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Published as doi: 10.1096/fj.07-098954.
 (The FASEB Journal. 2008;22:2465-2475.)
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Angiotensin II accelerates osteoporosis by activating osteoclasts

Hideo Shimizu*,{dagger},1, Hironori Nakagami{ddagger},1, Mariana Kiomy Osako*, Rie Hanayama*, Yasuo Kunugiza§, Takuji Kizawa§, Tetsuya Tomita§, Hideki Yoshikawa§, Toshio Ogihara{dagger} and Ryuichi Morishita*,2

* Division of Clinical Gene Therapy,

{dagger} Department of Geriatric Medicine,

{ddagger} Division of Gene Therapy Science, and

§ Department of Orthopedic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan

2Correspondence: Division of Clinical Gene Therapy, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: morishit{at}cgt.med.osaka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent clinical studies suggest that several antihypertensive drugs, especially angiotensin-converting enzyme inhibitors, reduced bone fractures. To clarify the relationship between hypertension and osteoporosis, we focused on the role of angiotensin II (Ang II) on bone metabolism. In bone marrow-derived mononuclear cells, Ang II (1x10–6 M) significantly increased tartrate-resistant acid phosphatase (TRAP) -positive multinuclear osteoclasts. Of importance, Ang II significantly induced the expression of receptor activator of NF-{kappa}B ligand (RANKL) in osteoblasts, leading to the activation of osteoclasts, whereas these effects were completely blocked by an Ang II type 1 receptor blockade (olmesartan) and mitogen-activated protein kinase kinase inhibitors. In a rat ovariectomy model of estrogen deficiency, administration of Ang II (200 ng/kg/min) accelerated the increase in TRAP activity, accompanied by a significant decrease in bone density and an increase in urinary deoxypyridinoline. In hypertensive rats, treatment with olmesartan attenuated the ovariectomy-induced decrease in bone density and increase in TRAP activity and urinary deoxypyridinoline. Furthermore, in wild-type mice ovariectomy with five-sixths nephrectomy decreased bone volume by microcomputed tomography, whereas these change was not detect in Ang II type 1a receptor-deficient mice. Overall, Ang II accelerates osteoporosis by activating osteoclasts via RANKL induction. Blockade of Ang II might become a novel therapeutic approach to prevent osteoporosis in hypertensive patients.—Shimizu, H., Nakagami, H., Osako, M. K., Hanayama, R., Kunugiza, Y., Kizawa, T., Tomita, T., Yoshikawa, H., Ogihara, T., Morishita, R. Angiotensin II accelerates osteoporosis by activating osteoclasts.


Key Words: hypertension • renin-angiotensin system • RANKL • ARB • TRAP activity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HYPERTENSION AND OSTEOPOROSIS are two common diseases in the elderly population, which are caused by the interaction of genetic and environmental factors. As 50% of the hypertensive population comprises postmenopausal women at high risk of osteoporosis, hypertension represents a considerable health problem in this population. Animal and epidemiological evidence suggests that high blood pressure is associated with abnormalities of calcium metabolism, leading to an increase in calcium loss, secondary activation of the parathyroid gland, and increased movement of calcium from bone, thereby increasing the risk of osteoporosis (1 , 2) . Indeed, clinical studies have shown that antihypertensive drugs such as thiazides decrease the risk of hip fracture by reducing renal calcium excretion (3 , 4) . However, other antihypertensive drugs [β-blockers and angiotensin-converting enzyme (ACE) inhibitors] are also associated with a reduced risk of fractures (5) , whereas calcium antagonists did not reduce the risk (5) . Recent clinical studies also support the benefit of ACE inhibitors to reduce risk of fractures or improve bone metabolism (6 , 7) . These data suggest that the renin-angiotensin system might be involved in bone metabolism.

Because the vasculature plays an important role in bone remodeling, the effect of the renin-angiotensin system on bone metabolism may be partially related to the regulation of blood flow. However, there is no report documenting a direct relation of the renin-angiotensin system with bone metabolism. Although previous reports showed that the receptor for Ang II is expressed in osteoblasts and osteoclasts, the effects of Ang II are controversial (8 , 9) . Therefore, in this study, we examined whether the renin-angiotensin system is directly involved in bone metabolism, focusing on osteoclast activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Bone marrow cells were obtained from 3-day-old neonatal white rabbits as described previously (10) . Briefly, rabbit bone marrow cells were flushed out from the femurs and tibiae, collected into tubes, and washed twice with PBS. The mononuclear cell-rich fraction was separated from marrow cells by density gradient centrifugation with Ficoll and cultured (1x105 cells/well of 24-well plate) in {alpha}-minimal essential medium ({alpha}-MEM) containing 10% FBS.

Osteoclast differentiation was also examined using a rat osteoclast culture system obtained from Hokudo Co. Ltd. (Sapporo, Japan). Rat osteoclast precursor cells seeded in a 24-well plate were incubated with macrophage colony-stimulating factor (M-CSF) (10 ng/ml) and receptor activator of NF-{kappa}B ligand (RANKL; also called OPGL, TRANCE, and ODF) (10 ng/ml) -containing medium or Ang II (1x10–6, 1x10–7, or 1x10–8 M) to examine the differentiation of osteoclasts. Human osteoblasts or osteoclast precursors were obtained from Cell Applications (San Diego, CA, USA) or Lonza (Walkersville, MD, USA), respectively.

Tartrate-resistant acid phosphatase (TRAP) staining
After treatment with 1,25-dihydroxyvitamin D3 (vitamin D3) (1x10–8 M) or Ang II (1x10–8, 1x10–7, or 1x10–6 M), mononuclear cells were fixed with 4.0% paraformaldehyde in PBS for 10 min at room temperature before being stained for TRAP. Rat osteoclasts were treated similarly after treatment with M-CSF and RANKL or Ang II (1x10–6, 1x10–7, or 1x10–8 M). Enzyme histochemical staining for TRAP and Hoechst 33526 nuclear staining were performed as reported previously (11) .

Real-time reverse transcription (RT) -polymerase chain reaction (PCR)
Human RANKL, receptor activator of NF-{kappa}B (RANK), and osteoprotegerin (OPG) expressions were measured by real-time RT-PCR. Total RNA of cells or tissue samples was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) or Isogen (Nippon Gene, Toyama, Japan). cDNA was synthesized using the Thermo Script RT-PCR System (Invitrogen, Carlsbad, CA, USA). Relative gene copy numbers of RANKL, OPG, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified by real-time RT-PCR using TaqMan Gene Expression Assays (human RANKL: Hs00243522, human OPG: Hs00900360, human RANK: Hs00921375, and human GAPDH: Hs99999905; Applied Biosystems, Foster City, CA, USA). The absolute number of gene copies was normalized using GAPDH and standardized by a sample standard curve.

Western blotting
Western blotting was performed for analysis of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and Akt expression using a phospho-specific antibody as described previously (12) . After treatment, cells were extracted with lysis buffer (50 mM Tris-Cl, 2.5 mM EGTA, 1 mM EDTA, 10 mM NaF, 1% Triton X-100, 1 mM PMSF, and 2 mM Na3VO4). Samples containing 20 µg of protein were separated on 10% sodium dodecyl sulfate (SDS) -polyacrylamide gels, transferred to nitrocellulose membranes (Hybond ECL; Amersham Biosciences Corp., Piscataway, NJ, USA), and incubated with a polyclonal antibody against phospho-specific or total ERK, phospho-specific or total p38 MAPK, and phospho-specific or total Akt (polyclonal rabbit IgG, 1:1000; Cell Signaling Technology Inc., Danvers, MA, USA) at 4°C overnight. The membranes were then washed and incubated with a 1:5000 dilution of anti-rabbit IgG horseradish peroxidase-conjugated antibody (Amersham Biosciences Corp.). Bound antibodies were detected by enhanced chemiluminescence (Amersham Biosciences Corp.) and Hyperfilm-MP (Amersham Biosciences Corp.).

Inhibition of ERK, Akt, and p38 MAPK
To examine the effect of ERK, p38 MAPK, and Akt in the regulation of RANKL expression, human osteoblasts were pretreated (30 min) by the inhibitors of MEK (U0126, 50 µM; Calbiochem, San Diego, CA, USA), p38 MAPK (SB203580, 10 µM; Cell Signaling Technology Inc.), and phosphatidylinositol 3-kinase (PI3K) (LY294002, 50 µM or wortmannin, 100 nM; Calbiochem) in preparation for assays.

Quantification of RANKL and OPG protein
Human osteoblasts seeded in a 24-well plate were incubated with vitamin D3 (1x10–8 M) or Ang II (1x10–6 M) with pretreatment (30 min) with olmesartan (1x10–5 M) or PD123329 (1x10–5 M) for 48 h. Soluble RANKL and OPG in the conditioned medium were measured according to the manufacturer’s instructions (Biomedica Medizinprodukte, Vienna, Austria). Western blotting was also performed for analysis of RANKL expression using an anti-human RANKL antibody (polyclonal goat IgG, 1:1000; R&D Systems, Minneapolis, MN, USA) and an anti-β-actin (monoclonal mouse IgG, 1:3000; Sigma-Aldrich Corp., St. Louis, MO, USA).

RNA Interference and oligodeoxynucleotides
The small interfering (si) RNA for human RANK or scramble siRNA was designed using the siSNIPER system (Genomidea, Inc., Ibaraki, Japan, and Mitsubishi Space Software Co., Ltd., Amagasaki, Japan). The sequence of human RANK (sense) was 5'-GUACCAGUGAGAAGCAUUATT-3' and the sequence of scramble siRNA (sense) was 5'-CGAGACCCGUUCACAUUGATT-3'. The siRNA oligonucleotides were transfected into human osteoclast precursors using a Human Macrophage Nucleofector Kit (Amaxa Biosystems, Cologne, Germany) according to the manufacturer’s instructions (13) .

