Angiotensin II Type 2 Receptor Blockade Increases Bone Mass
2008; Elsevier BV; Volume: 284; Issue: 8 Linguagem: Inglês
10.1074/jbc.m807610200
ISSN1083-351X
AutoresYayoi Izu, Fumitaka Mizoguchi, Aya Kawamata, Tadayoshi Hayata, Testuya Nakamoto, Kazuhisa Nakashima, Tadashi Inagami, Yoichi Ezura, Masaki Noda,
Tópico(s)Vitamin D Research Studies
ResumoRenin angiotensin system (RAS) regulates circulating blood volume and blood pressure systemically, whereas RAS also plays a role in the local milieu. Previous in vitro studies suggested that RAS may be involved in the regulation of bone cells. However, it was not known whether molecules involved in RAS are present in bone in vivo. In this study, we examined the presence of RAS components in adult bone and the effects of angiotensin II type 2 (AT2) receptor blocker on bone mass. Immunohistochemistry revealed that AT2 receptor protein was expressed in both osteoblasts and osteoclasts. In addition, renin and angiotensin II-converting enzyme were expressed in bone cells in vivo. Treatment with AT2 receptor blocker significantly enhanced the levels of bone mass, and this effect was based on the enhancement of osteoblastic activity as well as the suppression of osteoclastic activity in vivo. These results indicate that RAS components are present in adult bone and that blockade of AT2 receptor results in alteration in bone mass. Renin angiotensin system (RAS) regulates circulating blood volume and blood pressure systemically, whereas RAS also plays a role in the local milieu. Previous in vitro studies suggested that RAS may be involved in the regulation of bone cells. However, it was not known whether molecules involved in RAS are present in bone in vivo. In this study, we examined the presence of RAS components in adult bone and the effects of angiotensin II type 2 (AT2) receptor blocker on bone mass. Immunohistochemistry revealed that AT2 receptor protein was expressed in both osteoblasts and osteoclasts. In addition, renin and angiotensin II-converting enzyme were expressed in bone cells in vivo. Treatment with AT2 receptor blocker significantly enhanced the levels of bone mass, and this effect was based on the enhancement of osteoblastic activity as well as the suppression of osteoclastic activity in vivo. These results indicate that RAS components are present in adult bone and that blockade of AT2 receptor results in alteration in bone mass. Angiotensin II type 2 receptor blockade increases bone mass.Journal of Biological ChemistryVol. 284Issue 31PreviewVOLUME 284 (2009) PAGES 4857–4864 Full-Text PDF Open Access Osteoporosis is one of the major diseases associated with aging. This disease is based on the imbalance between the two major activities, i.e. bone formation and bone resorption. Systemic signals such as parathyroid hormone (PTH) 3The abbreviations used are: PTH, parathyroid hormone; RAS, renin angiotensin system; Ang II, angiotensin II; ACE, angiotensin-converting enzyme; AT1, angiotensin II type 1 receptor; AT2, angiotensin II type 2 receptor; BV/TV, bone volume per tissue volume; TRAP, tartrate-resistant acid phosphatase; PBS, phosphate-buffered saline; RT, reverse transcription; Tb.N, trabecular number; Tb.Spac, trabecular spacing; BS, bone surface; BFR, bone formation rate; MS/BS, mineralized surface per bone surface; Oc, osteoclast; Tb.N, trabecular number; μCT, micro x-ray computed tomography; MAR, mineral apposition rate; ENaC, epithelial sodium channels. and vitamin D are the major regulators of the maintenance of bone mass and blood calcium (1Kitahara K. Ishijima M. Rittling S. Tsuji K. Kurosawa H. Nifuji A. Denhardt D. Noda M. Endocrinology. 2003; 144: 2132-2140Crossref PubMed Scopus (52) Google Scholar, 2Girotra M. Rubin M. Bilezikian J. Rev. Endocr. Metab. Disord. 2006; 7: 113-121Crossref PubMed Scopus (119) Google Scholar, 3Ono N. Nakashima K. Schipani E. Hayata T. Ezura Y. Soma K. Kronenberg H. Noda M. J. Biol. Chem. 2007; 282: 25509-25516Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In addition, bone mass levels are also determined by a central nervous control through the activities of sympathetic tone on both bone formation and resorption sides (4Takeda S. Elefteriou F. Levasseur R. Liu X. Zhao L. Parker K.L. Armstrong D. Ducy P. Karsenty G. Cell. 2002; 111: 305-317Abstract Full Text Full Text PDF PubMed Scopus (1373) Google Scholar, 5Elefteriou F. Ahn J.D. Takeda S. Starbuck M. Yang X. Liu X. Kondo H. Richards W.G. Bannon T.W. Noda M. Clement K. Vaisse C. Karsenty G. Nature. 