Noboru Horiuchi

Induction of osteoblast differentiation indices by statins in MC3T3‐E1 cells

Statins inhibit 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase, which catalyzes conversion of HMG‐CoA to mevalonate, a rate‐limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10−7 M markedly increased mRNA expression for bone morphogenetic protein‐2 (BMP‐2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3‐E1), while suppressing gene expression for collagenase‐1, and collagenase‐3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase‐digestive proteins, and noncollagenous proteins was increased in the cells treated with 10−7 M simvastatin, or 10−8 M cerivastatin. In the culture of MC3T3‐E1 cells, statins stimulated mineralization; pretreating MC3T3‐E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin‐induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.

Leptin stimulates fibroblast growth factor 23 expression in bone and suppresses renal 1α,25-dihydroxyvitamin D3 synthesis in leptin-deficient ob/ob Mice

Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25‐hydroxyvitamin D3 1α‐hydroxylase (CYP27B1) gene through the leptin receptor (ObRb) by indirectly acting on the proximal tubules. This study focused on bone‐derived fibroblast growth factor 23 (FGF‐23) as a mediator of the influence of leptin on renal 1α‐hydroxylase mRNA expression in leptin‐deficient ob/ob mice. Exposure to leptin (200 ng/mL) for 24 hours stimulated FGF‐23 expression by primary cultured rat osteoblasts. Administration of leptin (4 mg/kg i.p. at 12‐hour intervals for 2 days) to ob/ob mice markedly increased the serum FGF‐23 concentration while significantly reducing the serum levels of calcium, phosphate, and 1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3]. Administration of FGF‐23 (5 µg i.p. at 12‐hour intervals for 2 days) to ob/ob mice suppressed renal 1α‐hydroxylase mRNA expression. The main site of FGF‐23 mRNA expression was the bone, and leptin markedly increased the FGF‐23 mRNA level in ob/ob mice. In addition, leptin significantly reduced 1α‐hydroxylase and sodium‐phosphate cotransporters (NaPi‐IIa and NaPi‐IIc) mRNA levels but did not affect Klotho mRNA expression in the kidneys of ob/ob mice. Furthermore, the serum FGF‐23 level and renal expression of 1α‐hydroxylase mRNA were not influenced by administration of leptin to leptin receptor–deficient (db/db) mice. These results indicate that leptin directly stimulates FGF‐23 synthesis by bone cells in ob/ob mice, suggesting that inhibition of renal 1,25(OH)2D3 synthesis in these mice is at least partly due to elevated bone production of FGF‐23.

Mouse Vitamin D-24-Hydroxylase: Molecular Cloning, Tissue Distribution, and Transcriptional Regulation by 1α,25-Dihydroxyvitamin D31

Vitamin D-24-hydroxylase (24-OHase) is a cytochrome P-450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 (25OHD3) and 1α,25-dihydroxyvitamin D3[ 1,25-(OH)2D3] to 24,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3, respectively. A full-length complementary DNA for mouse 24-OHase has now been characterized. The complementary DNA consists of 3309 bp and encodes a protein of 514 amino acids that shows 82% and 95% sequence identity with the human and rat enzymes, respectively. Northern blot analysis of tissues from mice injected with 1,25-(OH)2D3 (24 pmol/g) revealed that the 3.4-kb 24-OHase messenger RNA (mRNA) is most abundant in kidney and intestine, with smaller amounts present in skin, thymus, and bone. RT-PCR and Southern blot analysis detected 24-OHase mRNA in several other tissues including lung, testis, spleen, pancreas, and heart. Intraperitoneal injection of 1,25-(OH)2D3 induced dose- and time-dependent increases in both 24-OHase mRNA abundance and enzyme activity in mouse kidn...

Localization of 25-hydroxyvitamin D3-lα-hydroxylase activity in the mammalian kidney

Summary Intra-renal distribution of 25-hydroxyvitamin D 3 (25-OH-D 3 )-lα-hydroxylase activity was studied in single nephron segments prepared from New Zealand White rabbit fetuses (26th to 28th day of gestation). Fetal kidneys were treated with collagenase and the isolated nephrons were micro-dissected into five differrent parts. Each segment of the nephron was incubated with [ 3 H]-25-OH-D 3 . Metabolites of [ 3 H]-25-OH-D 3 were separated by high performance liquid chromatography. The results show that 25-OH-D 3 -1α-hydroxylase activity is localized only in the proximal tubule and that the pars recta of proximal tubule possesses higher activity than the proximal convoluted tubule. The findings indicate that the major and probably exclusive site of 1α,25-(OH) 2 -D 3 synthesis is the proximal tubule not only in birds, as reported by Brunette et al. (8) , but also in mammals.

