QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 16 October 2008
doi: 10.1242/dev.025825

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kamiya, N. Articles by Mishina, Y. Search for Related Content PubMed PubMed Citation Articles by Kamiya, N. Articles by Mishina, Y. Social Bookmarking                         What's this?

BMP signaling negatively regulates bone mass through sclerostin by inhibiting the canonical Wnt pathway

¹ Laboratory of Reproductive and Developmental Toxicology, NIEHS/NIH, Research Triangle Park, NC 27709, USA.
² School of Dentistry, University Michigan, Ann Arbor, MI 48109, USA.
³ School of Dentistry, University of Missouri-Kansas City, Kansas City, MO 64108, USA.
⁴ Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
⁵ Dental Research Center, University of North Carolina, Chapel Hill, NC 27599, USA.

View larger version (57K): [in this window] [in a new window]   Fig. 1. Tamoxifen (TM)-dependent and osteoblast-specific Cre activity using Cre-ERTM mice. (A) Cre-ERTM transgenic mice mated with ROSA26 Cre reporter mice (R26R). TM was injected daily into pregnant females for 3 days from E13.5 to E15.5. Cre-negative and -positive littermates were stained for β-gal at E18.5. (B) Cre activity in long bones. Forelimbs were stained for β-gal at E18.5. C, chondrocytes; black arrow, periosteum; red arrow, osteogenic center. Scale bars: 50 µm. (C) Cre recombination in calvariae at E15.5, E16.5 and E18.5 in whole head (lateral and overhead views) and histology of E18.5 calvariae. Broken line, areas of parietal bones (P). Scale bar: 20 µm. (D) BMPR1A as evaluated by immunohistochemistry using cKO calvariae at E18.5. BMPR1A, green; DAPI (nuclei), blue. Scale bars: 10 µm. (E) Immunoblotting for phosphorylated Smad 1/5/8 using E18.5 calvariae. The membrane was treated with polyclonal rabbit anti-phospho-Smad1/5/8 (1:1000) and monoclonal mouse anti-beta-actin (1:2000), and visualized by ECL.

View larger version (70K): [in this window] [in a new window]   Fig. 2. Increased bone mass in Bmpr1a cKO mice. (A) Skeletal preparation of the skull and long bones at E18.5 using Alizarin Red/Alcian Blue staining. Broken line, areas of parietal bones (P). (B) Bone volume over total tissue volume (BV/TV) obtained from µCT analysis on parietal bones of the skull (left) and femur (right) at E18.5. Values are expressed as mean±s.d. (wild type, n=5; cKO, n=4, Student's t-test; *P<0.01). (C) Hematoxylin and Eosin staining of E18.5 calvariae. Boxed areas in frontal sections were magnified. Scale bars: 25 µm. (D) Von Kossa staining for Ca2+ using E18.5 calvariae. Scale bars: 25 µm. (E) Hematoxylin and Eosin staining of E18.5 humerus. Boxed areas in sagittal sections are magnified. Scale bars: 100 µm. (F) BrdU incorporation using E18.5 calvariae. BrdU-positive cells (arrows) per total cells in bone were unchanged. Scale bars: 25 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Alteration of bone formation and resorption in Bmpr1a cKO mice. (A) QRT-PCR for bone formation markers (Runx2, Sp7, Ibsp, Akp2 and Bglap2 using E16.5 and E18.5 calvariae). Values are expressed relative to wild type at E16.5. (B) QRT-PCR for bone resorption markers expressed by osteoclasts (Mmp9, Ctsk and Trap), and Rankl and Opg expressed by osteoblasts using E18.5 and E19.5 calvariae. Values are expressed relative to wild type at E18.5. (C) Relative ratio of Rankl to Opg expression levels calculated from Fig. 3B. Values are expressed relative to wild type at E18.5. (D) Evaluation of osteoclast activity by TRAP staining using E18.5 calvariae. The positive cells localized randomly in cKO calvariae compared with wild type (left two panels, red arrows). The percent of TRAP-positive cells per total cells in bone area detected by DAPI was significantly reduced in cKO calvariae (wild type, 13.9%; cKO, 6.4%, right panel). Scale bars: 50 µm. (E) QRT-PCR for BMP type I receptors (Bmpr1b, Acvr1), type II receptors (Bmpr2, Acvr2a and Acvr2b) and potential ligands for these receptors (Bmp2, Bmp4, Bmp6 and Bmp7) using E18.5 calvariae. Values in A-E represent mean±s.d. from a minimum of three independent experiments using wild-type and cKO bones. Student's t-test; *P<0.05.

