Monika forex anna nagar

Monika forex anna nagar
5 Kasi Arcade 1st Floor 116, Sir Thyagaraya Road Pondy Bazaar, lojas Nr Rathna.
Chennai - 600017 Chennai, Tamil Nadu 600017.
Sr. Gajapathy / Sr. Raja V.
Escreva uma crítica para Maharaja Forex Pvt Ltd.
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R. P.ABACUS NETCOMM PRIVATE LIMITED.
Detalhes da Companhia.
R. P.ABACUS NETCOMM PRIVATE LIMITED.
Companhia limitada por Ações.
Sub Categoria da empresa.
Classe de empresa.
Data de incorporação.
12 anos, 8 meses, 15 dias.
Consultoria em hardware. [Esta classe inclui consultoria sobre tipo e configuração de hardware com ou sem aplicativo de software associado. (Atividades similares realizadas por unidades que vendem computadores são classificadas na classe 3000)].
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Número de Membros.
Nomes anteriores.
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CINS anterior.
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Capital social e número de empregados.
Pagou o capital.
Número de empregados.
Listagem e detalhes de conformidade anual.
Data da última assembléia geral anual.
30 de setembro de 2018.
Data do último balanço patrimonial.
Relatório Legal.
Veja todos os casos criminais e civis de R. P.ABACUS NETCOMM PRIVATE LIMITED.
Relatório financeiro.
Empréstimos a longo prazo.
Empréstimos de curto prazo.
Caixa e saldos bancários.
Receita Total (Volume de Negócios)
Despesas com benefícios ao empregado.
Lucro antes de impostos.
Lucro depois do imposto.
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Detalhes do contato.
ID de email: abacusnc @ vsnl.
G - 33, PLANTA BAIXA, ANNA NAGAR PLAZA, 2ª AVENIDA, ANNA NAGAR CHENNAI TN 600040 IN.
Detalhes do diretor.
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Atualmente, Ramanujam Prabakar não está associado a nenhuma outra empresa.
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U65922TN1990PTC018655.
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Detalhes do Ministério Público.
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Encargos / Detalhes de empréstimos.
Propriedade imobiliária ou qualquer interesse nele; Dívidas do livro; Carga flutuante; soberano Gold Bond.
Detalhes dos Estabelecimentos.
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Conversão de empresa pública em empresa privada ou empresa privada em empresa pública.
Alocação de capital (ESOP, captação de fundos, etc.)
Retorno em relação à recompra de valores mobiliários.
Carta de oferta.
Aviso de consolidação, divisão, etc., ou aumento de capital social ou aumento de número de membros.
Registo de encargos (novos empréstimos garantidos)
Retorno de depósitos.
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Mudanças nas posições acionárias dos promotores e dos dez maiores acionistas.
Relatório de processo de compra.
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Agentes no exterior.
A Universidade possui uma rede de representantes em todo o mundo. Eles fornecem aos potenciais estudantes internacionais informações sobre os programas da Universidade de Auckland e os regulamentos governamentais da Nova Zelândia.
Nossos agentes e representantes no exterior podem ajudar com sua aplicação. Eles também podem fornecer folhetos e informações para fazer uma escolha informada. Eles também podem dar-lhe informações sobre como se mudar para um novo país para estudar, incluindo como obter vistos de estudantes, atender aos requisitos de língua inglesa e encontrar alojamento.
Algumas agências podem ter filiais em países diferentes dos listados abaixo.
Para obter mais informações, clique no link do site da respectiva agência.
Representantes oficiais no Azerbaijão.
44 Jafar Jabbarli Street.
Caspian Plaza, 1º andar.
República do Azerbaijão.
Telefone: +994 12 437 1051.
Representantes oficiais na Argentina.
Paraguai 647 Piso 4 Of. 17/18.
Telefone: +54 11 4311 9828.
Fax: +54 11 4103 0355.
Representantes oficiais na Austrália.
Escritório 3 (Nível 2) / 145 Melbourne St.
Telefone: +61 4 3487 9216.
333 Collins Street.
Telefone: +61 3 9629 2677.
Fax: +61 3 9629 2611.
165 Blackburn Rd.
South Victoria 3030.
Telefone: +61 9893 3718.
Fax: +61 9893 3721.
Nível 4 Suite 403.
Telefone: +61 431 7398 12.
Representantes oficiais no Bahrein.
Isa Town Mall Shop # 20 (Behind Ahli United Bank)
Edifício 3324, Estrada 1012, bloco 810.
Isa Town - Barrain.
Telefone: +973 1768 9960.
Fax: +973 1768 9930.
Representantes oficiais na Bélgica.
Sint Pieternieuwstraat 105.
Telefone: +32 25 887 184.
Representantes oficiais no Brasil.
Rua Barão do Triunfo.
550 conj. 65 Brooklin.
Telefone: +55 11 5041 9277.
Rua Haddock Lobo, 578.
cj. 82, Jardim Paulista.
São Paulo 01414-000.
Telefone: +55 11 5095 3030.
Representantes oficiais no Camboja.
No 657, Kampuchea Krom Blvd.
Sangkat Teuk Laark 1, Khan Toul Kork,
Phnom Penh (caixa postal 860)
Telefone: +855 23 881 017.
Fax: +855 23 881 020.
Representantes oficiais no Canadá.
Representantes oficiais no Chile.
Carmencita 265, Las Condes.
Telefone: +56 2 26117818.
Fax: +56 2 232 6212.
Cruz del Sur 235, Las Condes.
Telefone: +56 2 228 4843.
Fax: +56 2 228 4843.
Representantes oficiais na China.
Quarto 804, Edifício GuoMao.
No. 700 Yangming West Rd, província de Zhejiang.
Ningbo City 315000.
Telefone: +86 574 6285 0125.
Suite 550 Shanghai Center.
1376 West Nanjing Road.
Telefone: +86 21 6289 8123.
Fax: +86 21 6289 8208.
2F, Edifício comercial Interchina.
No.33, Deng Shi Kou Street, Dong Cheng District.
Telefone: +861 065 229 780.
Fax: +861 065 129 608.
Nível 7, BM InterContinental Center.
No. 100 YuTong Road.
Telefone: +86 21 6353 4219.
Fax: +86 21 6353 6188.
11F Hong Kong Macau Office Tower.
No.2 Chao Yang Men Bei Street.
Contato: James Zhang.
Telefone: +86 10 5166 7188.
Fax: +86 10 6553 6201.
Suite 1111/1438 Shaanxi North Road.
Telefone: +86 21 6095 6826.
802, Torre B, Jian Wai SOHO, Edifício de escritórios.
Telefone: +86 10 5869 9445.
Fax: +86 10 58694171.
Quarto 8010 Tower B Century Plaza.
14 Zhongnan Road, distrito de Wuchang.
Telefone: +86 27 8726 9936.
Fax: +86 27 8726 9935.
719 Yang Ming East Road.
Província de Jiang Xi.
Telefone: +86 791 8621 6226.
Kunlunshangcheng, distrito de Nangang.
Telefone: +86 451 8230 7080.
Fax: +86 451 8230 7103.
2 / F Guangzhou Library.
42 Zhongshan Si Road.
Telefone: +86 20 2833 9966.
Fax: +86 20 8386 7652.
Sala 2610-2617, Nível 26, Bloco A, Cyber ​​Tower, nº 2.
Zhongguancun South Street, distrito de Haidian.
Outras agências localizadas em Chengdu, Guangzhou, Xangai, Zhengzhou, Nanjing, Shenzhen.
Telefone: +86 10 5278 8989.
Fax: +86 10 5278 999.
5F Mi Yang Tower, Yong An Dong Li.
Avenida Jianwai, distrito de Chaoyang.
Telefone: +86 10 6568 5656.
Fax: +86 10 6568 6116.
Sala 711, Secção B, EAC da Legend City.
No.18 Jiaogong Road.
Fax: +86 574 8732 1979.
Piso 6, nº 6 Haidian Central Street.
Telefone: +86 10 6260 5599.
Fax: +86 10 6260 5266.
Piso 13, Edifício Zhongtian.
No. 1063, Siping Road, distrito de Yangpu.
Telefone: +86 21 5031 5706.
Fax: +86 21 5031 5702.