Simvastatin Suppresses Leptin Expression in 3T3-L1 Adipocytes via Activation of the Cyclic AMP-PKA Pathway Induced by Inhibition of Protein Prenylation

Simvastatin inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyses conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. We demonstrated that simvastatin at 1 microM markedly inhibited adipocyte differentiation measured by Oil Red O staining in preadipocyte cells (3T3-L1), while expression of leptin, a marker of adipocyte differentiation, was suppressed by 1 muM simvastatin for up to 12 days of culture. Next, to elucidate mechanisms underlying the reduction of leptin expression induced by simvastatin, differentiated 3T3-L1 adipocytes were treated with various inhibitors with mevalonate or its metabolite in the presence or absence of simvastatin. Simvastatin time- and dose-dependently suppressed leptin mRNA expression. Heterogeneous nuclear RNA related to leptin mRNA was inhibited by 10 muM simvastatin, while stability of the mRNA was not changed by treatment with simvastatin in transcription-arrested 3T3-L1 cells. Simvastatin inhibition of leptin gene transcription was not abrogated by pre-treatment with cycloheximide, an inhibitor of protein synthesis. Addition of mevalonate or geranylgeranyl pyrophosphate (GGPP), a mevalonate metabolite, abolished simvastatin-induced inhibition of leptin expression in 3T3-L1 cells. Suppression of expression was observed upon addition of GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor. Expression was suppressed by treatment with hydroxyfasudil, a protein prenylation inhibitor. Treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, reduced leptin expression in 3T3-L1 cells. Simvastatin dose-dependently increased intra-cellular cyclic AMP (cAMP) concentrations in 3T3-L1 cells, with maximal stimulation obtained at 10 muM. Addition of GGPP abolished simvastatin-induced stimulation of cAMP accumulation and protein kinase A (PKA) activity. H89, an inhibitor of PKA, completely abolished simvastatin-induced suppression of leptin expression. These results suggested that simvastatin reduced geranylgeranylprotein prenylation followed by deactivation of PI3K, leading to cAMP accumulation and subsequent activation of PKA in differentiated 3T3-L1 adipocytes. Finally, PKA inhibited leptin gene transcription without new protein synthesis.

Dexamethasone enhances vitamin D-24-hydroxylase expression in osteoblastic (UMR-106) and renal (LLC-PK1) cells treated with 1α,25-dihydroxyvitamin D3

Chronic glucocorticoid therapy causes rapid bone loss and clinical osteoporosis. We previously found that dexamethasone, a potent glucocorticoid, increased renal expression of vitamin D-24-hydroxylase, which degrades such vitamin D metabolites as 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3). We therefore investigated the mechanisms of this increase in UMR-106 osteoblast-like cells and LLC-PK1 kidney cells. To induce 24-hydroxylase expression, 1,25(OH)2D3 (10−7M) and dexamethasone were added simultaneously to the medium of LLC-PK1 cells, and 24 h before dexamethasone treatment, 1,25(OH)2D3 was added to the medium of UMR-106 cells. Dexamethasone dose dependently increased 24-hydroxylase mRNA and enzymatic activity in 1,25(OH)2D3-treated LLC-PK1 and UMR-106 cells. Maximal stimulation was observed with 10−6M dexamethasone in both cell lines. The addition of 10−6M dexamethasone significantly increased the abundance of 24-hydroxylase mRNA by 24 and 8 h in 1,25(OH)2D3-treated LLC-PK1 and UMR-106 cells, respectively. Stimulation for dexamethasone in UMR-106 cells persisted for up to 48 h. Dexamethasone stimulation of 24-hydroxylase mRNA expression in UMR-106 cells was abolished by pretreatment with cycloheximide, an inhibitor of protein synthesis. Northern and Western analyses indicated that 10−6M dexamethasone markedly increased the abundance of c-fos mRNA at 20 min and c-fos protein concentration at 60 min in 1,25(OH)2D3-treated UMR-106 cells but only slightly induced the abundance of c-jun mRNA. The addition of phorbol 12-myristate 13-acetate increased mRNA expression for both c-fos and 24-hydroxylase in 1,25(OH)2D3-treated UMR-106 cells. The effect of dexamethasone on 24-hydroxylase mRNA expression was blocked by RO31-8220, a specific inhibitor of protein kinase C. Thus, dexamethasone in the presence of 1,25(OH)2D3 enhances expression of 24-hydroxylase in UMR-106 osteoblastic cells via new protein synthesis. The mechanism of this effect appears to involve activation of the AP-1 site by increased c-fos protein.

