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First published online May 28, 2004
doi: 10.1242/10.1242/jcs.01133

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Targeted expression of a dominant-negative N-cadherin in vivo delays peak bone mass and increases adipogenesis

Charlles H. M. Castro^1,3,*, Chan Soo Shin^1,4,*, Joseph P. Stains¹, Su-Li Cheng¹, Sharmin Sheikh¹, Gabriel Mbalaviele^1,5, Vera Lucia Szejnfeld³ and Roberto Civitelli^1,2,

¹ Division of Bone and Mineral Diseases, Department of Internal Medicine, Washington University School of Medicine, 216 S. Kingshighway Blvd, St Louis, MO 63110, USA
² Department of Cell Biology and Physiology, Washington University School of Medicine, 216 S. Kingshighway Blvd, St Louis, MO 63110, USA
³ Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua Botucatu 740, São Paulo-SP, Brasil
⁴ Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-774, Republic of Korea
⁵ Pfizer Incorporated, 700 Chesterfield Parkway West, Chesterfield, MO 63107, USA

View larger version (42K): [in a new window]   Fig. 1. Development of NcadC transgenic mice. (A) Linearized structure of the OG2-NcadC construct used for transgenic expression. The 1.1 kb PstI fragment was used as a probe in Southern analysis, and the two arrows identify the primers used for PCR genotyping. Both the cDNA probe and the 250 kb PCR product encompass the junction between the OG2 promoter and the 5' end of the NcadC reading frame, thus making them specific for the transgene. (B) Southern blot (upper panel) and PCR (lower panel) of genomic DNA of one mouse litter showing a 1.1 kb band or a 250 bp product, respectively, corresponding to the transgene. (C) Detection of transgene expression by reverse transcription and PCR of total RNA extracted from tissues of 2-month-old transgenic animals, showing a 195 bp band corresponding to NcadC mRNA only in extracts from calvaria and femurs, but not from other tissues. Control reactions were run either in the absence of reverse transcriptase (No RT) or in the presence of NcadC plasmid, as indicated. Amplification of GAPDH was used to control for mRNA integrity and PCR efficiency. (D) Total protein extracts of mouse calvaria isolated from either OG2-NcadC (Tg) or wild-type (WT) animals were immunoprecipitated (IP) using the PEP.1 antibody that recognizes the intracellular tail of Xenopus N-cadherin. Proteins in the whole-cell lysate (input) and in the immunoprecipitate (beads) were separated by SDS-PAGE and blotted (WB) using an anti-FLAG antibody. A band of 45 kDa, corresponding to the truncated NcadC, was detected in the immunoprecipitate from transgenic animals, but not from wild-type littermates. The higher molecular weight bands represent IgG heavy chain.

View larger version (14K): [in a new window]   Fig. 2. Low bone mass and high body fat in OG2-NcadC mice. Bone density and body composition were analyzed by dual energy X-ray absorptiometry in live animals at different times up to 4 months. (A) Total body bone mineral density (BMD) was significantly reduced in OG2-NcadC mice during the first 3 months of age–when BMD accumulates–compared with wild-type littermates, whereas percent body fat (B) and body weight (C) were significantly higher in OG2-NcadC mice compared with wild-type littermates during the first 3 months of age. *P<0.05, two-way ANOVA. Error bars represent standard deviation.

View larger version (49K): [in a new window]   Fig. 3. Reduced bone formation in OG2-NcadC transgenic mice: histologic and histomorphometric analysis. (A) Goldner's trichrome stained, 4 µm undecalcified sections of proximal tibiae from OG2-NcadC (Tg) or wild-type (WT). Micrographs on the right are enlargements (400x) of the rectangular areas shown on the left micrographs (200x). (B) Bone volume, expressed as a percentage of total tissue volume (BV/TV) in the entire area of the bone marrow cavity 175-875 µm from the growth plates was significantly reduced in transgenic mice compared with those of wild-type controls (n=8). (C-E) OG2-NcadC mice also exhibited a significantly lower trabecular number and thickness and higher trabecular separation compared with wild-type mice. (F) Fluorescence micrograph of calcein labeling in 8 µm unstained sections of cancellous bone, showing no double labels in sections of transgenic mice bone. Accordingly (G,H), dynamic parameters of bone formation, bone formation rate/tissue volume and mineral apposition rate were significantly reduced in transgenic mice compared with wild-type littermates. (I) Four-micrometer sections of proximal tibiae were stained for tartrate resistant acid phosphatase activity and counterstained with methyl green and thionin for identification of osteoclasts (Oc) and osteoblasts (Ob). Micrographs on the right are enlargements (400x) of the rectangular areas shown on the left micrographs (200x). (J,K) The number of osteoblasts expressed as osteoblast perimeter per bone perimeter (%) was reduced in OG2-NcadC mice compared with wild-type mice. There was no significant difference in the number of osteoclasts between transgenic and wild-type animals. In all panels, asterisks denote P<0.05, t-test.

