subcellular level, HCaRG had a nuclear localization. We cloned the human homolog of this gene. Sequence comparison revealed 80% homology between rats and humans at the nucleotide and amino acid sequences. Tissue distribution showed a preponderance in the heart, stomach, jejunum, kidney (tubular fraction), liver, and adrenal gland (mainly in the medulla). HCaRG mRNA was significantly more expressed in adult than in fetal organs, and its levels were decreased in tumors and cancerous cell lines. We observed that after 60-min ischemia followed by reperfusion, HCaRG mRNA declined rapidly in contrast with an increase in c-myc mRNA. Its levels then rose steadily to exceed base line at 48 h of reperfusion. HEK293 cells stably transfected with HCaRG exhibited much lower proliferation, as shown by cell count and [(3)H]thymidine incorporation. Taken together, our results suggest that HCaRG is a nuclear protein potentially involved in the control of cell proliferation.