4F Área A, Dragon Century Plaza.
No.1 Hangda Road.
Telefone: +86 571 8790 1122.
Fax: +86 571 8790 1099.
Representantes oficiais na Colômbia.
Carrera 19A No. 84 - 72.
Telefone: +57 1 636 3003.
Fax: +57 1 616 4998.
Calle 93B # 17-42 de 506.
Telefone: +57 1 257 0059.
Fax: +57 1 622 2487.
Calle 93 # 15-27 De 306.
Edificio Avante 93.
Tem também uma filial em Medellín.
Telefone: +57 4 772 772.
Avenida Roosevelt nº 52 A-45.
Oficina 1B, Centro Comercial Súper Rápido del Sur.
Telefone: +57 2 551 3167.
Fax: +57 2 513 1459.
Office 503 Business Center 93.
Telefone: +57 301 7878892.
Calle 113, nº 11A-44.
Telefone: +571 6 37 5227.
Telefone: +571 6 29 7187.
Representantes oficiais na Dinamarca.
Nikolaj Plads 26, 2º andar.
Copenhagen K. DK-1067.
Telefone: +45 70 202824.
Fax: +45 70 202837.
Telefone: +45 33 4807 20.
Fax: +45 33 32 32 69.
Representantes oficiais no Egito.
31 Omar Bekir St.
de Othman Bin Affan St.,
7º andar Escritório # 601, Heliopolis.
Telefone: +202 2638 9905.
Telefone: +202 2638 9731.
Representantes oficiais na Finlândia.
Telefone: +35 09 680 78250.
Representantes oficiais na França.
Le Guide de vos études en Austrália.
16 rue Charlemagne.
Telefone: +33 1 40 468476.
Fax: +33 1 43 37 92 21.
Nível 1, Suite 2.
13 - 15 Wentworth Avenue.
Telefone: +61 2 9264 9733.
Fax: +61 2 9475 4078.
133, Avenue Emile Zola.
Telefone: +33 (0) 1 43 38 86 95.
Fax: +33 (0) 1 71 18 25 34.
20 rue Jean-Baptiste.
Telefone: +33 0 171 199 647.
Representantes oficiais na Alemanha.
Telefone: +49 251 1498 9380.
Fax: +49 251 1498 9385.
Consultoria - Escritório principal.
Lange Strasse 54.
Telefone: +49 711 284 88 86.
Fax: +49 711 284 88 96.
Centro de Educação Online.
Telefone: +49 30 2045 8687.
Fax: +49 30 2045 8688.
Telefone: +49 201 252552.
Fax: +49 201 267553.
Representantes oficiais em Hong Kong.
Suite 1501 Two Grand Tower.
No.625 Nathan Road.
Mong Kok, Kowloon.
Telefone: +852 2323 3287.
Fax: +852 2457 7118.
913 Lippo Sun Plaza, 28 Canton Rd.
Telefone: +852 2311 2689.
Fax: +852 2311 6011.
Unidade 818-819, Star House3.
Telefone: +852 2377 9115.
Fax: +852 2377 0344.
Quarto 1229-1230, 12 / F Star House.
3 Salibury Road, Tsim Sha Tsui.
Telefone: +852 2730 2068.
Fax: +852 2730 2808.
Quarto 2807, 28º andar.
25 Harbour Road.
Telefone: +852 2827 6362.
Fax: +852 2827 9286.
Fortune Commercial Building.
362 Sha Tsui Road, Tsuen Wan.
Telefone: +852 6187 7084.
Representantes oficiais na Índia.
Lote no.45, enclave Akash.
J. J. Nagar Colony Post.
Andhra Pradesh 500087.
Telefone: +91 40 4003 1023.
88a Rajpur Road.
Oposto Centro Osho.
Telefone: +91 9 761 213 000.
Contato: Anuradha Mankotia.
Telefone: +91 172 5051 600.
1006 Torres Chiranjiv.
Nova Deli 110019.
Telefone: +91 11 4160 8466.
Fax: +91 11 2628 0361.
Flat G-C Palace Gardens.
20 Ramanathan Street, Kilpauk.
Telefone: +91 44 4285 7500.
Fax: +91 44 4285 7100.
2 Jer Mahal, piso térreo.
Dhobi Talao Junction.
Opp. Metro Cinema.
Telefone: +91 22 4081 3333.
Fax: +91 22 2200 3269.
B-1C / 11A, Janak Puri.
Nova Deli 110058.
Telefone: +91 844 7838 452.
27 Mansões Vaswani, 5º andar.
Dinshaw Vachha Road, Opp. K. C. College Churchgate.
Telefone: +91 22 4322 2333.
Fax: +91 22 2285 4453.
Serviços (Pvt) Ltd.
Edifício Neerazhi, Menon Lane.
59/1447 Ravipuram, Ernakulam, Kerala.
Telefone: +91 484 402 2224.
HS 27 Segundo andar, Colônia Kailash.
Telefone: +91 11 471414.
Fax: +91 11 4714144.
Global Reach - Sede central.
1st Floor Ambady Apartments (Acima de Pães Quentes)
Outros ramos localizados em Ahmedabad, Anand, Baroda, Bangalore, Bhubaneswar, Chennai, Chandigarh, Guwahati, Hyderabad, Indore, Kolkata, Kohima, Nova Deli, Nagpur, Patna, Pune, Raipur, Salt Lake, Siliguri.
Telefone: +91 88 9121 7999.
1st Floor Ambady Apartments (Acima de Pães Quentes)
Kochi, Kerala 682018.
Telefone: +91 88 9121 7999.
Apartamento nº 516-520, 5º andar,
Torre de Comércio Internacional,
Nova Deli 110019.
Outros ramos localizados em Ahmedabad, Amritsar, Bangalore, Chandigarth, Chennai, Coimbatore, Gurgaon, Jalandhar, Kochi, Kolkata, Ludhiana, Pune Vadodra, Hyderabad, Mumbai, Mumbai West.
Telefone: +91 124 441 1888.
5/217, 1st Floor, Subhash Nagar.
Perto de Cambridge Foundation School, Rajouri Garden.
Nova Deli 110027.
Telefone: +91 11 4567 2200.
Fax: +91 11 2540 4753.
Sethi Niwas, 4ª Estrada.
Khar (oeste), Opp. Banco de sindicatos.
Telefone: +91 022 26056083.
Fax: +91 22 5694 2030.
SCO 124-126 Setor 9C.
Telefone: +91 172 330 0300.
Fax: +91 172 395 0701.
Câmara de julgamento, 1º andar.
Opp Axis Bank, MKK Nair Road.
Kerala Pin 682 025.
Telefone: +91 484 234 4664.
Móvel: +91 98 4703 6665.
No. 112, primeiro andar, Barton Center.
Telefone: +91 80 2558 0426.
Fax: +91 80 2558 7144.
B-2/9, 1st Floor, Opp. Happy Model School.
Nova Deli 110058.
Telefone: +91 11 2557 2009.
Fax: +91 11 2550 9156.
3º andar, RSR Plaza.
50 & amp; 51 Arcot Road, Saligramam.
Telefone: +91 99 4118 1615.
Pty Ltd (VIEC) - sede.
LMR House, Building No.-16, Shopping Center.
Green Park Ext., Near Green Park Metro Station.
Nova Deli 110016.
Telefone: +91 11 4657 7550.
Fax: +91 11 4657 7558.
Representantes oficiais na Indonésia.
Jl. Warung Jati Barat.
Taman Margasatwa 19, Ragunan.
South Jakarta 12510.
Telefone: +62 21 780 5636.
Fax: +62 21 781 4827.
STC Senayan Lantai 1 No 79-82.
Jalan Asia Afrika Senayan 10270.
Telefone: +62 21 579 31532.
Rich Palace R-40.
Jl. Mayjen Sungkono 151.
Telefone: +62 21 3156 61188.
Fax: +62 21 3156 30103.
Grupo - Sede central.
Plaza Sentral 10th Floor.
Jl. Jendral Sudirman 47.
Telefone: +62 21 252 4568.
Fax: +62 21 252 4741.
Jl. Sultão Iskandar Muda No. 18-G.
Arteri Pondok Indah, Kebayoran Lama.
Jakarta Selatan 12240.
