Naomichi Matsukawa

Molecular cloning and expression of cDNAs encoding rat aldosterone synthase: Variants of cytochrome P-45011β

Abstract Two distinct forms of cDNA encoding rat aldosterone synthase were cloned from an adrenal capsular tissue cDNA library. The deduced amino acid sequences showed that one of the enzymes (P-45011β,aldo-1) had a long extension peptide composed of 34 amino acid residues while the other (P-45011β,aldo-2) had an extension peptide identical to that of rat P-45011β. Glu at the 320th position of P-45011β,aldo-1 was replaced with Lys in P-45011β,aldo-2. The amino acid sequence of the aldosterone synthase was highly homologous (81%) to rat P-45011β. Constructed expression vector containing the cDNA for extension peptide of P-45011β and the mature protein of P-45011β,aldo-1 was transfected into COS-7 cells. The cells converted 11-deoxycorticosterone into corticosterone, 18-hydroxycorticosterone, and aldosterone.

Molecular cloning and sequence analysis of cDNA encoding rat adrenal cytochrome P-45011β

A cDNA clone encoding cytochrome P‐45011β of rat adrenal has been cloned and sequenced using a bovine P‐45011β cDNA insert (pcP‐450(11β)‐2; (1987) J. Biochem. 102, 559–568) as a probe. The nucleotide sequence contains an open reading frame sufficient to encode the entire amino acid sequence of a P‐45011β precursor protein consisting of 499 amino acids including an extension peptide of 24 amino acids at the NH2‐terminus. The cDNA contains 1247 nucleotides at the 3′‐noncoding region including 51 nucleotides of poly A, but lacks the 5′‐noncoding region. The deduced amino acid sequence shows 61% similarity to that of bovine P‐45011β. Putative binding sites for heme and steroid are highly conserved among steroidogenic P‐450s of known structure.

Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone

The matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors that cause oncogenic hypophosphatemic osteomalacia (OHO). MEPE is also known as one of the bone-tooth matrix proteins and is associated with bone mineralization. We developed a rabbit polyclonal antibody directed against recombinant human MEPE (rhMEPE) after cloning its cDNA from the cDNA library of a nasal tumor tissue causing OHO. Using this antibody, we analyzed the distribution of MEPE in human bones by immunohistochemistry. In bone specimens from normal subjects, MEPE was predominantly expressed by osteocytes and not by osteoblasts. In bone specimens from patients with osteomalacia, however, MEPE was focally expressed by deeply located osteocytes. We also compared the MEPE positivity of osteocytes in mineralized bone and non-mineralized osteoid obtained from patients with osteomalacia and osteoporosis. Among osteomalacia patients, MEPE positivity was seen in 87.5 ± 8.6% of the osteocytes from mineralized bone compared with 7.8 ± 6.4% of those from osteoid. Among osteoporosis patients, MEPE positivity was found in 95.3 ± 0.5% of the osteocytes from mineralized bone compared with 4.9 ± 5.7% of those from osteoid. MEPE was mainly expressed by osteocytes embedded in the matrix of mineralized bone from patients with osteomalacia or osteoporosis. Our data provide the first histological evidence that MEPE is predominantly expressed by osteocytes in human bone, with significant expression by osteocytes within mineralized bone.

Upregulation of cAMP is a new functional signal pathway of Klotho in endothelial cells

We measured angiotensin I-converting enzyme (ACE) activity in a human endothelial cell to characterize the intracellular signal pathways of Klotho. COS-1 cells transfected with naked mouse membrane-form klotho plasmid DNA (pCAGGS-klotho) translated proper Klotho protein. This translated Klotho protein was secreted into the culture medium. Furthermore, ACE activity in human umbilical vein endothelial cells (HUVEC) was upregulated when HUVEC were co-cultured with COS-1 cells that were pre-transfected with pCAGGS-klotho. The conditioned medium from COS-1 cells pre-transfected with pCAGGS-klotho also dose-dependently upregulated ACE in HUVEC. In addition, the conditioned medium induced time- and dose-dependent enhancement of cAMP production in HUVEC. Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A (PKA), inhibited the upregulation of ACE by Klotho protein. Our results suggest that mouse membrane-form Klotho protein acts as a humoral factor to increase ACE activity in HUVEC via a cAMP-PKA-dependent pathway. These findings may provide a new insight into the mechanism of Klotho protein.

Klotho protein activates the PKC pathway in the kidney and testis and suppresses 25-hydroxyvitamin D3 1α-hydroxylase gene expression

Homozygous Klotho mutant (kl−/−) mice exhibit a variety of phenotypes resembling human aging, including arteriosclerosis, infertility, skin atrophy, osteoporosis, and short life span. Calcium abnormality, one of the phenotypes in kl−/− mice, is thought to be due to the elevated gene expression of 25-hydroxyvitamin D3 1α-hydroxylase in the kidney. We studied 25-hydroxyvitamin D3 1α-hydroxylase gene expression using a Klotho plasmid that we had previously constructed for Klotho protein production. It was found that Klotho protein medium upregulated cAMP and the PKC pathway, and suppressed 25-hydroxyvitamin D3 1α-hydroxylase in kidney cells. However, both cAMP and PKC are known to elevate 25-hydroxyvitamin D3 1α-hydroxylase gene expression, therefore, another unknown calcium regulation pathway using Klotho protein medium might exist. Furthermore, we found that activation of the PKC pathway by Klotho was observed only in the kidney and testis, where the Klotho gene is expressed, although activation of the cAMP pathway was observed in any kind of cell. These data suggest that calcium regulation through 25-hydroxyvitamin D3 1α-hydroxylase by Klotho depends on non-cAMP and a non-PKC pathway and that the Klotho protein may have different signaling pathways, depending on the Klotho gene expression in different cells and organs.