2005; 434: 514-520Crossref PubMed Scopus (1009) Google Scholar, 6Kondo H. Nifuji A. Takeda S. Ezura Y. Rittling S. Denhardt D. Nakashima K. Karsenty G. Noda M. J. Biol. Chem. 2005; 280: 30192-30200Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 7Katayama Y. Battista M. Kao W.M. Hidalgo A. Peired A.J. Thomas S.A. Frenette P.S. Cell. 2006; 124: 407-421Abstract Full Text Full Text PDF PubMed Scopus (1037) Google Scholar). In the local milieu of bone, two major types of cells, osteoblasts and osteoclasts, are located in close proximity, exchange their signals, and coordinately resorb and form bone matrix (8Zhao C. Irie N. Takada Y. Shimoda K. Miyamoto T. Nishiwaki T. Suda T. Matsuo K. Cell Metab. 2006; 4: 111-121Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar). Such events are controlled by molecules present in the local microenvironment. These include cytokines, their modulators, and matrix proteins secreted by osteoblasts and osteoclasts (9Miyazono K. Maeda S. Imamura T. Oncogene. 2004; 23: 4232-4237Crossref PubMed Scopus (139) Google Scholar, 10Usui M. Yoshida Y. Tsuji K. Oikawa K. Miyazono K. Ishikawa I. Yamamoto T. Nifuji A. Noda M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6653-6658Crossref PubMed Scopus (42) Google Scholar, 11Morinobu M. Nakamoto T. Hino K. Tsuji K. Shen Z. Nakashima K. Nifuji A. Yamamoto H. Hirai H. Noda M. J. Exp. Med. 2005; 201: 961-970Crossref PubMed Scopus (62) Google Scholar). However, the bone environment is quite heterogeneous, and there are also cells other than these two types. Microvasculatures are serving as a root to supply osteoclast progenitors, which are derived from hematopoietic lineage cells. Vasculatures in bone are also considered to give rise to progenitors for osteoblastic cells from their perivascular regions (12Sacchetti B. Funari A. Michienzi S. Cesare S.D. Piersanti S. Saggio I. Tagliafico E. Ferrari S. Robey P.G. Riminucci M. Bianco P. Cell. 2007; 131: 324-336Abstract Full Text Full Text PDF PubMed Scopus (1731) Google Scholar). In addition to the anatomical relationship between the vascular cells and bone cells, these cells may be functionally involved in the coordinate regulation of bone mass. Recent clinical studies indicated that beta blockers and anti-hypertension drugs would reduce the risk of bone fractures in the elderly populations (13Sanada M. Taguchic A. Higashi Y. Tsuda M. Kodama I. Yoshizumi M. Ohama K. Atherosclerosis. 2004; 176: 387-392Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Lynn H. Kwok T. Wong S.Y.S. Woo J. Leung P.C. Bone (New York). 2006; 38: 584-588Crossref PubMed Scopus (128) Google Scholar). This suggests a possible link between vascular and skeletal systems. Renin angiotensin system (RAS) is operating not only systemically but also locally in several tissues, and bone microenvironments have been studied in this regard (15Lavoie J. Sigmund C. Endocrinology. 2003; 144: 2179-2183Crossref PubMed Scopus (456) Google Scholar, 16Sakai K. Agassandian K. Morimoto S. Sinnayah P. Cassell M.D. Davisson R.L. Sigmund C.D. J. Clin. Investig. 2007; 117: 1088-1095Crossref PubMed Scopus (115) Google Scholar). Osteoblasts and osteoclasts express angiotensin II type 1 receptor in cell cultures (17Hatton R. Stimpel M. Chambers T. J. Endocrinol. 1997; 152: 5-10Crossref PubMed Scopus (114) Google Scholar, 18Hiruma Y. Inoue A. Hirose S. Hagiwara H. Biochem. Biophys. Res. Commun. 1997; 230: 176-178Crossref PubMed Scopus (71) Google Scholar, 19Shimizu H. Nakagami H. Osako M. Hanayama R. Kunugiza Y. Kizawa T. Tomita T. Yoshikawa H. Ogihara T. Morishita R. FASEB J. 2008; 22: 2465-2475Crossref PubMed Scopus (230) Google Scholar), suggesting the existence of local RAS in bone. However, whether RAS components are expressed in bone in vivo is not known. Angiotensin II (Ang II) acts via angiotensin II type 1 (AT1) and type 2 (AT2) receptors, which are members of the 7-transmembrane-spanning G-protein-coupled receptors. AT1 and AT2 receptors exhibit limited sequence homology (∼34% amino acid sequence identity) (20Senbonmatsu