Yoji Sato

Thyroid Hormone Targets Matrix Gla Protein Gene Associated With Vascular Smooth Muscle Calcification

Thyroid hormones have marked cardiovascular effects in vivo. However, their direct effects on vascular smooth muscle cells have been unclear. Because thyroid hormones play critical roles in bone remodeling, we hypothesized that they are also associated with vascular smooth muscle calcification, one of the pathological features of vascular sclerosis. To test this hypothesis, we examined the effects of 3′,3,5-triiodo-l-thyronine (T3) on the expression of calcification-associated genes in rat aortic smooth muscle cells (RAOSMCs). Quantitative RT-PCRs revealed that a physiological concentration of T3 (15 pmol/L free T3) increased mRNA level of matrix Gla protein (MGP), which acts as a potent inhibitor of vascular calcification in vivo, by 3-fold in RAOSMCs, as well as in cultured human coronary artery smooth muscle cells. In RAOSMCs transiently transfected with a luciferase reporter gene driven by the MGP promoter, T3 significantly stimulated luciferase activity. In addition, RNA interference against thyroid hormone receptor-&agr; gene diminished the effect of T3 on MGP expression. Aortic smooth muscle tissues from methimazole-induced hypothyroid rats (400 mg/L drinking water; 4 weeks) also showed a 68% decrease in the MGP mRNA level, as well as a 33% increase in calcium content compared with that from the control euthyroid animals, whereas hyperthyroidism (0.2 mg T3/kg IP; 10 days) upregulated MGP mRNA by 4.5-fold and reduced calcium content by 11%. Our findings suggest that a physiological concentration of thyroid hormone directly facilitates MGP gene expression in smooth muscle cells via thyroid hormone nuclear receptors, leading to prevention of vascular calcification in vivo.

Differential contribution of two serine residues of wild type and constitutively active β2-adrenoceptors to the interaction with β2-selective agonists

We have studied the difference in receptor binding activity between partial and full β2‐adrenoceptor agonists and the abilities of the agonists to interact with Ser204 and Ser207 in the fifth transmembrane region of the β2‐adrenoceptor, amino acid residues that are important for activation of the β2‐adrenoceptor. In the binding study with [125I]‐iodocyanopindolol, the Ki values of (±)‐salbutamol, (±)‐salmeterol, TA‐2005 and (−)‐isoprenaline for the β2‐adrenoceptor expressed in COS‐7 cell membranes were 3340, 21.0, 12.0 and 904nm, respectively. The β1/β2 selectivity of these agonists was in the order of (±)‐salmeterol (332 fold)>TA‐2005 (52.8)>(±)‐salbutamol (6.8)>(−)‐isoprenaline (1.1), and the β3‐/β2‐adrenoceptor selectivity of these agonists was in the order of TA‐2005 (150 fold)>(±)‐salmeterol (88.6)>(±)‐salbutamol (10.4)>(−)‐isoprenaline (3.2). The maximal activation of adenylyl cyclase by stimulation of the β1‐, β2‐ and β3‐adrenoceptors by TA‐2005 was 32, 100 and 100% of that by (−)‐isoprenaline, respectively, indicating that TA‐2005 is a full agonist at the β2‐ and β3‐adrenoceptors and a partial agonist at the β1‐adrenoceptor. (±)‐Salbutamol and (±)‐salmeterol were partial agonists at both β1‐ (8% and 9% of (−)‐isoprenaline) and β2‐ (83% and 74% of (−)‐isoprenaline) adrenoceptors. The affinities of full agonists, TA‐2005 and (−)‐isoprenaline, were markedly decreased by substitution of Ala for Ser204 (S204A) of the β2‐adrenoceptor, whereas this substitution slightly reduced the affinities of partial agonists, (±)‐salbutamol and (±)‐salmeterol. Although the affinities of full agonists for the S207A‐β2‐adrenoceptor were decreased, those of partial agonists for the S207A‐β2‐adrenoceptor were essentially the same as for the wild type receptor. The constitutively active mutant (L266S, L272A) of the β2‐adrenoceptor had an increased affinity for all four agonists. The affinities of full agonists were decreased by substitution of Ser204 of the constitutively active mutant, whereas the degree of decrease was smaller than that caused by the substitution of the wild type receptor. Although the affinities of (±)‐salbutamol and (±)‐salmeterol for the S207A‐β2‐adrenoceptor were essentially the same as those for the wild type β2‐adrenoceptor, the affinities of (±)‐salbutamol and (±)‐salmeterol for the constitutively active β2‐adrenoceptor were decreased by substitution of Ser207. These results suggest that Ser204 and Ser207 of the wild type and constitutively active β2‐adrenoceptors differentially interacted with β2‐selective agonists.

In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association.

Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor-coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K(d)) by 8-11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3alpha,7alpha-dihydroxy-5beta-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.

Molecular characterization of pharmacological properties of T-0509 for β-adrenoceptors

The pharmacological properties of T-0509, (-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol, were compared with those of isoproterenol. In the radioligand binding studies of [125I]iodocyanopindolol with COS-7 cell membranes that transiently expressed beta-adrenoceptor subtypes, T-0509 exhibited 11- and 97-fold greater Ki values for beta 2- and beta 3-adrenoceptors, respectively, compared with beta 1-adrenoceptors. Affinities of beta 2- and beta 3-adrenoceptors to isoproterenol were 1.4- and 28-fold lower than that of beta 1-adrenoceptors, respectively. The maximal stimulatory effects of T-0509 on adenylyl cyclase of CHO-K1 (chinese hamster ovary K1) cell membranes expressing beta 1- or beta 2-adrenoceptors were 85% or 96% of those produced by isoproterenol, respectively. These results indicate that T-0509 is a relatively specific beta 1-adrenoceptor agonist with a high intrinsic activity as compared with isoproterenol.

HX531, a retinoid X receptor antagonist, inhibited the 9-cis retinoic acid-induced binding with steroid receptor coactivator-1 as detected by surface plasmon resonance

HX531 is a retinoid X receptor (RXR) antagonist that inhibits 9-cis retinoic acid-induced neutrophilic differentiation of HL-60 cells. In order to elucidate the inhibitory mechanism of HX531, we have developed a novel ligand sensor assay for RXR in which the receptor-coactivator interaction is directly monitored using surface plasmon resonance (SPR) biosensor technology. A 20-mer peptide from steroid receptor coactivator-1 (SRC-1), containing nuclear receptor interaction motif LXXLL was immobilized on the surface of a BIAcore sensor chip. Injection of human recombinant RXR with or without 9-cis retinoic acid resulted in ligand-dependent interaction with the SRC-1 peptide. Kinetic analysis revealed dissociation constants (KD) of 9-cis RA-preincubated RXR to SRC-1 was 5.92 x 10(-8)M. Using this technique, we found that 1 microM HX531 reduced the ka value of liganded-RXR with SRC-1, suggesting that HX531 reduced the affinity of RXR to SRC-1. This SPR assay system was applied to obtain quantitative kinetic data of RXR ligand binding to the SRC-1 peptide and the alteration of these data by antagonists.