Telefone: +62 21 7233 001.
Fax: +62 21 2920 9025.
Jl. MI. Ridwan Rais No. 6.
Jakarta Pusat 10110.
Telefone: +62 21 344 0555.
Fax: +62 21 345 0488.
Ruko Financial Center BA2.
No 54 Gading Serpong.
Telefone: +6221 547 6600.
Wisma Budi, 5º andar, JI. HR Rasuna disse Kav C-6, Kuningan.
Outras agências localizadas em Bali, Bandung, East Java, Jakarta Pondok Indah, Makassar, Medan, Kelapa Gading, Semarang, Yogyakarta.
Telefone: +62 21 252 3291.
Edifício La Pasadena.
JL Ciputat Raya No.1 Pondok Pinang.
Jakarta Selatan 12310.
Telefone: +62 21 765 1808.
Gedung Graha Kencana, Nível 9, unidade A.
Jln. Raya Perjuangan No.88, Kebon Jeruk.
Jakarta Barat 11530.
Telefone: +62 21 5366 0001.
Fax: +62 21 5366 0012.
Representantes oficiais no Irã.
(IGEC) - Instituto RAD.
Unidade 1, 5º andar, Complexo Khorshid.
Rua Valiasr, Vanak SQ.
Telefone: +98 21 8888 6556.
Fax: +98 21 8888 6558.
Representantes oficiais na Itália.
Viale Europa 331.
Telefone: +39 34 8350 9043.
Representantes oficiais no Japão.
5º andar Edifício Da Vinchi Shinjuku.
4-3-17 Shinjuku, Shinjuku-ku.
Telefone: +81 3 5367 3315.
1F F-Nissei Ebisu Bldg.
Telefone: +81 3 6434 1315.
Fax: +81 3 3409 8180.
Edifício Mitsui Seimei, 1F,
Telefone: +81 3 5287 2941.
Fax: +81 3 5287 2943.
39F, Shinjuku Center Building.
1-25-1, Nishi-Shinjuku, Shinjuku-ku.
Telefone: +81 3 3340 5300.
Fax: +81 3 3340 5327.
JR Shinanomachi Bldg.
6F, 34 Shinanomachi.
Telefone: +81 3 5312 4430.
Fax: +81 3 5312 4469.
Representantes oficiais na Jordânia.
Jubiha, área, rua de Ahmed Al-Tarawneh.
Ao lado de Northern Gate of Jordan University.
2º Edifício de investimento da Universidade da Jordânia, 3º andar.
Telefone: +962 6533 8785.
Representantes oficiais no Cazaquistão.
126 Nauryzbai Batyr St. No.2.
Telefone: +7 7272 720988.
Fax: +7 7272 509050.
56 Ivanilova Street.
Telefone: +7 777 188 7 188.
Representantes oficiais na Coréia do Sul.
[135-080] Edifício Hoya de nível 3,
830 Yeoksam-dong Kangnam-ku.
Telefone: +82 70 7509 7281.
Fax: +82 2 556 1109.
Edifício Uhak Net (Edu Net).
834-27 Yoksam 1 Dong, Gangnam Gu.
Telefone: +82 2 3481 1217.
Fax: +82 2 3482 2951.
IDP Coreia do Sul.
11º FL, Edifício Gwanghwamun.
211 Sejongno, Jongno-gu.
Outras agências localizadas em Busan, Gangnam.
Telefone: +82 2 739 7246.
401 Doosan Bearstel.
1319-11 Seocho 2-Dong, Seocho-Gu.
Telefone: +82 2 569 1989.
Fax: +82 2 569 4247.
SiS International Education.
404 Hanseung Building.
423 Gangnamdaero, Seochogu,
Outras agências localizadas em Busan.
Telefone: +82 2 525 5570.
Fax: + 82 2 587 4787.
Representantes oficiais no Kuwait.
General Trading and Contracting Co.
Escritório # 205 Edifício Al-Rabea.
Salem Al-Mubarak Street.
Salmiya P O Box 35199.
Telefone: +965 2574 3843.
Fax: +965 25756241.
Representantes oficiais na Malásia.
6º andar, bloco ocidental.
Wisla Selangor Dredging.
142-C Jalan Ampang.
Kuala Lumpur 50450.
Outros ramos localizados em Johor Bahru, Kota Kinabalu, Kuching, Penang, Subang Jaya.
Telefone: +60 3 2162 3755.
Sdn Bhd - Head Office.
Selangor Darul Ehsan 47500.
Telefone: +60 3 5633 4732.
Fax: +60 3 5634 1944.
Sdn Bhd - Head Office.
Bloco E, nº 32 (2º e 3º andar) Centro Comercial Taman Sri Sarawak.
Jalan Tunku Abdul Rahman, Serawak.
Telefone: +60 82 246795.
Fax: +60 82 428636.
Nível 24, Unidade nº B1, Menara MARA.
Jalan Tuanku Abdul Rahman.
Kuala Lumpur 50100.
Telefone: +60 3 2693 9121.
Fax: +60 3 2698 9121.
Jalan Genting Klang.
Telefone: +603 4141 8180.
Lote 6623 Térreo.
Jalan Pendente, Serawak.
Telefone: +60 82 347910.
Fax: +60 82 489834.
Selangor Darul Ehsan 47500.
Telefone: +60 3 5631 0322.
Fax: +60 3 5631 0522.
Representantes oficiais na Maurícia.
4º andar, edifício Ken Lee.
20 Rua Edith Cavell.
Telefone: +230 210 1971.
Fax: +230 210 4035.
Representantes oficiais no México.
Escritório Euler # 152-306, esq. Torcuato Tasso.
Col. Chapultepec Morales (Polanco)
México, D. F. 11570.
Telefone: +52 55 5545 3131.
Fax: +52 55 5545 1221.
Av. Paseo de la Reforma 412, 7º andar.
Suite 729, Colonia Juarez,
Telefone: +01 800 774 4644.
Representantes oficiais em Myanmar.
1 Coleman Street.
O Adelphi, # 08-07.
Telefone: +65 98281078.
Representantes oficiais no Nepal.
Bagbazar, Opp Hotel Hardik.
Telefone: +91 85 0585 6868.
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Interrupção da sinalização de BMP em Osteoblastos Através do Receptor de Tipo IA (BMPRIA) Aumenta a Massa e Daga de Osso;
Nobuhiro Kamiya,
Laboratório de Toxicologia Reprodutiva e de Desenvolvimento, Instituto Nacional de Ciências da Saúde Ambiental, Institutos Nacionais de Saúde, Research Triangle Park, Carolina do Norte, EUA. Procure mais artigos deste autor.
Biologia Oral, Faculdade de Odontologia, Universidade de Missouri-Kansas City, Kansas City, Missouri, EUA. Procure mais artigos deste autor.
Tatsuya Kobayashi,
Unidade Endócrina, Hospital Geral de Massachusetts e Harvard Medical School, Boston, Massachusetts, EUA. Procure mais artigos deste autor.
Donald J Lucas,
Laboratório de Toxicologia Reprodutiva e de Desenvolvimento, Instituto Nacional de Ciências da Saúde Ambiental, Institutos Nacionais de Saúde, Research Triangle Park, Carolina do Norte, EUA. Procure mais artigos deste autor.
Yoshiyuki Mochida,
Centro de Pesquisa Odontológica, Universidade da Carolina do Norte em Chapel Hill, Chapel Hill, Carolina do Norte, EUA. Procure mais artigos deste autor.
Mitsuo Yamauchi,
Centro de Pesquisa Odontológica, Universidade da Carolina do Norte em Chapel Hill, Chapel Hill, Carolina do Norte, EUA. Procure mais artigos deste autor.
Henry M Kronenberg,
Unidade Endócrina, Hospital Geral de Massachusetts e Harvard Medical School, Boston, Massachusetts, EUA. Procure mais artigos deste autor.
Jian Q Feng,
Biologia Oral, Faculdade de Odontologia, Universidade de Missouri-Kansas City, Kansas City, Missouri, EUA. Procure mais artigos deste autor.
Yuji Mishina.
Publicado em: 4 de agosto de 2008 Histórico completo de publicação DOI: 10.1359 / jbmr.080809 Ver / salvar citação Citado por (CrossRef): 72 artigos Verifique se há atualizações.