T. Saito T. Landon E. Watanabe O. Price E.J. Roberts R. Imboden H. Fitzgerald T. Gaffney F. Inagami T. EMBO J. 2003; 22: 6471-6482Crossref PubMed Scopus (171) Google Scholar). Many actions of Ang II appear to be through AT1 receptors. In bone tissue, Ang II was reported to promote bone resorption via the AT1 receptor in cell culture system and in ovariectomized mice and rats (19Shimizu H. Nakagami H. Osako M. Hanayama R. Kunugiza Y. Kizawa T. Tomita T. Yoshikawa H. Ogihara T. Morishita R. FASEB J. 2008; 22: 2465-2475Crossref PubMed Scopus (230) Google Scholar). Expression of AT1 receptor was observed in cultured osteoblasts (21Bandow K. Nishikawa Y. Ohnishi T. Kakimoto K. Soejima K. Iwabuchi S. Kuroe K. Matsuguchi T. J. Cell. Physiol. 2007; 211: 392-398Crossref PubMed Scopus (90) Google Scholar), and Ang II inhibit differentiation and bone formation via the AT1 receptor in rat calvarial osteoblastic cells (22Hagiwara H. Hiruma Y. Inoue A. Yamaguchi A. Hirose S. J. Endocrinol. 1998; 156: 543-550Crossref PubMed Scopus (98) Google Scholar). In contrast to the AT1 receptor, no significant effect was observed in cells by the AT2 receptor blocker in rat calvarial cell (22Hagiwara H. Hiruma Y. Inoue A. Yamaguchi A. Hirose S. J. Endocrinol. 1998; 156: 543-550Crossref PubMed Scopus (98) Google Scholar) or in the co-cultures of human osteoblast and osteoclast precursor cells (19Shimizu H. Nakagami H. Osako M. Hanayama R. Kunugiza Y. Kizawa T. Tomita T. Yoshikawa H. Ogihara T. Morishita R. FASEB J. 2008; 22: 2465-2475Crossref PubMed Scopus (230) Google Scholar). In this study, we examined the expression of renin, angiotensin-converting enzyme (ACE), and Ang II receptors in bone in vivo. The effects of AT2 receptor blocker on bone mass were also investigated. These experiments revealed that AT2 receptor as well as renin and ACE were expressed in bone in vivo and that AT2 receptor blocker treatment enhanced bone mass through both enhancement of osteoblastic activity and suppression of osteoclastic activity in vivo. Animals—9-Week-old C57BL/6J male mice were purchased from Oriental Yeast Co. (Tokyo, Japan). Angiotensin II type 2 receptor-deficient mice were generated on a C57BL/6 background as described previously (23Ichiki T. Labosky P.A. Shiota C. Okuyama S. Imagawa Y. Fogo A. Niimura F. Ichikawa I. Hogman B.L.M. Inagami T. Nature. 1995; 377: 748-750Crossref PubMed Scopus (802) Google Scholar). The mice were kindly gifted from Dr. Tadashi Inagami, Vanderbilt University School of Medicine. We used 15-week-old male mice. All experiments were conducted according to the guidelines from the Animal Welfare Committee of Tokyo Medical and Dental University. Histochemistry for Tartrate-resistant Acid Phosphatase (TRAP)—The animals were deeply anesthetized with ether and perfused through the cardiac left ventricle with 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). Tibiae were removed and immersed in the same fixation buffer at 4 °C overnight. Then tissues were decalcified in 15% EDTA in 0.01 m phosphate buffer, pH 7.4, at 4 °C for 1 week. After dehydration through a graded series of ethanol at 4 °C, they were embedded in paraffin and sectioned at 3 μm. The histochemistry for TRAP was performed as described previously (24Ishijima M. Tsuji K. Rittling S.R. Yamashita T. Kurosawa H. Denhardt D.T. Nifuji A. Ezura Y. Noda M. J. Endocrinol. 2007; 193: 235-243Crossref PubMed Scopus (47) Google Scholar). Briefly, after the preincubation of the deparaffinized sections with a tartrate buffer solution (50 mm tartrate in 100 mm NaOAc, pH 5.0) for 5 min, the sections were incubated with the substrate solution for 20 min. The substrate solution was prepared by dissolving 10 mg of AS mix (Sigma) in 500 ml of dimethyl formamide together with 60 mg of fast red violet/LB in 100 ml of tartrate buffer. Finally, the sections were counterstained with hematoxylin. Immunohistochemistry—After preparation of tibia sections by the same procedure described above, the sections were processed for immunohistochemistry for AT1, and AT2 receptors were performed using antibodies against these molecules according to the manufacturer's instructions (Santa Cruz Biotechnology). Briefly, deparaffinized sections were treated with 0.3% hydrogen peroxidase for 30 min to inhibit