Osteoclast formation assay
An osteoclast formation assay was performed using cocultures of human osteoblasts and osteoclast precursors as described previously (14) . Human osteoblasts seeded in a 24-well plate were incubated with vitamin D3 (1x10–8 M) or Ang II (1x10–6 M) with pretreatment (30 min) with olmesartan (1x10–5 M) or PD123329 (1x10–5 M) for 48 h and fixed in PBS containing 1% paraformaldehyde for 8 min at room temperature. Human osteoclast precursors were also cultured for 6 days on the fixed cells in {alpha}-MEM containing 10% FCS and 10 ng/ml of human M-CSF in a 24-well plate. After treatment, the cells were subjected to TRAP staining and nuclear staining as described above.

Rat ovariectomy osteoporosis model
Female adult Wister rats (10 wk old) were purchased from SLC Japan (Shizuoka, Japan). After the rats were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (10 mg/kg), bilateral ovariectomy or sham operation was performed, and an osmotic minipump (Alzet model 2004; Alza, Palo Alto, CA) containing a subpresser dose of Ang II (200 ng/kg/min) or saline was implanted (15) . Female adult spontaneously hypertensive rats (SHRs) or Wistar-Kyoto rats (WKYs) (10 wk old) were also purchased from SLC Japan, and bilateral ovariectomy or sham operation was performed. In some groups of rats, an osmotic minipump containing olmesartan (0.5, 1, or 3 mg/kg/day) was implanted, and in another group of rats hydralazine (10 mg/kg/day) was administered with drinking water. The body weights of these rats were recorded for 4 wk. At 4 wk after operation, systolic blood pressure was then measured using the tail-cuff method (BP-98A; Softron Beijing Incorporated, Beijing, China), and rats were deeply anesthetized and sacrificed to collect femurs, tibiae, and blood for biochemical analysis. Both TRAP and alkaline phosphatase (ALP) activity were measured to evaluate the total balance of osteoclast and osteoblast activity in the process of osteoporosis. The proximal tibia and distal femur were excised and homogenized in 10 mM triethanolamine buffer (pH 7.5) for TRAP activity and diethanolamine buffer (pH 9.8) for ALP activity. Supernatants were subjected to measurement of TRAP activity as described previously (11) . For ALP activity, supernatants were incubated with p-nitrophenylphosphate as a substrate for 30 min at 25°C, and absorbance was measured at 405 nm. The urinary deoxypyridinoline level was measured by enzyme immunoassay (Metra Biosystems, Mountain View, CA, USA) on day 28 of the experiments.

Dual energy X-ray absorptiometry (DEXA) and microcomputed tomography
Bone density measurements were performed by DEXA bone densitometry (GE-Lunar DPX-IQ; Madison, WI, USA). High- and low-beam energies for all scans were 80 and 35 kV, respectively, at 0.5 mA as described previously (16) . Bone mineral density was obtained in g/cm2.

Bone microarchitecture was analyzed by using cone beam microcomputed tomography (X-ray computed tomography system, SMX-100CT-SV; Shimazu, Osaka, Japan) and software (TRI/3D-BON; RATOC System Engineering Co. Ltd., Tokyo, Japan), which serves as a valuable tool for evaluating both antiresorptive and anabolic agents in ovariectomized (OVX) mice (17) . Briefly, the proximal tibia metaphysis was scanned at the region of 0.65–2.35 mm under the growth plate. A total of 135 consecutive tomographic slices were obtained with a slice thickness of 12.8 µm at 8 µm resolution. After scanning, three-dimensional microstructual image data were reconstructed and structural indices were calculated using the three-dimensional trabecular bone analysis software TRI/3D-BON as described previously (18) . The gray-scale images were segmented using a median filter to remove noise and a fixed threshold to extract the mineralized bone phase. Subsequently, the isolated small particles in the marrow space and the isolated small holes in the bone were removed using a cluster-labeling algorithm. The trabecular bone was then separated and analyzed for structural indices. Bone volume was calculated using tetrahedrons corresponding to the enclosed volume of the triangulated surface. Total tissue volume was the volume of the entire scanned sample. Trabecular bone volume fraction was calculated from these values. Trabecular thickness and space were estimated as described previously (19) .

Five-sixths nephrectomy and OVX model
Angiotensin II type 1A receptor-deficient (AT1A KO) mice (20) (n=10; The Jackson Laboratory, Bar Harbor, ME, USA) and wild-type mice (n=10), 8 wk old, from the same genetic background (C57BL/6 mice) (Oriental Bioservice Co., Ltd., Kyoto, Japan) were used in the present study. Adult female mice, fed standard rat chow with free access to water, were subjected to subtotal renal ablation (21) . Infarction of the left kidney was produced by ligation of two segmental renal arteries, and 2 weeks later, right nephrectomy and ovariectomy were performed. After ablation, systolic blood pressure was measured by the tail-cuff technique.