Insulin-mediated regulation of the endothelial renin-angiotensin system and vascular cell growth.

Objective Insulin has a growth-stimulating effect for vascular tissue. At the tissue level, the vascular renin–angiotensin system (RAS) may be involved in the progression of atherosclerosis or vascular hypertrophy. We previously reported that the vascular RAS activity is activated in vascular smooth muscle cells (SMC) by insulin stimulation. However, the effect of insulin on the RAS in endothelial cells (EC) is not fully understood. Methods Cultured human EC were incubated with or without insulin. After incubation for 48 h, cellular angiotensinogen and renin mRNA expression and levels in the cells were quantified by slot-blot hybridization and radioimmunoassay. Angiotensin I converting enzyme (ACE) activity in EC homogenates was measured by modified Cushman and Cheung method. EC growth and SMC with or without EC using co-culture were assessed by 3H-thymidine uptake for evaluation of their growth. Results All doses of insulin (10, 100, 1000 μU/ml) decreased angiotensinogen and renin mRNA expression (angiotensinogen: 19.3%, P < 0.05; 25.4%, P < 0.01; 26.2%, P < 0.01, renin: 12.9%, P < 0.05; 21.3%, P < 0.01; 14.3%, P < 0.05, respectively). Both cellular angiotensinogen and renin level were also reduced by high levels of insulin. Neither 10 nor 100 μU/ml insulin increased cellular angiotensin converting enzyme (ACE) activity (2.17 to 3.48-folds, P = 0.077, 0.125, respectively) significantly, but 1000 μU/ml insulin strongly up-regulated ACE activity by 16.67-folds (P = 0.001) in cultured EC. For the co-culture with EC and SMC, 100 μU/ml insulin was not able to induce SMC but 1000 μU/ml insulin accelerated SMC growth in the co-culture. In contrast insulin that was over 100 μU/ml induced SMC growth in the sole culture of SMC. Conclusion Either low or high levels of insulin suppressed angiotensinogen and renin expression, however, high doses of insulin stimulated ACE activity in cultured human aortic EC. This may indicate that insulin regulates vascular cell growth and endothelial function via bifunctional modification of the vascular angiotensin generation.

Role of endothelin-1 induced by insulin in the regulation of vascular cell growth

Insulin is not only a growth factor for vascular cells, but also an inducer of other vasoactive substances such as endothelin-1 (ET-1) in vascular cells. The aim of the present study was to assess the role of endothelial cells (EC) in insulin mediated vascular smooth muscle cell (VSMC) proliferation. Cultured human aortic EC and VSMC were separately incubated. EC were stimulated with insulin (0 to 1000 microU/mL) for 24 h, in the presence or absence of anti-insulin-growth factor-1 (anti-IGF-1) receptor antibody (alphaIR(3)) or a nonselective ET-1 receptor antagonist (TAK044). Cell proliferation was measured by determining (3)H-thymidine uptake. Although 10 microU/mL insulin did not affect ET-1 production in the EC culture medium, a higher concentration of insulin stimulated it. Production of ET-1 in EC was activated by insulin via the IGF-1 receptor, inasmuch as alphaIR(3) blocked insulin mediated upregulation of ET-1. There was no significant difference in (3)H-thymidine incorporation in the presence of insulin (up to 1000 microU/mL) or TAK044. Culture medium from EC stimulated with insulin enhanced VSMC proliferation, which was almost totally suppressed by TAK044. Insulin induced VSMC growth dose dependently when VSMC were cultured alone. In contrast, insulin at concentrations of 100 microU/mL or lower failed to stimulate growth of co-cultured VSMC, but only at 330 microU/mL or higher concentrations stimulated VSMC growth in this system. Of interest, VSMC proliferation was greatest when L-NAME was added and co-cultured with EC. In summary, a severely hyperinsulinemic state may regulate VSMC and EC proliferation via activation of vasoactive substances such as ET-1 and nitric oxide induced by insulin.

Klotho gene delivery suppresses oxidative stress in vivo

Objective:  Mice deficient in the klotho gene exhibit a syndrome resembling premature human aging. A recent report also suggested that klotho transgenic mice exhibited a long lifespan, which shows that klotho is an antisenescence gene. Previously, klotho has been reported to improve endothelial dysfunction, and also to have a preventive effect against oxidative stress. In the present study, we investigated the effect of klotho gene delivery on blood pressure and oxidative stress in vivo.

Molecular nature of aldosterone synthase, a member of cytochrome P-45011β family

The molecular nature of the aldosterone synthesizing enzymes of cattle and rat is discussed. In bovine adrenal cortex, one molecular species of cytochrome P-450(11 beta) catalyzes aldosterone synthesis as well as 11 beta-hydroxylation. The intactness of the mitochondrial membrane surrounding P-450(11 beta) in the zonae fasciculata-reticularis is essential to keep the aldosterone synthesizing activity of the cytochrome in these zones latent. In rat adrenal cortex, two distinct molecules belonging to a P-450(11 beta) family exist. One is 11 beta-hydroxylase, and the other aldosterone synthase.