Partes do manuscrito foram apresentadas na 28ª Reunião Anual da Sociedade Americana de Pesquisas Ósseas e Minerais, Filadélfia, PA, EUA, 15 de setembro e 19 de dezembro de 2006.
Os autores afirmam que não têm conflitos de interesse.
As proteínas morogenéticas do osso (BMPs) são conhecidas como indutores de osso ectópicos. O FDA aprovou BMPs (BMP2 e BMP7) para uso clínico. No entanto, os efeitos diretos das BMP no metabolismo ósseo endógeno ainda não são bem conhecidos. Nós interrompermos de forma condicionada o receptor de BMP tipo IA (BMPRIA) em osteoblastos durante estádios de amamentação e adulto para mostrar o impacto da sinalização de BMP na modelagem e remodelação do osso endógeno. A recombinação creativa foi detectada em osteoblastos imaturos no periósteo, osteoblastos e osteócitos, mas não em condrócitos e osteoclastos após a administração de tamoxifeno. Os ratinhos knockout condicionais Bmpr1a (cKO) mostraram aumento da massa óssea principalmente no osso trabecular em P21 e 22 semanas, conforme determinado pela coloração H & amp; E. Vertebrae, cauda e costelas apresentaram maior radiodensidade a 22 semanas, consistente com um aumento significativo da DMO. Tanto a TC como a histomorfometria mostraram aumento da BV / TV trabecular e da espessura dos ossos adultos de CKO, enquanto o número de osteoclastos, a taxa de formação óssea e a taxa de aposição mineral diminuíram. Os níveis de expressão dos marcadores de formação óssea (Runx2 e Bsp), marcadores de reabsorção (Mmp9, Ctsk e Tracp) e Rankl diminuíram, e Opg foi aumentado em ossos adultos, resultando em uma redução na proporção de Rankl para osteoprotegerina (Opg) . A redução da osteoclastogênese através da via RANKL & ndash; OPG também foi observada nos estádios de cebola e reproduzida na cultura da calvária neonatal. Estes resultados sugerem que Bmpr1a cKO aumentou a massa óssea endógena principalmente em osso trabecular com diminuição da osteoclastogênese através da via RANKL & ndash; OPG. Concluímos que a sinalização BMPRIA em osteoblastos afeta a formação óssea e a reabsorção para reduzir a massa óssea endógena in vivo.
INTRODUÇÃO.
B uma proteína morfogenética (BMPs) são membros do TGF - e beta; superfamília e foram originalmente descobertos como indutores de osso ectópicos em tecidos moles. A função osteogênica das BMP foi amplamente examinada, principalmente usando osteoblastos em cultura. A FDA aprovou algumas BMPs (BMP2 e BMP7) para uso clínico em fraturas abertas de osso longo, fraturas não uniões e fusão espinhal. No entanto, apesar das evidências significativas de seu potencial de regeneração óssea em estudos animais e pré-clínicos, os dados clínicos atuais que sustentam sua eficácia não são robustos. Isso pode ser porque em parte da falta de compreensão dos efeitos variáveis, os BMPs têm in vivo em diferentes tipos celulares, incluindo condrócitos, osteoblastos e osteócitos.
BMP2, BMP4 e o seu receptor de BMP de receptor potente IA (BMPRIA ou ALK3) são abundantemente expressos no esqueleto; no entanto, os camundongos convencionais para nocaute (KO) para estes genes causam a letalidade embrionária precoce. Anteriormente resgatamos a letalidade da perda de Bmpr1a utilizando o sistema Cre-Lox P sob o controle de um promotor de osteocalcina e descobrimos que a massa óssea (BV / TV) avaliada por histomorfometria óssea foi reduzida nos camundongos mutantes aos 3 meses, mas foi aumentada em 10 mo. Esta evidência sugere que as BMPs têm efeitos diversos na massa óssea, formação óssea e homeostase de uma maneira dependente da idade.
Para entender as funções dependentes da idade da sinalização BMP no esqueleto, é importante interromper a sinalização BMP em diferentes idades de forma específica para osteoblastos. Aqui, utilizamos um sistema creatividade Creoxo-induzível com tamoxifeno (TM) sob o controle de um promotor de colágeno de tipo I de 3,2 kb (ratinhos Col1-CreER TM), que é ativado em osteoblastos, odontoblastos e fibroblastos tendinosos e interrompido Sinalização BMP através de BMPRIA no osso. Este sistema nos permite controlar o início da ruptura de Bmpr1a em osteoblastos em qualquer idade por administração de TM. Como o pico de massa óssea no mouse é alcançado após 20 semanas, primeiro nos concentramos em remodelação óssea e nos ratos analisados ​​22-wk, iniciando a administração de TM a 8 semanas. Além disso, para identificar os efeitos dependentes da idade da sinalização BMP, também estudamos modelagem óssea usando camundongos erva iniciando a administração de TM no pós-natal dia 2 (P2).
Neste estudo, descobrimos que a massa óssea foi aumentada pela perda de sinalização BMP em osteoblastos através da BMPRIA durante os dois estágios. A osteoclastogênese foi reduzida através da via RANKL & ndash; osteoprotegerina (OPG), mesmo que os marcadores de formação óssea tenham sido reduzidos ou inalterados nos camundongos mutantes, resultando em um aumento líquido na massa óssea. Esta evidência sugere que a sinalização de BMP nos osteoblastos contenha a massa óssea endógena, o que é inesperado e contrário à compreensão atual das BMPs como indutores ósseos.
MATERIAIS E MÉTODOS.
Administração de ratos e TM.
Os ratos que expressam a proteína de fusão cre-indutível Cre-ER TM TM sob o controle de um promotor de propollagen de rato de 3,2 kb e promotor alfa 1 (Col1-CreER TM) foram gerados por injeção pronuclear e cruzados com ratos Bmpr1a de floxed. Os ratos ROSA26 Cre Reporter (R26R) foram gentilmente fornecidos pelo Dr. Philippe Soriano. Os ratinhos Col1-CreER TM foram cruzados com ratos Bmpr1a floxed para gerar gatos manipulados como Col1-CreER TM +: Bmpr1a fx / fx e Col1-CreER TM & minus; : Bmpr1a fx / fx. TM (T5648; Sigma) foi dissolvido num pequeno volume de etanol, diluído com óleo de milho a uma concentração de 10 mg / ml, e armazenado a & minus; 20 ° C até à utilização. Após a administração de TM, a recombinação de Cre foi induzida em camundongos CKO de Bmpr1a (Col1-CreER TM +: Bmpr1a fx / fx), mas não em controles de littermate (Col1-CreER TM & minus;: Bmpr1a fx / fx). Em ratos machucados, TM (75 mg / kg) foi injetada intraperitonealmente em fêmeas lactantes a cada 3 dias de P2 até a dissecção em P14 ou P21 (Fig. 1B). Para estágios para adultos, a TM foi injetada intraperitonealmente duas vezes por semana a partir de 8 semanas até a dissecção às 10 ºC, 12 ou 22 semanas (Fig. 1C). Os resultados foram analisados ​​comparando os controles Bmpr1a cKO (Col1-CreER TM +, Bmpr1a fx / fx) e littermate (Col1-CreER TM & minus; Bmpr1a fx / fx), ambos recebendo TM. Não foram observados efeitos colaterais na morfologia óssea utilizando este regime de TM. O protocolo animal foi aprovado pelo Comitê Institucional de Cuidados e Uso Animal no Instituto Nacional de Ciências da Saúde Ambiental, NIH.
Figura FIG. 1 ..
