Structure, gene expression, and evolution of primate copper chaperone for superoxide dismutase.

Copper chaperone for superoxide dismutase (CCS) is essential for transporting copper ion to Cu,Zn-superoxide dismutase (Cu,Zn-SOD). We cloned cDNAs for six primate species' CCSs. The total number of amino acid residues of primate CCSs is 274. Similarities between primates were over 96%. Important residues for the CCS function were well conserved. A phylogenetic tree of CCSs and Cu,Zn-SODs from various organisms showed that these two proteins were derived from a common ancestor, diverging very early on during eukaryote evolution. The high frequency of nonsynonymous substitutions was found in the lineage to Old World monkeys and apes. Expression of the CCS gene in various tissues of Japanese monkey was found to be high in the liver and adrenal gland, followed by the kidney and small intestine. Such expressional pattern was similar with that of Cu,Zn-SOD gene (Fukuhara et al., 2002).

Physiological function of the angiotensin AT1a receptor in bone remodeling.

In order to determine whether the renin-angiotensin system (RAS) has any physiologic function in bone metabolism, mice lacking the gene encoding the major angiotensin II receptor isoform, AT1a, were studied using micro CT scanning, histomorphometric, and biochemical techniques. Three-dimensional (3D) micro CT analysis of the tibial metaphysis revealed that both male and female AT1a knockout mice exhibited an increased trabecular bone volume along with increased trabecular number and connectivity. Histomorphometric analysis of the tibial metaphysis indicated that the parameters of bone formation as well as resorption were increased, which was also supported by elevated serum osteocalcin and carboxy-terminal collagen crosslink (CTX) concentrations in the AT1a-deficient mice. Osteoclastogenesis and osteoblastogenesis assays in ex vivo cultures, however, did not reveal any intrinsic alterations in the differentiation potential of AT1a-deficient cells. Quantitative RT-PCR using RNA isolated from the tibia and femur revealed that the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio and the expression of stromal cell-derived factor (SDF)1α were increased, whereas that of SOST was decreased in AT1a-deficient bone, which may account for the increased bone resorption and formation, respectively. AT1a-deficient mice also displayed a lean phenotype with reduced serum leptin levels. They maintained high bone mass with advancing age, and were protected from bone loss induced by ovariectomy. Collectively, the data suggest that RAS has a physiologic function in bone remodeling, and that signaling through AT1a negatively regulates bone turnover and bone mass.

Activation of renin-angiotensin system induces osteoporosis independently of hypertension.

Hypertension and osteoporosis are two major age-related disorders; however, the underlying molecular mechanism for this comorbidity is not known. The renin-angiotensin system (RAS) plays a central role in the control of blood pressure and has been an important target of antihypertensive drugs. Using a chimeric RAS model of transgenic THM (Tsukuba hypertensive mouse) expressing both the human renin and human angiotensinogen genes, we showed in this study that activation of RAS induces high turnover osteoporosis with accelerated bone resorption. Transgenic mice that express only the human renin gene were normotensive and yet exhibited a low bone mass, suggesting that osteoporosis occurs independently of the development of hypertension per se. Ex vivo cultures showed that angiotensin II (AngII) acted on osteoblasts and not directly on osteoclast precursor cells and increased osteoclastogenesis-supporting cytokines, RANKL and vascular endothelial growth factor (VEGF), thereby stimulating the formation of osteoclasts. Knockdown of AT2 receptor inhibited the AngII activity, whereas silencing of the AT1 receptor paradoxically enhanced it, suggesting a functional interaction between the two AngII receptors on the osteoblastic cell surface. Finally, treatment of THM mice with an ACE inhibitor, enalapril, improved osteoporosis and hypertension, whereas treatment with losartan, an angiotensin receptor blockers specific for AT1, resulted in exacerbation of the low bone mass phenotype. Thus, blocking the synthesis of AngII may be an effective treatment of osteoporosis and hypertension, especially for those afflicted with both conditions.

Structure, gene expression, and evolution of primate glutathione peroxidases.

Glutathione peroxidases (GPxs) are a family of enzymes that scavenge peroxides generated in cells. We carried out molecular cloning for cDNAs of four GPx isozymes (GPx-1 through 4) in primate species. The essential residues for the function of these isozymes were well conserved. A phylogenetic tree of GPx isozymes of primates and other mammals showed that GPx-4 diverged first, followed by GPx-3, GPx-2, and GPx-1. Expression of mRNAs for GPx-2 through 4 in various tissues of Japanese monkey was analyzed by Northern blot hybridization. GPx-2 mRNA was detected at 1.7 kb, exclusively in the stomach and small intestine. GPx-3 mRNA was detected at 1.8 kb, intensively in the kidney and adrenal gland, and weakly in the cerebellum, heart, and lung. GPx-4 mRNA was detected at 1.1 kb, very intensively in the testis and weakly in lung, heart, and cerebellum. These results showed that GPx isozymes were expressed under tissue-specific regulations.

Tissue distribution, molecular cloning, and gene expression of cytosolic glutathione peroxidase in Japanese monkey.

Cytosolic glutathione peroxidase (GPX-1) is an important antioxidant enzyme that scavange hydrogen peroxide in mammalian cells. The level of GPX-1 activity in Japanese monkey (Macaca fuscata) tissues was determined and it was found to be high in the liver, kidney, and adrenal gland followed by the small intestine. We also cloned the GPX-1 cDNA that included the whole protein-coding region. The active-site selenocysteine was assumed to be encoded by a TGA codon. Compared to the GPX-1s of other mammalian species, essential residues in catalysis were well conserved in monkey GPX-1. Amino acid substitutions were frequent in the N- and C-terminal regions which are less essential in catalysis. Expression of GPX-1 mRNA was found to be high in the liver, kidney, and adrenal gland, in consistence with the tissue distribution of GPX-1 activity.

Structure, molecular evolution, and gene expression of primate superoxide dismutases.

Mn- and Cu,Zn-superoxide dismutase (SOD) cDNAs of eight primate species, Pan troglodytes, Pongo pygmaeus, Hylobates lar, Macaca fuscata, Macaca fascicularis, Macaca mulatta, Cebus apella, and Callithrix jacchus, were cloned. The whole protein-coding sequences were covered, comparing 198 and 153 (or 154) amino acids, for Mn- and Cu,Zn-SODs, respectively. Residues forming metal ligands were completely conserved in the two primate SODs and nucleotide/amino acid substitutions were more frequent in Cu,Zn-SODs than in Mn-SODs. Molecular evolutionary analyses showed Mn-SOD to have evolved at a constant rate and its phylogenetic tree well reflected primate phylogeny. Cu,Zn-SOD was shown to have evolved differently between primate lineages. The significant high ratio of a non-synonymous/synonymous rate was found in the lineage leading to great apes and humans, showing that this lineage underwent positive Darwinian selection. Southern hybridization suggested that the genes for primate Mn- and Cu,Zn-SOD exist as single copies. Northern analysis in various Japanese monkey tissues showed Mn- and Cu,Zn-SOD expression to be high in the liver, kidneys, and adrenal glands.