endogenous peroxidase and preincubated with 0.1% swine serum for 30 min at room temperature. Rabbit antisera against human AT1 and AT2 receptors (Santa Cruz Biotechnology) were applied to the sections, respectively, at 4 °C overnight. After treatment with the swine anti-rabbit immunoglobulin (DAKO A/S, Glostrup, Denmark), the sections were incubated in a streptavidin-biotin-peroxidase complex (sABC) (DAKO A/S). To visualize the antigen-antibody reaction, the sections were treated with 0.02% diaminobenzidine tetrahydrochloride and 0.005% H2O2. Finally, the sections were counterstained with hematoxylin. Immunohistochemistry for ACE was performed according to Ref. 25Loghman-Adham M. Soto C.E. Inagami T. Sotelo-Avila C. J. Histochem. Cytochem. 2005; 53: 979-988Crossref PubMed Scopus (26) Google Scholar. In brief, after deparaffinized sections were treated by inhibition of endogenous peroxidase, antigen retrieval was performed with 0.01 m citrate buffer, pH 6.0, at 65 °C for 30 min. Following the similar process of AT1 and AT2 receptor antibodies, the sections were preincubated with 0.1% swine serum and incubated with rabbit antiserum against human ACE in sequence. In Situ Hybridization—Bone and kidney were removed and processed as described above. For the paraffin sections, the samples were dehydrated through a graded series of ethanol solutions at 4 °C, embedded in paraffin, and sectioned at 3 μm. For the detection of renin mRNA, a 508-bp fragment of renin cDNA was amplified by the PCR method using the primer pair (5′-GAA CCA GAT GGA CAG GAG GA-3′,5′-CAC AGT GAT TCC ACC CAC AG-3′), designed according to the reports of the mouse renin sequences. For experiments with riboprobes, digoxigenin-labeled antisense and sense RNA probes were prepared using a digoxigenin RNA labeling kit (Roche Applied Science) according to the manufacturer's instructions. In situ hybridization was performed at 45 °C on the deparaffinized sections. Hybridized riboprobes were detected with an alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Applied Science), and visualized with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science). PCR Analysis—For RT-PCR analysis, kidney, bone marrow, and primary osteoblastic cells obtained from mouse neonatal calvarial cells were used. Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Reverse transcription (RT) was performed using 1 μg of total RNA, containing (dT)12–18 primers for quantitative real time PCR and SuperScriptII transcriptase (Invitrogen). Complementary DNA was amplified in reaction mixture containing 2.5 mm deoxynucleotide triphosphate mix, 10 mm specific primers, and rTaq DNA polymerase (Takara, Ohotsu, Japan) under the following conditions: 95 °C for 30 s, 55 or 60 °C for 30 s, and 72 °C for 30 s for 40 cycles in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA). After PCR amplification, the PCR products were sequenced. The primer sequence was as follows: 5′-CAC CTA TGT AAG ATC GCT TC-3′ and 5′-GCA CAA TCG CCA TAA TTA TCC-3′ for AT1 receptor; 5′-GAA GGA CAA CTT CAG TTT TGC-3′ and 5′-CAA GGG GAA CTA CAT AAG ATG C-3′ for AT2 receptor; 5′-CAG AGG CCA ACT GGC ATT AT-3′ and 5′-CTG GAA GTT GCT CAC GTC AA-3′ for ACE; and 5′-ACC ACA GTC CAT GCC ATC AC-3′ and 5′-TCC ACC ACC CTG TTG CTG TA-3′ for glyceraldehyde-3-phosphate dehydrogenase gene (gapdh). renin primer was described above. Treatment with an AT1 or AT2 Receptor Blockers—Animals were divided into two groups (eight in each group). One group was treated with an oral administration of an AT1-R blocker (losartan potassium; LKT Laboratories, Inc., St. Paul, MN) or water daily for 2 weeks. The other group was treated with intraperitoneal injection of AT2-R blocker (PD123319; Sigma) or phosphate-buffered saline (PBS) daily for 2 weeks. Mice were treated with losartan and PD123319 at 10 mg/kg body weight. A number of AT1 receptor antagonists exist. Among those losartan is the most widely used antagonist for AT1 in experimental studies as reported in the literature; therefore, we chose losartan. The dosages were chosen based on preliminary three-dimensional mCT analysis as well as the hypertension experiments (data not shown) (26Nagai N. Oike Y. Izumi-Nagai K. Urano T. Kubota Y. Noda K. Ozawa Y. Inoue M. Tsubota K. Suda T. Ishida S. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2252-2259Crossref PubMed Scopus (117) Google Scholar). These animals were anesthetized using the method described above. Femora and tibiae were removed and immersed the 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4) at 4 °C overnight. Femora were stored in 70% ethanol followed by histomorphometric analysis. Tibiae were decalcified and embedded in paraffin for TRAP staining. Three-dimensional Micro X-ray Computed Tomography (μCT) Analysis—Three-dimensional μCT analysis was conducted using Scan-Xmate-E090 (Comscan Techno Co., Ltd., Sagamihara, Japan) and TRI/3D-Bon (Ratoc System Engineering Co., Ltd., Tokyo, Japan) as computer software. Bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular spacing (Tb.Spac), and other microarchitectural parameters were analyzed in the secondary trabecular regions from 0.2 to 1.2 mm away from the chondro-osseous junction. Histomorphometric Analysis—Calcein labeling was conducted to estimate the levels of newly formed bone within a unit time period according to the methods described elsewhere (27Mizoguchi F. Mizuno A. Hayata T. Nakashima K. Heller S. Ushida T. Sokabe M. Miyasaka N. Suzuki M. Ezura Y. Noda M. J. Cell. Physiol. 2008; 216: 47-53Crossref PubMed Scopus (97) Google Scholar). Briefly, calcein (10 mg/kg body weight) was injected intraperitoneally 2 and 7 days before sacrifice. Femora were fixed in 4% paraformaldehyde and rinsed in 0.1 m phosphate buffer. Then tissues were embedded in 4% carboxymethylcellulose and sectioned into 5 mm with a cryostat. Sagittal histological sections were prepared, and the calcein bands were observed by fluorescent microscopy. Single labeled bone surface, double labeled bone surface, and total bone surface (BS) were separately measured. Mineralizing surface (MS) per BS was calculated as (double labeled bone surface + single labeled bone surface/2)/BS. The distance between parallel calcein lines was measured to yield mineral apposition rate (MAR (μm/day)). Bone formation rate (BFR) was calculated as MAR multiplied by MS/BS. Histomorphometric analysis was performed by focusing the area where the same region of femora were analyzed by μCT analysis. Osteoclast number per bone surface (N.S/BS, N/mm) and osteoclasts surface per bone surface (Oc.S/BS, %) were analyzed by TRAP staining in sagittal sections of tibiae. Mineralized Nodule Analysis—Bone marrow cells were seeded to 12-well plates (2.0 cm2/well) at a density of 2 × 106 cells/well. The bone marrow cells were cultured in a standard growth medium containing 50 μg/ml ascorbic acid and 10 mm β-glycerophosphate. The medium was changed every 2 days. The cultures were stained with alizarin red solution on day 21. The area of mineralized nodules/total dish was measured with ImageJ analysis program. Primary Osteoblast Culture—Primary mouse osteoblastic cells were obtained by sequential enzyme digestion of excised calvarial bones from 2-day-old neonatal mice using 0.5% trypsin and 1% collagenase in PBS for 15 min. The first three digests were discarded, and the cells were resuspended in α-minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and antibiotics (Invitrogen). Osteoblasts were initially seeded at a density of 5 × 103 cells/96-well plate. When confluent, we changed to the growth medium containing 50 μg/ml ascorbic acid and 10 mm β-glycerophosphate. The medium was changed every 2 days. The cells were collected on days 0, 3, 7, 10, 14, 21, and 28 after changing the growth medium. The collected cells were used for the analysis of real time PCR and Western blotting. Western Blotting Analysis—To confirm the expression of AT1 and AT2 in osteoblasts at the developmental stage, primary osteoblastic cells were lysed in RIPA buffer (150 mm NaCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris-HCl (pH 8.0)) supplemented with complete mini (Roche Applied Science), as a protein inhibitor and protein extracts were cleared by centrifugation. The proteins were eluted in SDS sample buffer with heating at 95 °C for 2 min. The proteins were electrophoresed in SDS-PAGE. AT1 and AT2 receptors were detected by rabbit polyclonal anti-human AT1 and AT2 receptors (Santa Cruz Biotechnology), respectively, using ECL Plus Western blotting Detection System (GE Healthcare). β-Actin