Atorvastatin enhances bone density in ovariectomized rats given 17β-estradiol or human parathyroid hormone(1–34)

We investigated the in vivo effect of atorvastatin on bone mineral density (BMD) in ovariectomized (OVX) rats. Eight-week-old female rats underwent either a sham operation or ovariectomy, and treatments with vehicle, atorvastatin, 17β-estradiol (E2) and human parathyroid hormone(1–34) [hPTH(1–34)] were initiated 6 wk after the surgery. E2 (10 µg/kg) treatment for 12 wk significantly increased lumbar BMD (L2–L4), whereas atorvastatin did not affect lumbar BMD. The combination of atorvastatin (2 mg/kg) and E2 significantly enhanced the BMD of lumbar vertebrae (L2–L4) and femoral metaphyseal area (2/10–4/10 segments from the most proximal point) compared to that of either E2 or atorvastatin alone. A low dose 1 µg/kg of hPTH (1–34) did not alter lumbar or femoral BMD, whereas a high dose 17.5 µg/kg of the peptide significantly increased BMD. Concomitant injections of atorvastatin (2 mg/kg) with hPTH(1–34) (1 µg/kg) for 8 wk significantly enhanced the BMD of lumbar vertebrae and the metaphyseal area of the femur in OVX rats. These findings demonstrate that chronic administration of atorvastatin appears to modestly enhance the BMD of the lumbar vertebrae and femoral metaphysis of OVX rats treated with submaximal doses of E2 and hPTH(1–34).

Induction of vitamin D 24-hydroxylase messenger RNA and activity by 22-oxacalcitriol in mouse kidney and duodenum: Possible role in decrease of plasma 1α,25-dihydroxyvitamin D3

The synthetic analog of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], 22-oxacalcitriol (OCT), retains most of the properties of 1,25(OH)2D3 but exhibits much less hypercalcemic action than the parent compound. The effects of OCT on plasma calcium, phosphorus, and 1 alpha,25-dihydroxyvitamin D [1,25(OH)2D] concentrations were examined in mice. Administration of a single dose (24 pmol/g body wt, i.p.) of OCT had no effect on plasma calcium for up to 48 hr, significantly increased plasma phosphorus at 4 and 8 hr and significantly reduced the concentration of 1,25(OH)2D in plasma between 4 and 48 hr. Both OCT and 1,25(OH)2D3 at 24 pmol/g body wt (i.p.) induced a single, 3.4-kb mRNA encoding vitamin D 24-hydroxylase (24-OHase), the cytochrome P450 enzyme responsible for 1,25(OH)2D3 degradation, in kidney and duodenum. The OCT-induced increase in 24-OHase mRNA and an increase in enzyme activity were marked at 2 and 4 hr in both tissues. In kidney, mRNA abundance had decreased by 8 hr but remained above basal values for up to 30 hr; activity remained relatively high for up to 48 hr. In duodenum, 24-OHase mRNA abundance returned virtually to control values by 8 hr after OCT treatment; activity remained at nearly maximal levels for up to 30 hr but was decreased at 48 hr. The effects of OCT and 1,25(OH)2D3 on 24-OHase mRNA abundance and enzyme activity were dose-dependent in kidney and duodenum. Whereas the dose-response relations for the effects of both compounds on 24-OHase mRNA were similar, OCT was slightly more potent than 1,25(OH)2D3 in stimulating 24-OHase activity in both tissues. These results suggest that the OCT-induced decrease in plasma 1,25(OH)2D3 is attributable, at least in part, to an increased degradation of 1,25(OH)2D3, which results from an increase in 24-OHase abundance and enzyme activity.

Effect of Leptin on Regulation of Renal 25-hydroxyvitamin D3 Metabolism and Maintenance of Calcium Homeostasis

Abstract Leptin, a hormone secreted by adipocytes, acts on the hypothalamus to reduce appetite and inhibit body weight gain. Leptin has pleiotropic actions on bone, gonadal hormone secretion, the pancreas, immune tissues, and vascular organs. Leptin-deficient ( ob / ob ) mice, used in many studies defining leptin actions, have low bone mineral density (BMD) and retarded femoral growth. Loss of leptin function contributes to aberrant regulation of renal 25-hydroxyvitamin D 3 metabolism by increasing the gene expression of renal 1α-hydroxylase, which catalyzes 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] synthesis and thus elevates serum 1,25(OH) 2 D 3 concentrations. Excess 1,25(OH) 2 D 3 results in increased calcium absorption in the intestine and stimulates bone resorption, resulting in reduced BMD, especially in long bones. Finally, leptin deficiency increases serum calcium concentrations; excess calcium in blood is excreted in urine.

Dexamethasone Suppresses Parathyroid Hormone-related Protein Expression by Human Oral Squamous Carcinoma Cells (HSC-3)

Abstract We examined the effect of dexamethasone on parathyroid hormone-related protein (PTHrP) mRNA expression and PTHrP production in HSC-3 cells, a human oral squamous carcinoma cell line. Dexamethasone suppressed PTHrP mRNA expression and PTHrP production in a dose-dependent manner. A time course study demonstrated a significant decrease in PTHrP mRNA from as early as 6 h after the initiation of exposure to 10 −1 M dexamethasone, and the suppressive effect was stronger at 24 h. Dexamethasone-induced suppression of PTHrP mRNA expression and PTHrP production was abolished in the presence of RU486, a glucocorticoid receptor antagonist. Dexamethasone decreased the transcriptional activity of the PTHrP promoter. These findings show that dexamethasone acts through binding to functional glucocorticoid receptors and down-regulates PTHrP gene expression via a transcriptional mechanism in HSC-3 cells.