In vitro and in vivo synergistic interactions between the Runx2/Cbfa1 transcription factor and bone morphogenetic protein-2 in stimulating osteoblast differentiation - PubMed (original) (raw)

In vitro and in vivo synergistic interactions between the Runx2/Cbfa1 transcription factor and bone morphogenetic protein-2 in stimulating osteoblast differentiation

Shuying Yang et al. J Bone Miner Res. 2003 Apr.

Abstract

Bone regeneration requires interactions between a number of factors including bone morphogenetic proteins (BMPs), growth factors, and transcriptional regulators such as Runx2/Cbfal (Runx2). Because each component may provide a unique contribution to the overall osteogenic response, we hypothesized that bone formation may be enhanced by using combinations of complimentary factors. As an initial test of this concept, interactions between BMP2 and Runx2 were examined using adenovirus-based expression vectors (AdCMV-Runx2, AdCMV-BMP2) in the pluripotent C3H10T1/2 cell line. Cells transduced with AdCMV-Runx2 strongly expressed osteoblast markers, such as alkaline phosphatase and osteocalcin, but formed only a weakly mineralized extracellular matrix in vitro, whereas cells transduced with AdCMV-BMP2 exhibited higher levels of mineralization, but only expressed low levels of Runx2 and osteocalcin mRNA. Significantly, when cells were transduced with optimal titers of both viruses, osteoblast differentiation was stimulated to levels that were 10-fold greater than those seen with either AdCMV-Runx2 or AdCMV-BMP2 alone. To measure in vivo osteogenic activity, virally transduced cells were subcutaneously implanted into immunodeficient mice. Cells transduced with control virus produced only fibrous tissue while those with AdCMV-Runx2 produced limited amounts of both cartilage and bone. In contrast, cells transduced with either AdCMV-BMP2 alone or AdCMV-BMP2 plus AdCMV-Cbfal generated large ossicles containing cartilage, bone, and a marrow cavity. However, ossification in the AdCMV-BMP2 plus AdCMV-Cbfal group was more extensive in that both mineral content and fractional bone area were greater than that seen in the AdCMV-BMP2 group. Thus, the increased osteoblast differentiation observed with combined adenovirus treatment in vitro is also manifested by increased bone formation in vivo. These results suggest that Runx2 and BMP2 have distinct, but complementary, roles in osteogenesis and that their combined actions may be necessary for optimal bone formation.

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Figures

FIG. 1

FIG. 1

Effect of adenovirus titer on Runx2 expression and induction of osteoblast differentiation in C3H10T1/2 cells. C3H10T1/2 cells were transduced with the indicated titer of AdCMV-LacZ (AdLacZ) or AdCMV-Runx2 (AdRunx2) as described in the Materials and Methods section. After 24 h, cells were fed with complete medium containing 10% FBS, 50 _μ_g/ml ascorbic acid, and 5 mM _β_-glycerol phosphate. (A) On day 3 after viral transduction, Runx2 protein levels were measured in whole cell extracts by Western blotting. (B) On day 6, cells were harvested for measurement of ALP activity. (C) A separate set of cultures was stained for mineralization by method of von Kossa after 12 days in culture.

FIG. 2

FIG. 2

Time course of osteoblast differentiation in AdRunx2-transduced C3H10T1/2 cells. Cultures were transduced with AdLacZ (~) or AdRunx2 (r,p) at a titer of 250 pfu/cell. After 24 h, one-half the cells in each group were fed with basal medium (_α_-MEM, 10% FBS, open symbols) and one-half were fed with basal medium supplemented with 50 _μ_g/ml ascorbic acid (closed symbols). Cells were harvested at the times indicated and assayed for (A) ALP activity, (B) mRNA levels, or (C) Runx2 protein levels. (D–G) These panels show mRNA levels after imaging and normalization to 18S rRNA.

FIG. 3

FIG. 3

Effect of Runx2 expression on AdBMP2-dependent induction of ALP and mineralization. Cells were transduced with the indicated titer of AdCMV-BMP2 in the presence (▴) or absence (●) of AdRunx2 at a titer of 100 pfu/cell. Total viral titer was held constant at 250 pfu/cell by addition of the appropriate titer of AdCMV-lac Z control virus. The cells were harvested (A) at day 6 for ALP assays or (B) at day 9 for measurement of mineralization by von Kossa staining.

FIG. 4

FIG. 4

Time course of osteoblast differentiation in C3H10T1/2 cells transduced with AdBMP2 in the presence or absence of AdRunx2. Cells were transduced with the following combinations of adenovirus vectors: AdLacZ alone (200 pfu/cell), ●; AdLacZ (100 pfu/cell) plus AdBMP2 (100 pfu/cell), ●; AdLacZ (100 pfu/cell) plus AdRunx2 (100 pfu/cell), ▵; and AdBMP2 (100 pfu/cell) plus AdRunx2 (100 pfu/cell), ▴. Cells were harvested at the times indicated for measurement of (A) ALP activity, (B) calcium, (C) osteoblast marker mRNA expression, or (D) total Runx2 or BMP2 protein. Normalized mRNA levels are shown for (E) Runx2 mRNA and (F) OCN mRNA. (G) Replicate plates of cells were also stained for mineral by the method of von Kossa.

FIG. 5

FIG. 5

In vivo bone formation by virally-transduced C3H10T1/2 cells. Cells were transduced with indicated adenoviruses and implanted into immunodeficient mice as described in Methods. After 4 weeks, transplants were harvested for determination of (A) total wet weigh, (B) ALP activity, or (C) calcium. (D) A morphometric analysis of histological sections from each treatment group. Results are expressed as the ratio of total bone area/total implant area. Statistical analysis: a, significantly different from AdLacZ (p < 0.001); b, significantly different from AdBMP2 (p < 0.05). (E) Histological sections of each treatment group. Implants of cells transduced with control virus contained residual Gelfoam carrier (g) and fibrous tissue (f), but no bone or cartilage. Cells transduced with AdRunx2 alone contained small areas of both bone (b) and cartilage (c) as well as a small marrow cavity (m). Both BMP2 alone and BMP2 plus Runx2 treated groups formed large ossicles with clearly defined cortical and trabecular bone as well as a marrow cavity.

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