detected by mouse monoclonal anti-actin antibody (Sigma) was used as internal control. Organ Culture—Organ culture was examined as described previously (28Ihara H. Denhardt D.T. Furuya K. Yamashita T. Muguruma Y. Tsuji K. Hruska K.A. Higashioi K. Enomoto S. Nifuji A. Rittling S.R. Noda M. J. Biol. Chem. 2001; 276: 13065-13071Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Briefly, newborn mice (1 day after birth, P1) were sacrificed with ether, and the radii and ulnae were dissected out under a binocular microscope. The removed bones were rinsed in PBS with antibiotics and then placed in a 96-well plate (Corning Glass, Corning, NY) containing 50 ml of α-modified minimum essential medium (Sigma) supplemented with 10% bovine serum (Invitrogen). The bones were subsequently cultured in fresh media for 5 days under an atmosphere of 5% CO2, at 37 °C, changing the medium every day. In each experiment, bones from the right and left side of the P1 mice were collected. The bone from one side was used as an experimental group and treated with either PD123319 (10–8 m) plus angiotensin II (10–8 m), and the other was used as control with PD123319 plus PBS. The experiments were conducted with four samples from each group. The samples were then fixed in 4% paraformaldehyde and embedded in paraffin. The paraffin sections were stained for TRAP activity. TRAP-positive cells were counted in six sections per each sample. Statistical Analysis—To test the histological and histomorphometric parameter effects of AT1 or AT2 receptor blockers, Student's t test was used to compare between control (water or PBS) group and the experimental group treated with these blockers. The difference was judged to be statistically significant when p values were less than 0.05. All the numeral data in the results were presented as means ± S.D. Expression of Angiotensin II Type 2 (AT2) Receptor, Renin, and ACE in Bone in Vivo—To find out whether bone cells in vivo are the targets of Ang II, we first examined the expression of Ang II receptor proteins in the bones of adult mice. Immunohistochemistry was conducted using antibodies against these molecules. Osteoblasts were recognized as cuboidal mononuclear cells lining the trabecular bone, and these cells were positive for AT2 receptor staining (Fig. 1A, arrowheads). Examination of serial sections revealed that multinucleated cells, which attached to the bone matrix surface and expressed AT2 receptors (Fig. 1A, arrows), were also positive for TRAP (Fig. 1B, arrows), suggesting that osteoclastic cells or chondroclastic cells expressed AT2 receptor. These osteoblasts and osteoclasts also expressed AT1 receptor (Fig. 1C, arrowheads and arrows, respectively). Ang II, a ligand for these receptors, is generated by ACE cleavage of angiotensin I. Therefore, we examined whether ACE was expressed in bone cells. ACE signals were detected in osteoblasts and osteoclasts (Fig. 1D, arrowheads and arrows, respectively), the distribution of which was consistent with those expressing AT2 and AT1 receptors. As antibody against renin was not available, in situ hybridization was conducted. renin mRNA was expressed in the cells adjacent to trabecular bone (Fig. 1E, arrow). These cells exhibited vacuole-like morphology. The cells expressing renin were neither osteoblasts nor osteoclasts, although they were present in the bone microenvironment. These observations indicated that AT2 receptor as well as ACE and renin mRNA were expressed in the cells within a close proximity in the bone microenvironment. As positive control, renin signal was detected in juxtaglomerular cells in kidney (Fig. 1F). Furthermore, AT1, AT2, ACE, and renin mRNAs were expressed in primary cultures of osteoblastic cells derived from mouse neonatal calvarial cells and adult bone marrow based on RT-PCR analysis (Fig. 2). Thus, components of RAS are expressed locally in bone microenvironment. Real time PCR using RNA from bone marrow of tibiae indicated the expression of mRNAs encoding osteoclastic markers (Trap, Nfatc1, and Rank) as well as osteoblastic markers (alkaline phosphatase, osteocalcin, type I collagen, and Runx2) (data not shown). Time course experiments were conducted to examine the expression levels of AT1 and AT2 in primary cultures of osteoblasts