View larger version (31K): [in a new window]   Fig. 4. NcadC expression in vivo retards osteoblast differentiation. (A) Calvaria cells (CLVC) were cultured in 24-well plates up to 3 weeks after confluence, and alkaline phosphatase activity was measured in the cell layer at weekly intervals, and normalized to cellular protein content. Enzyme activity was reduced in CLVC from newborn OG2-NcadC mice compared with wild-type (WT) cells at 1 and 2 weeks post-confluence. *P<0.001, t-test (mean±s.e.m.). (B) Von Kossa staining of CLVC cultures isolated from newborn OG2-NcadC transgenic (Tg) or wild-type (WT) mice, after 2 or 3 weeks in culture in the presence of AA (50 µg/ml) and ß-GP (10 mM). There is no apparent difference in the mineralized (dark stain) area between the transgenic and wild-type cells at either time-point. Expression of the transgene NcadC was detected at the corresponding time-points by RT-PCR of total RNA extracted from parallel cultures, with GAPDH as control for RNA integrity and abundance (lower panels). (C) Total RNA was extracted from CLVC grown for 2 weeks after confluence. The amount of mRNA for osteoblast products was determined by real-time PCR and expressed as mRNA abundance in OG2-NcadC relative to wild-type (WT) cells. Type I collagen expression levels were increased 1.8 times in transgenic animals compared with wild-type controls, whereas osteoprotegerin, osteopontin, Cbfa1 and RANK-L mRNA abundance was significantly reduced. (D) Whole-cell lysates obtained from transgenic (Tg) and wild-type (WT) CLVC grown for 3 weeks post-confluence were separated by SDS-PAGE and blotted using antibodies that recognize the intracellular tail of Xenopus N-cadherin (PEP.1) or the N-terminus of N-cadherin (N19; Santa Cruz). Two bands of 100 and 120 kDa were detected in both cases, corresponding to endogenous N-cadherin and possibly other cross-reacting cadherins. A band of 45 kDa, corresponding to the truncated NcadC, was detected by the PEP.1 antibody only in CLVC from transgenic animals, but not from wild-type littermates (left panel). This membrane was stripped and blotted again with an anti-GAPDH antibody to control for loading.

View larger version (38K): [in a new window]   Fig. 5. Reduced osteoblastogenesis and increased adipogenesis of OG2-NcadC bone marrow cells. (A) Bone marrow stromal cells (BMSC) isolated from 2-month-old mice were cultured in 96-well plates in the presence of AA (50 µg/ml) and ß-GP (10 mM) for 4 weeks at low density (3.2x103 cell/cm2) for limiting dilution analysis of CFU-Osteoblast, determined by Von Kossa staining. Relative to wild-type cells (WT), there was a lower number of von Kassa-positive wells in BMSC cultures isolated from OG2-NcadC (Tg) mice. (B) BMSC from OG2-NcadC and wild-type mice were cultured in the presence of an adipogenic cocktail (0.5 mM IBMX, 60 µM indomethacin, and 0.5 µM hydrocortisone) for 2 weeks and the number of CFU-Adipocyte was determined by Oil red O staining. A higher abundance of lipid droplet-positive cells was detectable even in cultures seeded at high density in cells from transgenic mice (105 cells/cm2). Representative photomicrographs of wild-type and OG2-NcadC BMSC cultures stained with Oil Red O are shown on the right. (C) The number of CFU-Osteoblast was significantly lower, and the number of CFU-Adipocyte was significantly higher in transgenic compared with wild-type BMSC cultures, assessed by limiting dilution analysis (n=5); *P<0.05, t-test. (D) The abundance of PPAR-2, adipsin and Cbfa1 mRNA was assessed by real-time RT-PCR in BMSC cultures after 2 weeks of culture in the absence of stimulators. There was a significant increase in PPAR-2 and adipsin mRNA abundance in cells derived from OG2-NcadC animals compared with wild-type littermates, whereas Cbfa1 mRNA was reduced 40%. (E) Expression of the transgene, NcadC was detected after 2 weeks of culture by RT-PCR of total RNA extracted from parallel cultures, with GAPDH as control for RNA integrity and abundance (lower panels). No PCR products were obtained in the absence of reverse transcriptase (RT), thus excluding DNA contamination.

View larger version (31K): [in a new window]   Fig. 6. Altered ß-catenin subcellular distribution and effect of viral transduction of constitutively active ß-catenin in OG2-NcadC cells. (A) The cytosolic/membrane and nuclear fractions of calvaria cells (CLVC) isolated from newborn OG2-NcadC (Tg) or wild-type (WT) mice (left panel) or of bone marrow stromal cells (BMSC) isolated from 4-month-old mice (right panel) were separated as described in Materials and Methods, and blotted using an antibody that recognizes the cytoplasmic tail of N-cadherin. Note that OG2-NcadC-expressing cells exhibited more abundant ß-catenin in cytosol/membrane compartments and lower ß-catenin abundance in the nuclear fraction, relative to wild-type cells. The distribution of ß-catenin between cytosol/membrane and nuclear compartments is similar in transgenic and wild-type cells. (B) OG2-NcadC and wild-type CLVC were transduced with retroviral vectors expressing either the stable, constitutively active ß-catenin construct, N151 or LacZ, and cultured for 2 weeks before determination of alkaline phosphatase activity. Transduction of ß-catenin increased enzyme activity in OG2-NcadC CLVC to levels similar to those present in wild-type cells, whereas no effect was seen in either nontransduced cells or cells exposed to LacZ retrovirus. (C) OG2-NcadC and wild-type BMSC were transduced with the N151 or LacZ retroviral vectors, as just described, and grown for 2 weeks for CFU-Adipocyte determination. Transduction of ß-catenin reduced the number of CFU-Adipocyte in OG2-NcadC BMSC to levels similar to those present in wild type cells, while transduction of LacZ was ineffective; *P<0.05, t-test (mean±s.e.m.). All data are representative or the average of at least three different experiments.

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