View larger version (100K): [in this window] [in a new window]   Fig. 4. Upregulation of canonical Wnt signaling in Bmpr1a cKO mice. (A) Canonical Wnt signaling assessed by TOPGAL mice using E17.5 calvariae (upper panel, lateral view; lower panel, top view). Two independent parietal bones from two littermates in each group after dissection are shown in the right-hand panel. Red arrow indicates enhanced Wnt signaling. Broken line, areas of parietal bones (P). (B) Histological analysis assessed by TOPGAL mice using E17.5 calvariae. Scale bars: 50 µm. (C) Relative cell number of DAPI and β-gal positive cells. Cells were counted in 50 fields of E17.5 calvariae from cKO (n=4) and wild type (n=4). Total cell number was obtained by counting DAPI-positive nuclei in bone. Cell number of wild type is set as 1.0. There were 2.3 times more total cells in cKO calvariae in a given section than in wild type owing to the thicker tissues, but the number of β-gal-positive cells was 19.7 times greater, resulting in an 8.5-fold increase in the proportion of β-gal positive cells in cKO mice. (D) Canonical Wnt signaling assessed by TOPGAL mice using humerus, ulna and radius at E17.5. Red arrow indicates enhanced Wnt signaling. (E) Histological analysis assessed by TOPGAL mice using radius at E17.5. Asterisks, growth plates. Scale bars: 100 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 5. Inhibition of canonical Wnt signaling by sclerostin in Bmpr1a cKO mice. (A) QRT-PCR analysis for Sost, Dkk1, Dkk2 and Lrp5 using calvariae at E16.5, E17.5 and E18.5. Values are expressed relative to wild type at E16.5. Student's t-test; *P<0.05. (B) QRT-PCR analysis for Wnt target gene Axin2 and Ctgf using E18.5 calvariae. Expression levels of Axin2 and Ctgf were significantly increased in cKO calvariae. Student's t-test; *P<0.05. (C) Immunohistochemical staining of sclerostin (brown) counterstained with Hematoxylin (blue) using E17.5 calvariae. Broken line, osteoblasts. Scale bars: 50 µm, left panel; 10 µm, right panel. (D) Detection of BMPR1A (green), canonical Wnt signaling (blue) and sclerostin (red) using E17.5 calvariae. Canonical Wnt signaling was assessed by β-gal staining using TOPGAL mice. Nuclei were stained with DAPI (blue). Broken line, osteoblasts. Scale bars: 20 µm. (E) Immunohistochemical staining for sclerostin (red) in primary osteoblasts from Bmpr1a cKO and wild-type control. Nuclei were stained with DAPI (blue). Scale bars: 20 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 6. Suppressed expression of sclerostin by loss of BMP signaling ex vivo. (A) Newborn mouse calvariae were cultured for 5 days treated with 4OH-TM (100 ng/ml). (a) Confirmation of Cre activity in the calvariae from CreER:R26R mice assessed by β-gal staining. (b) Upregulation of canonical Wnt signaling in the calvariae from CreER:Bmpr1afx/fx:TOPGAL mice assessed by β-gal staining. Broken lines, areas of parietal bones (P). (c) Expression of Wnt inhibitors Sost, Dkk1, Dkk2 and Lrp5 assessed by QRT-PCR. (d) Expressions of bone resorption markers Mmp9, Ctsk, TRAP and Bmpr1a. Values are expressed relative to those of wild type (Cre-, TM-). (B) Sclerostin treatment of wild-type (WT+Scl) and cKO (cKO+Scl) calvariae ex vivo. The ratio of β-gal to DAPI-positive cells was evaluated from 50 fields in frontal sections (n=3, in each condition). Broken line, parietal bones. Scale bars: 50 µm. (C) Expression of bone resorption markers Rankl and Opg, and relative ratio of Rankl to Opg using wild-type and cKO calvariae in sclerostin treated versus untreated groups. Values are expressed relative to those of wild type without sclerostin treatment. (D) TRAP staining of cKO calvariae treated with sclerostin and non-treated ex vivo. Values in A-C represent mean±s.d. from three independent experiments. Student's t-test; *P<0.05.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Enhanced BMPR1A signaling upregulates both sclerostin and osteoclastogenesis. (A) Constitutively active Bmpr1a (caBmpr1a) mouse fetuses at E18.5 induced daily by TM injection from E13.5. Hematoxylin and Eosin staining showed moderately reduced thickness in the caBmpr1a calvariae (Cre+, caBmpr1a+) where levels of phosphorylated Smad1/5/8 (brown) were enhanced compared with littermate controls (Cre-, caBmpr1a+). Scale bars: 50 µm. (B) Expressions of Sost and the bone resorption markers Rankl and Opg assessed by QRT-PCR using caBmpr1a and control calvariae at E18.5. Values are expressed relative to control. (C) Relative ratio of Rankl to Opg calculated based on the expression levels in Fig. 7B. (D) Expressions of Sost, bone resorption markers, Rankl and Opg by QRT-PCR using rescued (black bar: Cre+, caBmpr1a+, Bmpr1a fx/fx) and littermate cKO (gray bar: Cre+, caBmpr1a-, Bmpr1a fx/fx) calvariae at E18.5. Values of rescued mice are expressed relative to littermate cKO mice. (E) Relative ratio of Rankl to Opg calculated based on expression levels in Fig. 7D. (F) Hematoxylin and Eosin staining of rescued and cKO calvariae at E18.5. Scale bars: 100 µm. Values in B-E are mean±s.d. from three independent experiments. Student's t-test; *P<0.05.

View larger version (24K): [in this window] [in a new window]   Fig. 8. A model of the relationship between BMPR1A and canonical Wnt signaling in mouse bone. BMPR1A signaling upregulates sclerostin expression, leading to an inhibition of canonical Wnt signaling and a decrease in bone mass by upregulating osteoclastogenesis through the RANKL-OPG pathway. Sclerostin, the SOST gene product, acts as a downstream effector of BMPR1A signaling, an inhibitor of canonical Wnt signaling and a bone mass-determining factor. Broken line indicates another possibility: that BMP signaling directly upregulates osteoclastogenesis through the RANKL-OPG pathway.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter