Guanine nucleotide binding proteins (G proteins) are key regulators of signal transduction that couple information from a myriad of hormonal and physical stimuli to a more limited number of intracellular pathways. Recently, activating mutations of Gs-alpha have been described in disorders associated with skeletal lesions of fibrous dysplasia. In fibrous dysplasia, differentiation of osteoblasts from their mesenchymal precursors appears to be deficient, impairing bone formation. Conversely, in Albright's Hereditary Osteodystrophy, a disorder of Gs-alpha deficiency, the opposite effect on bone is seen. Bone formation occurs prematurely in the epiphyses of these patients and heterotopic bone formation is observed. These findings suggest that G proteins perform an important function in regulating the course of mesenchymal differentiation toward the osteoblast phenotype. Here the investigators hypothesize that the G protein isoforms Gs-alpha and Gi2-alpha act as pivotal regulators of osteoblast differentiation, Gs-alpha as an inhibitor and Gi2-alpha as a stimulator. In support of this hypothesis, they present preliminary data that inhibition of Gs-alpha expression with antisense oligonucleotides promotes differentiation of pre-osteoblasts to osteoblasts and stimulates membranous bone formation in vitro. The application's Specific Aims to test this hypothesis are to determine the effect of perturbation of Gs-alpha and Gi2-alpha activity on the differentiation of osteoblasts in vitro with: 1) antisense oligonucleotides and antisense adenovirus vectors that inhibit the expression of Gs-alpha or Gi2-alpha; 2) infection of fetal calvaria pre-osteoblasts with adenovirus vectors expressing constitutively active Gs-alpha or Gi2-alpha; 3) to show that pre-osteoblasts from Gs-alpha gene knock-out mice (in a mouse line already available) exhibit accelerated in vitro differentiation relative to those of wild type littermates; and 4) to determine if intracellular cAMP mediates Gs-alpha and Gi2-alpha effects on osteoblast differentiation.
These Aims are to be accomplished using the diploid fetal calvaria cell culture model, antisense techniques with hybrid phosphorothioate oligonucleotides, transient transfections with high efficiency adenovirus expression vectors, and treatment of fetal calvaria cells with long-acting stimulatory or inhibitory cAMP analogues. Osteoblastic differentiation will be determined by measuring skeletal-specific gene expression, formation of mineralized bone nodules in culture, and secretion of osteoblast-specific osteocalcin. This project is intended to identify an important therapeutic target for the treatment of bone disease.
| Gilbert, L; He, X; Farmer, P et al. (2000) Inhibition of osteoblast differentiation by tumor necrosis factor-alpha. Endocrinology 141:3956-64 |
| Farmer, P K; He, X; Schmitz, M L et al. (2000) Inhibitory effect of NF-kappaB on 1,25-dihydroxyvitamin D(3) and retinoid X receptor function. Am J Physiol Endocrinol Metab 279:E213-20 |
| Fernandez-Martin, J L; Kurian, S; Farmer, P et al. (1998) Tumor necrosis factor activates a nuclear inhibitor of vitamin D and retinoid-X receptors. Mol Cell Endocrinol 141:65-72 |