Statistical analysis
All values are expressed as means ± SE. Analysis of variance with subsequent Bonferroni/Dunnet’s test was used to determine the significance of differences in multiple comparisons. Values of P < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Ang II on osteoclast differentiation
To clarify the direct effects of Ang II on osteoporosis, we initially focused on the differentiation of osteoclasts in two different cell culture systems. In rabbit bone marrow-derived mononuclear cells, which may include both osteoblasts and osteoclasts, treatment with Ang II (1x10–6 M) and with vitamin D3 (1x10–8 M) induced osteoclast differentiation as assessed by Hoechst 33258 nuclear staining and TRAP staining. These effects were significantly abolished by cotreatment with an Ang II type 1 receptor blocker, olmesartan, but not with an Ang II type 2 receptor blocker, PD123329 (Fig. 1 A). To address the target of Ang II in osteoclast differentiation, we used a rat osteoclast culture system that was dependent on treatment with recombinant RANKL and M-CSF without coculture of osteoblasts. Unexpectedly, treatment with Ang II and with 1,25-dihydroxyvitamin D3 (vitamin D3) did not increase TRAP-positive multinuclear cells (Fig. 1B ), although the treatment with RANKL and M-CSF increased TRAP-positive multinuclear cells. These results suggest that the osteoclast itself is not the direct target of Ang II in the process of osteoclast differentiation.


Figure 1
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  		Figure 1. Effect of Ang II on the differentiation of osteoclasts. A) Ang II-induced differentiation of bone marrow mononuclear cells to multinuclear cells (bottom panel) and number of multinuclear cells in osteoclasts (top panel) Representative phase-contrast microscopic views showing the differentiation of bone marrow cells induced by vitamin D3 (Vit.D3) (1x10–8 M) and Ang II (1x10–6 M) after 7 days, with TRAP staining (top panel) and Hoechst 33258 staining (bottom panel) (x40). ARB, treatment with an Ang II type 1 receptor blocker, olmesartan (1x10–5 M); PD, treatment with an Ang II type 2 receptor blocker, PD123329 (1x10–5 M). B) Effect of Ang II on TRAP activity in RANKL- and M-CSF-dependent rat osteoclasts. Representative phase-contrast microscopic views showing the differentiation of rat preosteoclast induced by vitamin D3 (1x10–8 M), Ang II (1x10–6 M), and RANKL (10 ng/ml RANKL+10 ng/ml M-CSF) after 7 days, with TRAP staining (top panel) and Hoechst 33258 staining (bottom panel) (x40).

Therefore, we examined the effect of Ang II on osteoblasts. Cell viability (assessed by the MTS assay) was not significantly changed by treatment with Ang II (1x10–6 M) (data not shown). However, stimulation with Ang II (1x10–6 M) led to an increase in RANKL mRNA expression by 8-fold and in OPG (decoy receptor for RANKL) by 3-fold as quantified by real-time PCR (Fig. 2 A). We confirmed that soluble RANKL protein was up-regulated by Ang II (1x10–6 M), as shown in Fig. 2B , consistent with the results of Western blotting (Fig. 2C ). Furthermore, we also confirmed that OPG protein was up-regulated by Ang II (1x10–6 M), as shown in Fig. 2D . These effects of Ang II in osteoblasts were significantly abolished by pretreatment with an Ang II type 1 receptor blocker, olmesartan, but not by an Ang II type 2 receptor blocker, PD123329. These results suggest that Ang II directly induced RANKL expression in osteoblasts through the activation of the Ang II type 1 receptor, leading to osteoclast activation.


Figure 2
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  		Figure 2. Effect of Ang II on RANKL expression in human osteoblasts. A) Quantification of RANKL and OPG mRNA expression by real-time PCR. Human osteoblasts were treated with Ang II (1x10–6 M) with or without an Ang II type 1 receptor blocker (ARB) (olmesartan; 1x10–5 M) or an Ang II type 2 receptor blocker[PD123329 (PD); 1x10–5 M] for 24 h. *P < 0.01 vs. no treatment; {dagger}P < 0.01 vs. Ang II; n = 6–8 per group. B, C) Effect of Ang II on RANKL expression in human osteoblasts, assessed by (B) soluble RANKL concentration and (C) Western blotting with anti-RANKL antibody. Human osteoblasts were treated with vitamin D3 (Vit.D3) (1x10–8 M) or Ang II (1x10–6 M) with or without an Ang II type 1 receptor blocker (olmesartan; 1x10–5 M) or an Ang II type 2 receptor blocker (PD123329; 1x10–5 M) for 48 h. *P < 0.01 vs. no treatment; {dagger}P < 0.01 vs. Ang II; n = 6–8 per group. D) Effect of Ang II on OPG expression in human osteoblasts. Human osteoblasts were treated with vitamin D3 (1x10–8 M) or Ang II (1x10–6 M) with or without an Ang II type 1 receptor blocker (olmesartan; 1x10–5 M) or an Ang II type 2 receptor blocker (PD123329; 1x10–5 M) for 48 h. *P < 0.01 vs. no treatment; {dagger}P < 0.01 vs. Ang II; n = 6 per group. E) Ang II activated ERK, p38 MAPK, and Akt in human osteoblasts. Representative Western blot of phospho-specific (P) -ERK, ERK, P-p38 MAPK, p38 MAPK, P-Akt, or Akt in human osteoblasts. Human osteoblasts were treated with Ang II (1x10–6 M) with or without pretreatment (1 h before) with an Ang II type 1 receptor blocker (olmesartan; 1x10–5 M) or an Ang II type 2 receptor blocker (PD123329; 1x10–5 M) for 10 min. F) Inhibition of ERK, p38 MAPK, and Akt by specific inhibitors on soluble RANKL concentration in human osteoblasts. Effect of an Ang II type 1 receptor blocker (olmesartan; 1x10–5 M), an MEK inhibitor (U0126; 50 µM), a p38 MAPK inhibitor (SB203580; 10 µM), and a PI3K inhibitor (wortmannin; 100 nM) in a soluble RANKL concentration in human osteoblasts. *P < 0.01 vs. control; {dagger}P < 0.01 vs. Ang II; n = 8 per group. G) Evaluation of Ang II-induced RANKL up-regulation on TRAP activity in a coculture system with human osteoblasts and osteoclast precursors. Representative phase-contrast microscopic views show the differentiation of osteoclasts induced by vitamin D3 (1x10–8 M) or Ang II (1x10–6 M) with or without an Ang II type 1 receptor blocker (olmesartan; 1x10–5 M), an MEK inhibitor (U0126; 50 µM), a p38 MAPK inhibitor [SB203580 (SB); 10 µM], and a PI3K inhibitor [wortmannin (Wor); 100 nM], with TRAP staining (top panel) and Hoechst 33258 staining (bottom panel) (x100). H) Evaluation of Ang II-induced RANKL up-regulation on TRAP activity in a coculture system with human osteoblasts and osteoclast precursors transfected with siRNA for RANK. Representative phase-contrast microscopic views show the differentiation of osteoclasts induced by Ang II (1x10–6 M) with siRNA for scramble or RANK, with TRAP staining (top panel) and Hoechst 33258 staining (bottom panel) (x40). Con, control.

We clarified the cellular signaling of Ang II, leading to up-regulation of RANKL in osteoblasts. Treatment with Ang II rapidly increased phosphorylation of ERK, p38 MAPK, and Akt, whereas this activation was blocked by pretreatment with an angiotensin receptor blocker but not with PD123329 (Fig. 2E ). Pretreatment with an angiotensin receptor blocker also blocked the up-regulation of Ang II-induced RANKL expression. Similarly, pretreatment with U0126 (MEK inhibitor) attenuated Ang II-induced up-regulation of RANKL protein, whereas SB203580 (p38 MAPK inhibitor) and wortmannin (PI3K inhibitor) did not (Fig. 2F ). To confirm the function of Ang II-induced up-regulation of RANKL protein, we examined the differentiation of osteoclasts using a coculture system with osteoblasts and osteoclast precursors. Treatment with Ang II and with vitamin D3 induced the number of TRAP-positive multinuclear cells. Pretreatment with an angiotensin receptor blocker or U0126 attenuated the Ang II-induced increase of the number of TRAP-positive multinuclear cells, whereas SB203580 and wortmannin did not (Fig. 2G ). These results suggest that the ERK pathway might be important for the up-regulation of RANKL protein in osteoblasts.

To further confirm the involvement of the RANKL-RANK pathway in Ang II-induced osteoclasts differentiation, we designed the siRNA for RANK to knockdown its expression. We successfully inhibited its expression (87% inhibition) in human osteoclast precursors by Nucleofector transfection (13) . To clarify the contribution of RANK in Ang II-induced osteoclast differentiation, we cocultured the siRNA transfected-osteoclast precursors with human osteoblasts with or without treatment with Ang II. Indeed, Ang II-induced TRAP-positive multinuclear cells were completely abolished in RANK siRNA transfected cells (Fig. 2H ). These results suggest that Ang II-induced osteoclast differentiation may be mediated by the RANK-RANKL system.

