ABSTRACT
Osteoporosis is caused by decreased bone mineral density (BMD) and new treatments for this disease are desperately needed. Bone morphogenetic protein 2 (BMP2) is crucial for bone formation. The mimetic peptide CK2.3 acts downstream of BMP2 and increases BMD when injected systemically into the tail vein of mice. However, the most effective dosage needed to induce BMD in humans is unknown. We developed a mathematical model for CK2.3-dependent bone mineralization. We used a physiologically based pharmacokinetic (PBPK) model to derive the CK2.3 concentration needed to increase BMD. Based on our results, the ideal dose of CK2.3 for a healthy individual to achieve the maximum increase of mineralization was about 409 µM injected in 500 µL volume, while dosage for osteoporosis patients was about 990 µM. This model showed that CK2.3 could increase the average area of bone mineralization in patients and in healthy adults.
Subject(s)
Bone Density/drug effects , Bone Morphogenetic Protein 2/administration & dosage , Models, Biological , Models, Theoretical , Osteoporosis/drug therapy , Peptide Fragments/administration & dosage , Adult , Bone Density/physiology , Humans , Osteoporosis/metabolism , Treatment OutcomeABSTRACT
Heterotopic ossification is a common complication after total hip replacement. Clinical studies showed the effectiveness of radiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to study an analogue model showing a time- and dose-dependent effect of radiation. Using cells of the defined embryonic mouse cell line C2C12, the influence of ionizing radiation on the Bmp-induced signal cascade leading to osteogenic differentiation was analyzed. Binding of iodinated Bmp2 to the receptors, Smad1 activation, and alkaline phosphatase (ALP) activity were determined in cells with or without irradiation. The cytotoxic effect of radiotherapy was evaluated using viability tests. Radiotherapy reduced formation of the Bmp2/Bmp receptor complex. This effect was dependent on dose. The phosphorylation (activation) of Smad1 decreased after irradiation in a time-dependent manner, whereas the level of total Smads was not influenced by radiotherapy. The ALP activity decreased after radiotherapy. A dose of 7 Gy delivered 6 h before or after incubation with Bmp resulted in about a 30% decrease in ALP activity. No signs of cytotoxic effects were observed within the time window studied using doses of 0 to 20 Gy. The time- and dose-dependent effect of radiotherapy for prevention of heterotopic ossification known from the results of clinical studies has an analogue in the C2C12 cell model. The primary mechanism of radiotherapy seems to be an influence on cellular responsiveness to the Bmp2-induced osteoblastic differentiation. The results suggest a down-regulation of the Bmp2/receptor complex.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Mesoderm/radiation effects , Signal Transduction/radiation effects , Transforming Growth Factor beta , Alkaline Phosphatase/metabolism , Animals , Blotting, Western , Bone Morphogenetic Protein 2 , Cell Division , Cell Line , Cobalt , Cross-Linking Reagents , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Radiation , Down-Regulation , Electrophoresis, Polyacrylamide Gel , Mice , Models, Biological , Ossification, Heterotopic , Osteoblasts/metabolism , Precipitin Tests , Smad Proteins , Smad1 Protein , Time Factors , Trans-Activators/metabolismABSTRACT
The bone morphogenetic proteins (BMPs) play important roles in embryogenesis and normal cell growth. The BMP receptors belong to the family of serine/threonine kinase receptors, whose activation has been investigated intensively for the transforming growth factor-beta (TGF-beta) receptor subfamily. However, the interactions between the BMP receptors, the composition of the active receptor complex, and the role of the ligand in its formation have not yet been investigated and were usually assumed to follow the same pattern as the TGF-beta receptors. Here we demonstrate that the oligomerization pattern of the BMP receptors is different and is more flexible and susceptible to modulation by ligand. Using several complementary approaches, we investigated the formation of homomeric and heteromeric complexes between the two known BMP type I receptors (BR-Ia and BR-Ib) and the BMP type II receptor (BR-II). Coimmunoprecipitation studies detected the formation of heteromeric and homomeric complexes among all the BMP receptor types even in the absence of ligand. These complexes were also detected at the cell surface after BMP-2 binding and cross-linking. Using antibody-mediated immunofluorescence copatching of epitope-tagged receptors, we provide evidence in live cells for preexisting heteromeric (BR-II/BR-Ia and BR-II/BR-Ib) and homomeric (BR-II/BR-II, BR-Ia/ BR-Ia, BR-Ib/ BR-Ib, and also BR-Ia/ BR-Ib) oligomers in the absence of ligand. BMP-2 binding significantly increased hetero- and homo-oligomerization (except for the BR-II homo-oligomer, which binds ligand poorly in the absence of BR-I). In contrast to previous observations on TGF-beta receptors, which were found to be fully homodimeric in the absence of ligand, the BMP receptors show a much more flexible oligomerization pattern. This novel feature in the oligomerization mode of the BMP receptors allows higher variety and flexibility in their responses to various ligands as compared with the TGF-beta receptors.