derived from neonatal calvariae. In these cultures, we observed an increase in the levels of alkaline phosphatase and osteocalcin mRNAs. Real time PCR data indicated that mRNA expression levels of AT1 and AT2 were increased with time with a peak on day 7 for AT2 and day 10 for AT1, respectively (Fig. 2B). Western blotting of AT1 and AT2 revealed that the expression levels were increased with time (Fig. 2C). We also examined the effects of AT2 blocker on Trap mRNA in bone marrow and observed that AT2 blocker treatment suppressed Trap mRNA levels in the bone marrow (data not shown). Angiotensin II Type 2 Receptor Blockade Increases Bone Mass—To examine the functional role of the angiotensin II receptors in bone, mice (9-week-old male) were treated with an AT2 receptor blocker (AT2-B, PD123319) or control (PBS) daily for 2 weeks. AT2 receptor blockade resulted in crowded patterning of the trabecular bone in these mice compared with control as shown in three-dimensional μCT pictures (Fig. 3A). Analysis of morphological parameters based on three-dimensional μCT revealed that AT2 receptor blocker treatment increased BV/TV by about 20% (Fig. 3B). In addition, AT2 receptor blocker treatment also increased the Tb.N by about 10% (Fig. 3C). On the other hand, Tb.Spac was reduced by about 10% (Fig. 3D). Other morphological parameters such as the thickness and separation of trabecular bone showed a similar trend, but the differences were not statistically significant (data not shown). As AT1 receptor was expressed in the bone cells, we also conducted oral treatment with a blocker for AT1 receptor (losartan) or control (water). In contrast to the blockade of AT2 receptor, AT1 blockade did not affect bone mass in terms of the patterning of the trabecular bone as well as the levels of BV/TV, Tb.N, and Tb.Spac in mice (Fig. 3, E–H). Thus, AT2 receptor blockade specifically enhances bone mass in adult mice. AT 2 Receptor Blockade Enhances Bone Formation Activity—Because AT2 receptor blockade enhanced bone volume, we examined the effects of this blocker on the dynamic metabolic parameters in bone. Bone formation parameters were examined based on the analysis using calcein double labeling as described under “Experimental Procedures.” AT2 receptor blockade significantly increased BFR (Fig. 4A). AT2 receptor blockade did not significantly alter MAR levels, although there were trends for enhancement. MS/BS also tended to be increased by the treatment with AT2 receptor blocker, although the difference was not statistically significant (Fig. 4, B and C). Nodule formation assay in vitro revealed no major difference between the bone marrow cells obtained from mice after 2 weeks of treatment with AT2 blockade compared with control (data not shown). These observations indicated that AT2 receptor blockade enhanced bone volume at least in part through the enhancement of the bone formation activity in vivo. AT2 Receptor Blockade Suppresses Bone Resorption—Increase in the bone volume would be either due to the activation of bone formation or the suppression of bone resorption or both. Therefore, we examined the osteoclastic parameters in vivo as well. AT2 receptor blockade reduced the presence of the TRAP-positive area in the histological sections (Fig. 5A). Quantification of osteoclast number/BS in the secondary trabecular region indicated that AT2 receptor blocker treatment decreased the level by about 20% (Fig. 5B). AT2 receptor blocker treatment also decreased the levels of osteoclast surface per bone surface (Oc/BS) (Fig. 5C). These observations indicated that AT2 receptor blockade suppressed bone resorption activities in vivo. AT2 Receptor Blockade Suppresses Increasing Number of Osteoclasts by Angiotensin II in Organ Culture—We looked into whether RAS has any critical function in bone directly. To determine the effects of RAS in bone, we performed organ cultures using newborn mouse (1 day after birth, P1). Ulnae and radii were removed from the mice and were placed in 96-well plates (one bone per well) containing Ang II and/or AT2 blocker (AT2B, PD123319). We found that angiotensin II treatment increased the number of TRAP-positive osteoclasts in the cultured bone. Such increase in osteoclasts in
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