In vivo effects of Ang II on osteoporosis in rat ovariectomy model
To further clarify the effect of Ang II on the differentiation of osteoclasts, we established a rat ovariectomy model of estrogen deficiency as a model of osteoporosis with or without systemic administration of Ang II at a subpresser dose (200 ng/kg/min). At 28 days after bilateral ovariectomy, the serum estradiol level was significantly decreased in the ovariectomy group, whereas there was no significant difference in body weight, consistent with our previous report (11) . We examined both TRAP and ALP activity to evaluate the total balance of osteoclast and osteoblast activity in the process of osteoporosis. TRAP activity was significantly increased in the tibiae of ovariectomized rats with systemic administration of Ang II (Fig. 3 A). Indeed, the TRAP-positive stained area was also increased in the tibiae of OVX rats with systemic administration of Ang II (Fig. 3B ). Although ALP activity was also increased in the tibiae of OVX rats by Ang II (Fig. 3C ), the ratio of ALP to TRAP was significantly decreased in the tibiae of OVX rats by Ang II (Fig. 3D ). These results suggest that Ang II accelerated the turnover of bone metabolism, which is similar to the typical pattern in elderly postmenopausal women who are at high risk for osteoporosis. Of importance, bone density as assessed by DEXA was significantly decreased in the tibiae of OVX rats by Ang II (Fig. 3E ). These results were accompanied by a change in urinary deoxypyridinoline, which is released from bone by the processing of tissue collagen. Treatment with Ang II significantly induced the ovariectomy-induced increase in urinary deoxypyridinoline (Fig. 3F ). These results suggest that Ang II directly accelerated estrogen deficiency-induced osteoporosis independent of blood pressure.


Figure 3
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  		Figure 3. Effects of Ang II infusion in ovariectomy-induced osteoporosis rat model. A) TRAP activity. B) TRAP staining. C) ALP activity. D) Ratio of ALP to TRAP activity. E) Bone marrow density by DEXA. F) Urinary deoxypyridinoline after 28 days of each treatment. Sham, sham operation; Ang II, infusion of Ang II (200 ng/kg/min); OVX, bilateral ovariectomy; OVX + Ang II, bilateral ovariectomy and treatment with Ang II (200 ng/kg/min); U, release of 1 µmol of p-nitrophenol/min. Urinary deoxypyridinoline was adjusted for urinary creatinine concentration. *P < 0.05 vs. sham; n = 6–10 per group.

Ang II type 1 receptor blocker ameliorates ovariectomy-induced osteoporosis
To clarify the role of Ang II and high blood pressure in bone metabolism further, we used an ovariectomy model of estrogen deficiency in a hypertensive model, SHRs. At 28 days after bilateral ovariectomy, TRAP activity was significantly increased in the tibiae of OVX SHRs compared with sham-operated SHRs (Fig. 4 A, B). Because ALP activity was not changed in the tibiae of OVX SHRs (Fig. 4C ), the ratio of ALP to TRAP was significantly decreased in the tibiae of OVX SHRs compared with that in sham-operated rats (Fig. 4D ). Of importance, TRAP and ALP activities were not increased in ovariectomized normotensive WKYs (TRAP activity, sham: 0.074±0.006, ovariectomy: 0.076±0.008; ALP activity, sham: 2.108±0.121, ovariectomy: 2.203±0.183). These results indicate that osteoclasts would be activated in SHRs but not in WKYs with ovariectomy, leading to worse osteoporosis.


Figure 4
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  		Figure 4. Effects of an Ang II type 1 receptor blocker (ARB), olmesartan, in the ovariectomy-induced osteoporosis SHR model. A) TRAP activity. B) TRAP staining. C) ALP activity. D) Ratio of ALP to TRAP activity. E) Bone mineral density by DEXA. F) Urinary deoxypyridinoline after 28 days of each treatment. Sham, sham operation; OVX, bilateral ovariectomy; OVX + ARB (0.5), bilateral ovariectomy and treatment with olmesartan (0.5 mg/kg/day); OVX + ARB (1), bilateral ovariectomy and treatment with olmesartan (1 mg/kg/day); OVX + ARB (3), bilateral ovariectomy and treatment with olmesartan (3 mg/kg/day); OVX + HYD, bilateral ovariectomy and treatment with hydralazine (10 mg/kg/day in drinking water); U, release of 1 µmol of p-nitrophenol/min. Urinary deoxypyridinoline was adjusted for urinary creatinine (Cre) concentration. *P < 0.05 vs. sham; {dagger}P < 0.05 vs. OVX; n = 6–10 per group.

Because tissue Ang II is well known to be increased in SHRs, we further examined whether an Ang II type 1 receptor blocker, olmesartan, would ameliorate ovariectomy-induced osteoporosis in SHRs. Blood pressure was decreased with continuous administration of olmesartan (0.5, 1, or 3 mg/kg/day by osmotic pump) and hydralazine (10 mg/kg/day in drinking water), as shown in Table 1 . Ovariectomy-induced TRAP activity in SHRs was significantly ameliorated by continuous administration of olmesartan but not hydralazine (Fig. 4A ), whereas both drugs lowered blood pressure to the same level. Indeed, the TRAP-positive stained area was also increased in the tibiae of OVX SHRs, whereas treatment with olmesartan significantly decreased the TRAP-positive stained area (Fig. 4B ). ALP activity was not changed with ovariectomy and only increased by treatment with high-dose olmesartan (3 mg/kg/day) (Fig. 4C ). The ratio of ALP to TRAP in the tibiae of ovariectomized SHRs was normalized by treatment with olmesartan but not hydralazine (Fig. 4D ). Of importance, these results were accompanied by a significant increase in bone mineral density, as assessed by DEXA, in the tibiae of OVX SHRs (Fig. 4E ). The increase in urinary deoxypyridinoline induced by ovariectomy in SHRs was consistently significantly attenuated by olmesartan but not by hydralazine (Fig. 4F ). These results suggest that an Ang II type 1 receptor blocker attenuated osteoporosis induced by estrogen deficiency and high blood pressure.


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  		Table 1. Physiological parameters of each group in SHRs

Furthermore, we used AT1A KO mice to examine the contribution of Ang II in the process of osteoporosis. It was reported that there was no gross abnormality in bone development or osteoporosis in AT1A KO mice, although arterial pressure was reduced (20) . Because amounts of circulating and tissue Ang II are elevated in the five-sixths nephrectomy hypertensive model with a significant increase in systolic blood pressure, we developed an Ang II-dependent osteoporosis model by ovariectomy and five-sixths nephrectomy. In the evaluation of physical characteristics, AT1A KO mice showed a decrease in arterial blood pressure compared with wild-type mice, whereas treatment with ovariectomy and five-sixths nephrectomy increased arterial blood pressure in wild-type mice but not in AT1A KO mice (Table 2 ). In ovariectomized and five-sixths nephrectomized mice, computed tomography indicated that bone volume and bone thickness were significantly decreased, whereas bone trabecular space was significantly increased compared with that in sham-operated mice (Fig. 5 A, B). In contrast, in AT1A KO mice, there was no difference in these markers between sham operation and ovariectomy/nephrectomy (Fig. 5A, B ). The histology of the proximal tibia showed that TRAP-positive cells were significantly increased in OVX/nephrectomized wild-type mice but not in AT1A KO mice (Fig. 5C ). The increase in urinary deoxypyridinoline induced by ovariectomy/nephrectomy was consistently observed in wild-type mice but not in AT1A KO mice (Fig. 5D ).


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  		Table 2. Physiological parameters of each group in wild-type and AT1R KO mice


Figure 5
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  		Figure 5. Effects of hypertension on bone metabolism in OVX wild-type (Wild) mice and AT1A KO mice. A) Microcomputed tomography three-dimensional image of the trabecular architecture of the proximal tibia metaphysic in sham operation (Sham) and ovariectomy and five-sixths nephrectomy (OVX+KC) of wild-type or AT1A KO mice. B) Change of trabecular bone parameters of mouse proximal tibia metaphysic analyzed by microcomputed tomography in sham operation and ovariectomy and five-sixths nephrectomy of wild-type or AT1A KO mice. *P < 0.05 vs. sham; n = 6 per group. C) TRAP staining. D) Quantification of the TRAP-positive staining area in cancerous bone under the growth plate. E) Urinary deoxypyridinoline in sham operation and ovariectomy and five-sixths nephrectomy of wild-type or AT1A KO mice. *P < 0.05 vs. sham; n = 6 per group.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Clinical epidemiological evidence has demonstrated that high blood pressure is associated with an increase in bone loss, especially in elderly women. Systemic blood pressure is a significant predictor of bone mineral loss in the femoral neck. It is known that high blood pressure is associated with abnormalities of calcium metabolism, including an increase in urinary calcium excretion for a given sodium intake and evidence of a secondary increase in parathyroid gland activity (22) . Sustained hypercalciuria in patients with high blood pressure leads to an increased risk of bone mineral loss, with a negative association between blood pressure and bone mineral density (23 , 24) . Thiazides are thought to protect against age-related bone loss byreducing urinary calcium excretion (25) . In the kidney, thiazides act at the distal convoluted tubule by blocking the coupled resorption of Na and Cl through the thiazide-sensitive Na/Cl cotransporter. This effect triggers a Na/Ca exchanger promoting calcium influx and sodium efflux, leading to a decrease in parathyroid hormone, a mild increase in serum calcium level, and a decrease in bone turnover. In a prospective cohort study, use of thiazides for more than 365 days was associated with a decreased risk of hip fracture (4) . Although this association may reflect calcium loss associated with high blood pressure, there are few reports clarifying the role of hypertension in osteoporosis. From this viewpoint, we focused on the role of Ang II in osteoporosis. Interestingly, subanalysis of a retrospective case-control study in a large population (30,601 fractures and 120,819 controls) of men and women with ages ranging from 30 to 79 yr has recently shown that use of ACE inhibitors significantly reduced the risk of fractures. However, calcium channel blockers had no effect on the risk of fractures in the present study, and thus far there has been no evidence of the value of calcium channel blockers for treatment of osteoporosis associated with hypertension. Thus, it is noteworthy that the present study demonstrated that Ang II regulated the bone metabolism of osteoblasts and osteoclasts, which potentially contributes to osteoporosis in hypertensive patients.