Ratinhos transgênicos Col1-CreER TM. (A) proteína de fusão Cre-ER TM, induzível a TM, sob o controle de um promotor de propollagen e alfa 1 (I) de 3,2 kb (Col1-CreER TM). (B) Horário de administração de TM durante o estágio de cebola (P21). A TM foi injetada intraperitonealmente em fêmeas lactantes de P2 a P20 a cada 3 dias. Ambos os ratos de controle (Cre & minus;, Bmpr1a fx / fx) e ratinhos cKO (Cre +, Bmpr1a fx / fx) receberam TM. (C) Programação de administração de TM durante o estágio adulto (22 semanas). A TM foi injetada intraperitonealmente em adultos de 8 a 22 semanas, duas vezes por semana. Ambos os controles e o cKO receberam TM. (D) RT-PCR quantitativa em tempo real mostra expressão reduzida de Bmpr1a em femora e vértebras lombares de camundongos e femores de 10 a 12 semanas e costelas de ratinhos P14 após recombinação. * Diferença estatisticamente significativa entre cKO e controle de três experimentos independentes (média e mais, SD, t - test; * p & lt; 0,01). Resultados semelhantes foram obtidos do fêmur em P21. (E) proteína BMPRIA em fêmeas de ratos de 10 a 12 semanas avaliados utilizando anticorpos policlonais de coelho contra BMPRIA expostos com DAB (marrom). Os núcleos foram corados com hematoxilina. Células DAB + por células totais são mostradas. * Diferença estatisticamente significativa entre cKO (8%) e controle (48%) em dados agrupados de três experimentos independentes (média e mais, SD, t - test; * p & lt; 0,01). Resultados semelhantes foram obtidos a partir de vértebras lombares no mesmo estágio e fêmur em P21. Bares, 50 m. (F) Os ratos ROSA26 Cre Reporter mostraram atividade Cre em osteoblastos, mas não em condrócitos na P21. A recombinação de DNA dependente de Cre foi detectada pela coloração de beta-galactosidase ("beta" - gal) (a). O osso cortical (b) e o osso trabecular (c) são mostrados. Osteoclasts foram negativos para & beta; - gal (d). Barras: 1 mm (a), 50 μ m (b e c) e 20 μ m (d). (G) Os ratos ROSA26 Cre Reporter mostraram atividade de Cre em osteoblastos e osteócitos na área óssea trabecular (a) e área óssea cortical (b) a 22 semanas. Barras: 200 (a) e 100 μ m (b).
Para manchas de H & amp; E, fêmora, tíbia, úmero, cauda, ​​vértebras lombares, costelas e calvária de ratinhos P21 e 22-wk foram fixados em paraformaldeído a 4%, descalcificados com 10% de EDTA e incorporados em parafina. As secções de parafina foram cortadas em planos sagital e coronal a 8 μ m. A imunocoloração foi realizada utilizando anticorpos policlonais de coelho contra BMPRIA (1:50; Orbigen). Posteriormente, um kit ABC (Santa Cruz Biotechnology) e DAB foram utilizados para detecção. Para a coloração de beta-galactosidase ("beta"), os ossos descalcificados foram preparados em 30% de sacarose antes da seção congelada. As secções foram coradas com X-gal para atividade beta e beta e contra-coloridas com eosina.
Histomorfometria óssea estática e dinâmica.
Os ratos adultos receberam calceína (10 mg / g; Sigma) em NaHCO3 a 2% por via intraperitoneal 7 dias antes da morte a 22 semanas e laranja de xilenol (90 mg / kg, Sigma) por via intraperitoneal 2 dias antes da morte. Femora e vértebra (L 2 e L 3) foram dissecadas e fixadas em paraformaldeído a 4%. A femora e as vértebras distal não condicionadas foram desidratadas e incorporadas em metacrilato de metilo. As secções sagital e coronal longitudinais de cinco milímetros foram cortadas em um microtomo Polycut S (Reichert-Jung). As secções foram tiradas do meio da femora e as vértebras foram coradas com tricromo de Masson modificado. As medidas histomorfométricas foram feitas de forma cega e sem via, utilizando o sistema de análise de imagem computadorizada OsteoMeasure (OsteoMetrics) interligado com um microscópio Optiphot Nikon (Nikon) em uma ampliação de & times; 20. Todas as medidas femorais foram confinadas ao spongiosa secundário e restringidas a uma área entre 400 e 2000 μm distal à placa de crescimento e junção metafisária do fêmur distal.
Para a coloração com TRACP, foram preparadas secções de parafina lombalas descalcificadas (L 3) 5 e como se descreveu para a coloração H & amp; E. Sections were stained using the leukocyte acid phosphatase kit (Sigma) and counterstained with hematoxylin. Osteoclasts were identified as multinucleated, TRACP + cells lining the trabeculae. All measurements were made as described for bone histomorphometry.
X-ray, μCT, and DXA analyses.
X-ray images of P21 and 22-wk mice were taken using Faxitron X-ray system (Faxitron X-Ray). Femora and vertebrae (L 3 ) were scanned using a μCT (Scanco Medical) system at 12 μm of thickness, 55 kV of energy, and 145 μA of intensity. The data were reconstructed to produce 2D and 3D images. BMD was determined by DXA using the Lunar PIXImus2 densitometer (GE).
Quantitative real-time RT-PCR.
RNA was isolated from ribs and femora from weanling mice (P14 and P21) and femora and lumbar vertebrae from adult mice (10–12 wk) using the MicroFastTrack 2.0 Kit (Invitrogen). cDNA was synthesized using the SuperScript Preamplification System (Invitrogen). PCR reactions, data quantification, and analysis were performed according to the manufacturer's protocol (Applied Biosystems). Taqman primers and probes used in this study were as follows: Bmpr1a , Mm00477650_m1 (64 bp); runt-related transcription factor 2 ( Runx2 ), Mm00501578_m1 (115 bp); osterix ( Sp7 ), Mm00504574_m1 (137 bp); bone sialoprotein ( Bsp ), Mm00492555_m1 (98 bp); alkaline phosphatase ( Akp2 ), Mm00475831_m1 (141 bp); osteocalcin ( Bglap2 ), Mm01741771_g1 (77 bp); metalloproteinase-9 ( Mmp9 ), Mm00600163_m1 (107 bp); cathepsin K ( Ctsk ), Mm00484036_m1 (84 bp); TRACP ( Tracp ), Mm00475698_m1 (79 bp); RANKL ( Rankl ), Mm00441908_m1 (69 bp); osteoprotegerin ( Opg ), Mm00435452_m1 (119 bp). Values were normalized to Gapdh using TaqMan Rodent GAPDH Control Reagents (Applied Biosystems). All measurements were performed in triplicate and analyzed using the 2 −ΔΔCt method.
Ex vivo culture of calvarial bone.
Untreated cKO calvaria were collected from newborn mice ( Cre + , Bmpr1afx/fx ), bisected at the sagittal suture, and cultured in modified BGJ (Invitrogen) supplemented with ascorbic acid (50 mg/ml; Sigma) and 5% FBS (Life Technologies) for the first 24 h in culture. One hemicalvaria from each pair was treated with 4-hydroxyl (4-OH) TM (100 ng/ml; Sigma) for 5 days. The medium was changed daily.
Serum examination.
Serum was collected from 22-wk-old mice. Circulating serum levels of osteocalcin (BT-470; Biomedical Technologies), pyridinoline (8019; Quidel), RANKL, and OPG (MBN-41K-1RANKL and MBN-41K-1OPG, respectively; Millipore) were measured according to the manufacturer's instructions.
Statistical analysis.
All results are expressed as mean ± SD. Student's t - tests were used to compare data between control and cKO mice; p < 0.05 indicates significance.
Tissue specificity and efficiency of Cre recombinase in Col1-CreER TM mice.
Both developmental and remodeling stages are crucial for understanding bone biology. In this study, we chose two different stages, weanling (P21) and adult (22 wk), and conditionally knocked out BMP signaling through BMPRIA in osteoblasts using a TM-inducible Cre - lox P system under the control of a 3.2-kb type I collagen promoter ( Col1-CreER TM ; Figs. 1A–1C).
Expression levels of Bmpr1a in cKO bones as determined by quantitative RT-PCR were significantly reduced >70% both in the weanling and adult stages (Fig. 1D). BMPRIA in cKO 22-wk femora decreased 84% in osteoblasts and osteocytes by immunostaining (Fig. 1E). Similar results were obtained from 22-wk lumbar vertebrae and P21 femora (data not shown). Cre efficiency and the specificity of Col1-CreER TM expression were confirmed by β-gal staining using R26R mice. In P21 femora, β-gal staining was observed in trabecular bone, as shown in the epiphysis and metaphysis (Fig. 1F, a), and in cortical bone (Fig. 1F, b). Staining was detected in immature periosteal cells, osteoblasts, and osteocytes (Fig. 1F, b) but not in chondrocytes (Fig. 1F, c) or soft tissues (Fig. 1F, a). Cre negative controls administered TM ( Col1-CreER TM − , TM+ ) and Cre-positive mice not given TM ( Col1-CreER TM + , TM− ) showed no β-gal staining (data not shown). In addition, no Cre activity was detected in osteoclasts as shown by staining for β-gal and H&E (Fig. 1F, d). These results show that the TM administration delivered through milk specifically and efficiently disrupts BMPRIA in osteoblasts and osteocytes in weanling stages. Similarly, Cre activity was also detected in osteoblasts and osteocytes in trabecular and cortical bone at 22 wk (Fig. 1G, a and b, respectively).
Increased bone mass in Bmpr1a cKO mice.
Morphologically, cKO mice appeared normal both at P21 and 22 wk with no significant difference in body weight or length compared with controls (data not shown). At P21, X-ray analyses showed rib flaring (Fig. 2A, a) and a modest increase in radiodensity of tail trabeculae (Fig. 2A, b) and femur metaphysis (Fig. 2A, c). H&E staining showed that trabecular bone in the epiphysis and metaphysis of femur, tibia, humerus, and vertebrae (tail) were dramatically increased in cKO mice (Fig. 3A). Intramembranous calvarial bones of cKO mice were thicker than those of controls with increased woven bone (Fig. 3A, f and g). BMD measured by DXA was significantly increased in ribs (control: 0.0152 g/cm 2 , cKO: 0.0163 g/cm 2 ; p < 0.05), consistent with the X-ray image (Fig. 2A, a). These results show increased bone mass in both endochondral (femur, tibia, humerus, and tail) and intramembranous (calvaria) cKO bones at P21 (Figs. 2A and 3A).
Figure FIG. 2..
Relative BMD shown by X-ray at P21 (A) and 22 wk (B). (A) White arrows indicate rib flaring (a) and increased radiodensity of metaphysis in femora (c) in P21 cKO mice. (B) Radiodensity of cKO bones was notably increased in spines, tails, and ribs but not in femora and skulls at 22 wk. White arrows indicate calvarial bones where sections were prepared (Fig. 3B, c and d).
Figure FIG. 3..
Bone histology in Bmpr1a cKO mice at P21 (A) and 22 wk (B). (A) H&E staining of femora (a), tibia (b), humerus (c), epiphysis of humerus (d), tails (e), and calvaria (f and g). Boxes in f are magnified in g. Bone mass was increased in cKO bones. Bars: 2 mm (a, b, and e), 1 mm (c and f), 500 μm (d), and 200 μm (g). (B) H&E staining of femora cortical bone (a), lumbar (L 3 ) trabecular bone (b), calvaria (c and d), and ribs (e). Bone mass was increased in L 3 trabecular bone but not in femoral cortical bone. Boxes in c are magnified in d. Bars: 1 mm (a, b, and e), 500 μm (c), and 250 μm (d).
At 22 wk, rib flaring was also noted by X-ray in cKO mice (Fig. 2B) similar to P21 mice (Fig. 2A), and radiodensity in cKO bones including spine, tail, and rib cage was dramatically increased (Fig. 2B), consistent with a significant increase in BMD (Fig. 4A). H&E staining showed increased bone mass in vertebrae and ribs (Fig. 3B, b and e, respectively) where radiodensity was increased (Fig. 2B). However, the cKO femora appeared to be similar to controls by X-ray analysis (Fig. 2B), H&E staining (Fig. 3B, a), and DXA (Fig. 4A). Although cKO calvaria appeared unchanged in X-rays (Fig. 2B), H&E staining showed a smaller bone marrow cavity in cKO calvaria compared with controls (Fig. 3B, c), which probably resulted in the significant increase in BMD observed (Fig. 4A). These results show that bone mass is also increased in cKO mice during adult stages with some variation by site.
Figure FIG. 4..
Increased bone mass in cKO bones at 22 wk. (A) BMD was determined by DXA using lumbar (L 3 ), tails, ribs, femora, and skulls. Values are expressed as mean ± SD (control, n = 8; cKO, n = 10). *Statistically significant difference between control and cKO (mean ± SD, t - test; * p < 0.01). (B) Reconstructions of bone structure from μCT. Cortical bone in femora and trabecular bone in lumbar vertebrae (L 3 ) are shown. Trabecular bone was dramatically increased in cKO vertebrae (L 3 ).
We further examined trabecular and cortical bone independently by μCT. In vertebral trabecular bone, in addition to a significant increase in tissue density (Table 1), bone volume (BV/TV) and trabecular thickness of cKO bones significantly increased 90% and 104% compared with controls, respectively (Table 1), which is shown by the 3D reconstruction (Fig. 4B). In cKO femora, tissue density of trabecular bone was significantly increased by a small amount (Table 1), suggesting that trabecular bone is increased in cKO mice to varying degrees by site. On the other hand, in cKO femoral cortical bones, there were statistically significant differences in the adult cKO femora in cortical porosity (increased), apparent density (decreased), and tissue density (decreased) as noted in Table 2. These differences are apparent in the 2D reconstruction (Fig. 4B) and by H&E staining (Fig. 3B, a). Because Cre recombination is observed in cortical bones (Fig. 1G, b), these results suggest that a decrease in BMPRIA signaling may be leading toward a “trabecularization” of cortical bone. Taken together, P21 and 22-wk data suggest that Bmpr1a deficiency in osteoblasts increased bone mass mainly by increasing trabecular bone during both modeling and remodeling phases with some variation by site.
Reduced bone formation and resorption in Bmpr1a cKO bones.
Bone mass is determined by the balance between formation and resorption. To further study the trabecular bone phenotype in cKO mice, these two factors were measured by static and dynamic histomorphometry. We chose 22-wk trabecular bones in femora and vertebrae because peak bone mass and strength in the mouse are achieved after 20 wk, with dynamic changes in both material and geometric properties. Dynamic analysis showed a reduction of bone formation (BFR/BS) and mineral apposition rate (MAR) in trabecular bone of cKO femora (Fig. 5A). Dye deposition showed double lines in trabecular bone of control femora but did not in cKO (Fig. 5B). No significant decrease of N. Oc/T. Ar was observed in the femora trabeculae (control: 123.1 ± 30.8, cKO: 119.1 ± 15.6). Bone volume (BV/TV) and trabecular thickness (Tb. Th) were more increased in cKO vertebrae than femora by static bone histomorphometry (data not shown), consistent with the results from μCT analysis (Table 1). Osteoclast number per bone area (N. Oc/T. Ar) as determined by static analysis with TRACP staining was significantly reduced in vertebrae (L 3 ) of cKO mice (Fig. 5C). These results suggest that both bone formation and resorption were reduced by loss of BMPRIA signaling with some variation by site.
Figure FIG. 5..
Morphometric markers for bone formation and resorption at 22 wk. (A) Dynamic (BFR/BS and MAR) analyses were performed using trabecular bone of femora and lumbar vertebrae (L 3 ) from control ( n = 10) and cKO ( n = 10) mice. *Statistically significant difference between control and cKO ( t - test; * p < 0.05; ** p < 0.005; BFR/BS, bone formation rate over bone surface; MAR, mineral apposition rate). (B) Dynamic histomorphometric analysis of cKO bones by double label staining of trabecular bone in femora with calcein and xylenol orange. Dotted and random staining pattern was shown in cKO. White arrows indicate merging of double labeled staining in cKO bone. Bars: 100 μm. (C) Static histomorphometric analysis of L 3 trabecular bone with TRACP staining. Values are expressed as mean ± SD (control, n = 5; cKO, n = 5). *Statistically significant difference between control and cKO (mean ± SD, t - test; * p < 0.05). N. Oc/T. Ar, osteoclast number per bone area. Bars: 100 μm.
We next examined formation and resorption by measuring expression and serum levels of markers. RNA was extracted from weanling (P14) or adult (10–12 wk) mice (Figs. 1B and 1C). In adult vertebrae, expression levels of formation markers, Runx2 ( Cbfa1 ) and Bsp , were significantly reduced by ∼60% (Fig. 6A), and resorption markers Mmp9 , Ctsk , and Tracp were all significantly reduced >50% (Fig. 6B), consistent with the reduction in osteoclast number stained by TRACP (Fig. 5C). However, formation markers Sp7 (osterix) and Akp2 (alkaline phosphatase) were unchanged. Similar results were obtained from femora at this stage (data not shown). In weanling ribs where increased radiodensity was observed (Fig. 2A, a), bone formation markers were unchanged significantly (data not shown); however, Tracp significantly decreased by 40% (Fig. 6D). Osteoblasts regulate osteoclastogenesis by producing RANKL, an osteoclast differentiation factor, and osteoprotegerin (OPG), a decoy receptor for RANKL. During adult stages, the expression of Rankl and Opg were significantly decreased and increased, respectively (Fig. 6C), resulting in a significant reduction of the ratio of Rankl to Opg by 70% (Fig. 6C). Similar results were obtained from weanling stages, beause the expression of Opg significantly increased by 50%, and the ratio of Rankl to Opg significantly decreased by 35% (Fig. 6D). Because RANKL stimulates osteoclastogenesis, whereas OPG inhibits it, these results suggest that osteoclastogenesis is decreased both in weanling and adult stages. Serum levels of the formation marker osteocalcin were significantly increased in adult cKO mice (data not shown), consistent with dramatically increased radiodensity at 22 wk (Fig. 2B). On the other hand, the resorption marker pyridinoline, RANKL, and the ratio of RANKL/OPG were nonsignificantly reduced in older mice (data not shown), partly because TM administration confounds straightforward interpretation of serum markers. It is also possible that timing of collecting serum is crucial for monitoring biological changes, because expression levels of resorption markers, Rankl , and Opg changed at earlier stages (10–12 weeks). Overall data suggest that resorption markedly decreases in cKO bones even though formation is somewhat reduced or unchanged, resulting in a net increase in bone mass.
Figure FIG. 6..
Expression levels of bone formation and resorption markers during adult and weanling stages. RNA was extracted from vertebrae of 10- to 12-wk mice for adult stages and ribs of P14 for weanling stages. (A) Quantitative RT-PCR for bone formation markers ( Runx2 , Sp7 , Bsp , Akp2 , and Bglap2 ) during adult stages. Expression levels of both Runx2 and Bsp were significantly reduced in cKO. (B) Quantitative RT-PCR for bone resorption markers expressed by osteoclasts ( Mmp9 , Ctsk , and Tracp ) during adult stages. These expression levels were all reduced over 50% in cKO. (C) Quantitative RT-PCR for Opg and Rankl expressed by osteoblasts and relative ratio of Rankl to Opg expression levels during adult stages. The expression of Rankl was significantly reduced by 60%, and Opg was significantly increased by 80%, resulting in a significant reduction in the ratio of Rankl to Opg by 70% in cKO. (D) Quantitative RT-PCR for bone resorption markers ( Mmp9 , Ctsk , and Tracp ) and RANKL–OPG pathway ( Opg and Rankl ), and relative ratio of Rankl to Opg expression levels during weanling stages. The expression of Tracp decreased by 40% and Opg increased by 50%, both significantly. The ratio of Rankl to Opg expression levels significantly decreased 35%. Similar results were obtained from femora during adult stages. *Statistically significant difference between cKO and control from three independent experiments in A–D (mean ± SD, t - test; * p < 0.05; ** p < 0.001).
To confirm the effect of BMP signaling on osteoclastogenesis through the RANKL–OPG pathway observed both in weanling and adult stages, we next set up a tissue culture system using newborn calvaria. Untreated cKO calvarial bones were collected from newborn mice ( Cre + , Bmpr1afx/fx ), bisected, and cultured with or without 4-OH TM (100 ng/ml). Cre-dependent DNA recombination using this method was confirmed by β-gal staining of calvarial bones from R26R mice (Fig. 7A), and expression of Bmpr1a was significantly reduced 78% in cKO ( Cre + , Bmpr1afx/fx , TM + ) compared with controls ( Cre + , Bmpr1afx/fx , TM − ; Fig. 7B). Expression levels of resorption markers Mmp9 , Ctsk , and Tracp were significantly reduced >60% in cKO compared with controls (Fig. 7C). Rankl expression was reduced 41%, and Opg was significantly increased 12%, resulting in a 50% reduction of the ratio of Rankl to Opg in the treated group, which was significant (Fig. 7D). There were no significant differences in these expression levels between TM-treated (Cre − , Bmpr1afx/fx, TM + ) and nontreated Cre negative bone (Cre − , Bmpr1a fx/fx, TM − ) using littermate controls (data not shown). These results suggest that Bmpr1a - deficient osteoblasts reduced osteoclastogenesis through the RANKL–OPG pathway ex vivo, which corroborates in vivo results.
Figure FIG. 7..
Reduced osteoclastogenesis by loss of BMPRIA ex vivo. (A) Cre activity was assessed using R26R . When Cre-positive calvarial bones from newborn mice were treated with 4-hydroxyl TM (100 ng/ml) for 5 days, Cre-dependent DNA recombination was detected by β-gal staining (Cre + , TM + ) compared with no staining in untreated bones (Cre + , TM − ). Histological analysis showed that osteoblasts were positive for β-gal. Stained nuclei were circled with dotted line. (B) Quantitative RT-PCR for Bmpr1a using calvarial bones (Cre + , Bmpr1afx/fx) with or without 4-hydroxyl TM. Calvarial bones were collected from newborn mice, which did not receive TM before death. Bmpr1a expression was significantly reduced after TM treatment (TM + ) compared with nontreated (TM − ). (C) Quantitative RT-PCR for bone resorption markers expressed by osteoclasts ( Mmp9 , Ctsk , and Tracp ). (D) Quantitative RT-PCR for Opg and Rankl expressed by osteoblasts, and relative ratio of Rankl to Opg expression levels. There were no significant differences in these expression levels between TM-treated ( Cre − , Bmpr1afx/fx , TM + ) and nontreated ( Cre − , Bmpr1afx/fx , TM − ) bones using littermate controls (data not shown). *Statistically significant difference between TM + and TM − from three independent experiments in B–D (mean ± SD, t - test; * p < 0.05).
DISCUSSION.
Osteoblasts, BMP signaling, and bone mass.
By using a 3.2-kb mouse procollagen α1(I) promoter, we showed that reduction in BMP signaling through BMPRIA in osteoblasts increased bone mass both in P21 and 22-wk mice. We also observed increased bone mass in another conditional Bmpr1a knockout mouse (data not shown) using Col13.6-Cre mice. These bone phenotypes are consistent with our previous report that showed increased bone volume (BV/TV) at 10 mo by loss of BMP signaling in osteoblasts using Og2-Cre mice, but it is not consistent with the 3-mo mice, which showed decreased bone mass. The bone phenotype observed in Bmpr1a - deficient mice with Og2-Cre was milder than that with Col1-CreER TM . These discrepancies may be caused by differences in the timing of recombination between promoters. Aubin described characteristics of osteoblasts that change along with their maturation; thus, the effects of disrupting BMPRIA signaling may be influenced by the stages of osteoblastic maturation. In Col1-CreER TM mice, Cre activity was detected in immature periosteum wrapping growth plates, in osteogenic centers, and in bone collars during embryonic stages (data not shown). Therefore, Col1-CreER TM mice can induce recombination earlier in osteoblastogenesis, including in pre-osteoblasts, compared with Og2-Cre mice, which could explain why the Bmpr1a - deficient mice using Og2-Cre mice did not change expression levels of early osteogenic markers Runx2 and Bsp . These in vivo experiments taken together support in vitro evidence and imply that the response of osteoblasts to BMP signaling differs both by age and differentiation stage. Further studies of embryonic and older mice using Col1-CreER TM mice and comparisons of the bone phenotype in Bmpr1a deficiency among different osteoblast-specific Cre mice are desired to address the diverse function of BMP signaling.
BMP signaling and osteoclastogenesis.