The local renin-angiotensin system plays an important role in the regulation of tissue remodeling in several tissues. Ang II has been postulated to be able to act on the cells involved in bone metabolism through receptors located in osteoblasts and osteoclasts or regulation of flow in bone marrow capillaries. Hatton et al. (8) indicated that Ang I and II were potent stimulators of osteoclastic bone resorption. On the contrary, Ang II stimulates the proliferation of osteoblast-rich populations of cells (9) . Osteoclasts originate from monocyte/macrophage lineage multinucleated cells, which can also be the target of Ang II. Osteoblasts/stromal cells express RANKL in response to several bone-resorbing factors including vitamin D3 to support osteoclast differentiation from their precursors. Osteoclast precursors, which express RANK, recognize RANKL through cell-to-cell interactions with osteoblasts/stromal cells and differentiate into mature osteoclasts in the presence of M-CSF. Targeted disruption of either RANKL or RANK in mice causes a lack of osteoclasts and an osteopetrotic phenotype (26) . Of note, the present study clearly demonstrated that Ang II indirectly promoted the differentiation and activation of osteoclasts responsible for bone resorption via up-regulation of RANKL in osteoblasts. We also confirm that treatment with Ang II activated NF{kappa}B in osteoclasts (data not shown). Thus, the renin-angiotensin system could be involved in the regulation of osteoclast activation. As osteoclast differentiation is regulated by a variety of hormones, local factors, and inflammatory cytokines, such as interleukin-1 and tumor necrosis factor-{alpha} (26 , 27) , the renin-angiotensin system is a novel component of the osteoclast differentiation system (Fig. 6 ). On the other hand, locally elevated extracellular calcium levels have been suggested to play roles in regulation of bone remodeling (28 , 29) , and MAPK pathways mediate the modulation of the cellular responses by high extracellular calcium (30) . Previous reports demonstrated that ERK would be involved mainly in induction of RANKL by high extracellular signaling, but not PI3K or p38 MAPK, by using these specific inhibitors (31) . Although it was known that intracellular signaling of Ang II via the Ang II type 1 receptor might be coupled with calcium reflex, especially inducing release of intracellular calcium (32) , our results also suggested that treatment of Ang II in osteoblasts up-regulated RANKL expression through the ERK pathway, which led to osteoclast activation and differentiation. Alternatively, Ang II also regulates local blood flow in bone marrow capillaries. As hypertension decreases local blood flow in microvessels, blockade of the renin-angiotensin system may improve tissue blood flow in bone marrow capillaries, enhancing bone marrow formation.


Figure 6
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  		Figure 6. Summary scheme of angiotensin II-induced osteoclastogenesis. Osteoclast precursors with RANK (receptor) recognize RANKL (ligand) of osteoblast by cell-cell contact and differentiate into mature osteoclasts. The transcription factor, NF{kappa}B, plays a pivotal role in the activation of nuclear factor of activated T cells c1 (NFATc1), which regulates osteoclastogenesis assessed by TRAP activity. Ang II increases RANKL expression in osteoblasts, whereas the angiotensin receptor blockade (ARB) ameliorates osteoclastogenesis through down-regulation of RANKL expression.

In this study, we established two rat models and one mouse model with ovariectomy as osteoporosis models corresponding to elderly women in a state of estrogen withdrawal. Interestingly, continuous administration of Ang II accelerated osteoclast activation induced by estrogen deficiency. It has been reported that estrogen antagonizes the bioactive effect of Ang II through signaling cross-talk in vascular smooth muscle cells (33 , 34) . Previous reports suggested that administration of an ACE inhibitor, enalapril, and an angiotensin II antagonist, losartan, had no effect on bone metabolism in normal rats (35) . The difference from the present study might be due to the different model (normotension vs. SHR or Ang II infusion) or high affinity to the Ang II receptor (losartan vs. olmesartan). More strong evidence about the contribution of Ang II to osteoporosis is that both hypertension and ovariectomy induced the symptoms of osteoporosis in wild-type mice but not in AT1A KO mice.

As osteoporosis is the main cause of bone fractures in postmenopausal women and elderly individuals and is associated with pain, deformity, and loss of independence (36) , the present study suggests new therapeutic aspects of antihypertensive drugs, Ang II receptor blockers, to treat elderly hypertensive patients, especially the female population. Because of the increasing number of elderly people and the increase in the prevalence of osteoporosis, the need for focused preventive strategies has become a public health priority. Peak bone mass attained in the first 20 yr of life and the rate at which bone is lost in later years are the most important factors influencing the occurrence of osteoporosis. Because Ang II caused osteoclast activation, leading to accelerated osteoporosis, angiotensin receptor blockers could lessen the risk of osteoporosis in elderly people, possibly beyond their blood pressure-lowering effect. Further clinical trials using Ang II receptor blockers are necessary to confirm this concept.


   ACKNOWLEDGMENTS
 
This work was partially supported by the National Institute of Biomedical Innovation, by a grant-in-aid from the Ministry of Public Health and Welfare, by a grant-in-aid from Japan Promotion of Science, and through Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. We thank Miss Natsuki Yasumasa for technical support.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication October 9, 2007. Accepted for publication January 10, 2008.


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