Mouse genetic models using BMP ligands and antagonists instead of receptors were recently reported. Overexpression of Bmp4 in osteoblasts reduced bone mass at E18.5 with upregulation of osteoclastogenesis. Overexpression of Noggin , an antagonist of BMP2 and BMP4, in osteoblasts increased bone mass with reduced osteoclast number (N. Oc/BS) and osteoclastogenesis both at E17.5 and 3 wk. These studies are consistent with our findings, because BMP2 and BMP4 are potent agonists of BMPRIA. Taken together with our results, these studies suggest that BMP signaling in osteoblasts negatively regulates bone mass by upregulating osteoclastogenesis.
Reduction in osteoclast number (Fig. 5C) and expression levels of functional markers ( Mmp9 , Ctsk , and Tracp ) (Fig. 6B) showed downregulated osteoclastogenesis in cKO mice, which may direct rib flaring (Fig. 2). The reduction of osteoclastogenesis in cKO mice is presumably caused by the defect in osteoblasts, because the 3.2-kb type I collagen promoter directed Cre recombination in osteoblasts but not osteoclasts (Fig. 1F). Osteoblasts regulate osteoclast by expressing RANKL, an osteoclast differentiation factor, and OPG, a decoy receptor for RANKL. The link between BMP signaling and the RANKL–OPG pathway has not been studied in vivo, because this signaling was unchanged in mice overexpressing Noggin or Bmp4 in osteoblasts using the 2.3-kb type I collagen promoter and knocking out BMPRIA using osteocalcin promoter ( Og2-Cre mice). This study is the first in vivo evidence showing that loss of BMP signaling reduces osteoclastogenesis by downregulating RANKL and upregulating OPG. Because the 2.3-kb type I collagen and osteocalcin promoter are first activated in mature osteoblasts, it is possible that immature osteoblastic stages, where the 3.2-kb type I collagen promoter is active, are crucial for regulating osteoclastogenesis through the RANKL–OPG pathway.
BMP signaling, bone formation, and mineralization.
Bone formation rate (BFR/BS) in femoral trabecular bone was reduced in 22-wk mice by loss of function of BMPRIA in osteoblasts (Fig. 5A), consistent with the previous report. Overexpression of Noggin in osteoblasts also reduced the formation rate at P21. These facts suggest that BMP signaling is critical for bone formation in vivo, which supports in vitro evidence that BMPs induce osteogenesis. However, in Bmpr1a cKO mice, the reduction of formation rate in femora was more severe than that in vertebrae, and mineral apposition rate (MAR) was reduced in femora but not in vertebrae. These results suggest that the effect BMP signaling has on bone formation may differ in a site-specific manner in vivo, which can explain variations in changes in BMD and volume in cKO bones as determined by X-ray, DXA, and μCT. Further study is necessary to understand the mechanism of the site specificity in vivo, which would be difficult to assess in vitro.
In addition, changes noted in the cortices were not consistent with findings in the trabeculae. One of the major contextual differences that could contribute to the unique osteotropic response to BMP signaling in the trabeculae compared with other sites such as cortical bone, perichondrium, and skeletal muscle (ectopic) is the immediate juxtaposition of trabecular osteoblasts to the hematopoietic lineage. The 3.2-kb promoter used in Col1-CreER TM mice is active in pre-osteoblasts, osteoblasts, periosteum, and osteocytes widely throughout all bones examined. Site-specific promoters for Cre recombination could be developed to answer these questions.
BMD as determined by X-ray, DXA, and μCT was increased in cKO ribs, vertebrae, and tails at 22 wk (Figs. 2–4). These results indicate that loss of BMP signaling enhances bone mineralization. Interestingly, two in vitro studies have data that support our findings. One showed an increase in mineralization by overexpressing truncated BMPRIA without a kinase domain to block BMP signaling. Another showed an inhibition of mineralization by adding exogenous BMP2 into culture. Neither interpreted these results in their discussions. However, these facts suggest that BMP signaling negatively regulates mineralization in vivo. Another possibility is that loss of BMPRIA signaling causes a decrease in bone resorption. The existing matrix ages and continues to mineralize, similar to the proposed mechanism for the increase in BMD observed in humans on bisphosphonate therapy. In addition, serum levels of osteocalcin increased (data not shown), indicating an increased number of osteoblast and osteocytes, which is consistent with increased bone mass in Bmpr1a cKO mice. This fact may also contribute to increased mineralization in cKO bones. Further study is necessary to address the mechanism by which BMP signaling controls bone mineralization.
BMP signaling, endogenous bone, and ectopic bone.
Osteoblasts and chondrocytes both affect bone mass and are the predominant cell types in bone and cartilage, respectively. However, the difference in molecular mechanisms by which BMP signaling regulates these cell types to control bone mass is largely unknown, partly because of a lack of appropriate mouse genetic models. In this study, we found that osteoblasts lacking Bmpr1a fail to support osteoclastogenesis, leading to an increase in endogenous bone mass. Thus, we propose that BMP signaling in osteoblasts has dual functions to balance bone mass; it enhances bone formation by osteoblasts and enhances bone resorption by supporting osteoclastogenesis. However, studies focusing on BMP receptors in chondrocytes suggest that these cells respond to BMP signaling by increasing bone mass during the endochondral formation process. Mice overexpressing Bmp4 in mesenchymal cells and chondrocytes increase bone mass, whereas overexpression of Noggin in those cells decreases bone mass. In addition, recent mouse studies in which Cre expression is driven by a promoter in mesenchymal cells and chondrocytes ( Prx1-Cre ) showed that the disruption of BMP ligands in these cells impaired osteogenesis, resulting in reduced bone mass, and blocked fracture healing, which follows endochondral bone formation. Taken together, we propose that BMP signaling in chondrocytes increases bone mass but BMP signaling in osteoblasts restrains endogenous bone mass.
BMPs given subcutaneously induce ectopic bone formation. Human genetics showed that enhanced BMP signaling by a point mutation in ACVR1 , another BMP type I receptor, causes fibrodysplasia ossificans progressiva (FOP) with ectopic bones in muscle. These ectopic bones in muscle may form primarily because of the effect of BMP signaling on chondrocytes as we discussed above, because ectopic bone formation follows an endochondral formation pattern. The FDA approved BMP2 and BMP7 for clinical use to improve fracture healing. However, our finding of increased bone mass by loss of BMPRIA is unexpected and contrary to the rationale behind clinical applications of BMPs in orthopedics. Current clinical data supporting their use is underdeveloped ; this may be in part because of a lack of understanding of the variable effects BMPs have on different cell types in the skeleton including chondrocytes, osteoblasts, and osteoclasts. In addition, species differences might also play a role in the lack of efficacy in human clinical trials. Whereas much of the in vitro and in vivo analyses have been done on mouse cells and mouse models, demonstration of the effects of BMPs on human cells in vitro are modest because of species differences.
Future work will focus on the mechanisms by which loss of BMPRIA signaling in osteoblasts impacts remodeling. This may best be performed in older animals, perhaps challenged with ovariectomy, orchiectomy, fracture, or immobilization.
In conclusion, this study shows that loss of BMP signaling by BMPRIA directs osteoblasts to increase bone mass primarily in trabecular bone in part by downregulating osteoclastogenesis through the RANKL–OPG pathway. In osteoblasts, BMPRIA is a signaling molecule that directs both bone formation and resorption to reduce endogenous bone mass in vivo.
Agradecimentos.
We thank Dr Douglas J Adams for μCT measurement, Dr Gloria A Gronowicz and Felicia Ledgard for bone histomorphometry, Toni Ward for mouse dosing, Gregory Travlos and Bridget Garner for serum biochemistry, and Naomasa Keiko, Mikiko Kamiya, and Dr Katsuji Shimizu for encouragement. This work was supported by NIH Grants P01 DK56246 (to HK), R01 AR051587 (to JF), R21 AR052824 (to MY), and the Intramural Research Program of the NIEHS, NIH (to YM, ES071003-10). The NIEHS Fellowship in Environmental Medicine supported DL. The Lilly Fellowship Foundation supported NK.
Informações do artigo.
Formato disponível.
Direitos autorais e cópia; 2008 ASBMR.
bone morphogenetic protein; knockout; modeling and remodeling; osteoblasts; rodent.
História da publicação.
Issue online: 4 December 2009 Version of record online: 4 August 2008 Manuscript Accepted: 31 July 2008 Manuscript Revised: 29 May 2008 Manuscript Received: 26 February 2008.
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