Changes in osteoblast, chondrocyte, and adipocyte lineages mediate the bone anabolic actions of PTH and small molecule GSK-3 inhibitor

Parathyroid hormone (PTH) and glycogen synthase kinase-3 (GSK-3) inhibitor 603281-31-8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281-31-8 in rat osteopenia model. Female 6-month-old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1-38) at 10 microg/kg/day s.c. or 603281-31-8 at 3 mg/kg/day p.o. for 60 days. Twenty-four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (P<0.05) were subjected to pathway analysis to delineate relevant bio-processes involved in skeletal biology. Genes involved in morphogenesis, cell growth/differentiation, and apoptosis were significantly altered by Ovx and the treatments. Analysis of morphogenesis genes showed an overrepresentation of genes involved in osteogenesis, chondrogenesis, and adipogenesis. A striking finding was that Ovx decreased several markers of osteogenesis/chondrogenesis and increased markers of adipogenesis/lipid metabolism. Treatment with either PTH or the GSK-3 inhibitor reversed these effects, albeit at different levels. Histological analysis confirmed that osteopenia in Ovx animals was associated with three-fold increase in marrow adiposity. PTH and GSK-3 inhibitor restored bone volume, and reversed or normalized marrow adiposity. Ex vivo studies showed that PTH and GSK-3 inhibitor increased the ratio of colony forming marrow stromal progenitors (CFU-fs) that were alkaline phosphatase positive (putative osteoblasts). Our results suggest that the bone anabolic actions of PTH and GSK-3 inhibitor in vivo involve concerted effects on mesenchymal lineages; osteoblasts, chondrocytes, and adipocytes.     

Topoisomerase II expression in osseous tissue

The molecular mechanisms that mediate the transition from an osteoprogenitor cell to a differentiated osteoblast are unknown. We propose that topoisomerase II (topo II) enzymes, nuclear proteins that mediate DNA topology, contribute to coordinating the loss of osteoprogenitor proliferative capacity with the onset of differentiation. The isoforms topo II-α and -β, are differentially expressed in nonosseous tissues. Topo II-α expression is cell cycle-dependent and upregulated during mitogenesis. Topo II-β is expressed throughout the cell cycle and upregulated when cells have plateaued in growth. To determine whether topo II-α and -β are expressed in normal bone, we analyzed rat lumbar vertebrae using immunohistochemical staining. In the tissue sections, topo II-α was expressed in the marrow cavity of the primary spongiosa. Mature osteoblasts along the trabecular surfaces did not express topo II-α, but were immunopositive for topo II-β, as were cells of the marrow cavity. Confocal laser scanning microscopy was used to determine the nuclear distribution of topo II in rat osteoblasts isolated from the metaphyseal distal femur and the rat osteosarcoma cells, ROS 17/2.8. Topo II-α exhibited a punctate nuclear distribution in the bone cells. Topo II-β was dispersed throughout the interior of the nucleus but concentrated at the nuclear envelope. Serum starvation of the cells attenuated topo II-α expression but did not modulate expression of the β-isoform. These results indicate that the loss of osteogenic proliferation correlates with the downregulation of topo II-α expression. J. Cell. Biochem. 67:451–465, 1997. © 1997 Wiley-Liss, Inc.     

Data-driven analysis approach for biomarker discovery using molecular-profiling technologies.

High-throughput molecular-profiling technologies provide rapid, efficient and systematic approaches to search for biomarkers. Supervised learning algorithms are naturally suited to analyse a large amount of data generated using these technologies in biomarker discovery efforts. The study demonstrates with two examples a data-driven analysis approach to analysis of large complicated datasets collected in high-throughput technologies in the context of biomarker discovery. The approach consists of two analytic steps: an initial unsupervised analysis to obtain accurate knowledge about sample clustering, followed by a second supervised analysis to identify a small set of putative biomarkers for further experimental characterization. By comparing the most widely applied clustering algorithms using a leukaemia DNA microarray dataset, it was established that principal component analysis-assisted projections of samples from a high-dimensional molecular feature space into a few low dimensional subspaces provides a more effective and accurate way to explore visually and identify data structures that confirm intended experimental effects based on expected group membership. A supervised analysis method, shrunken centroid algorithm, was chosen to take knowledge of sample clustering gained or confirmed by the first step of the analysis to identify a small set of molecules as candidate biomarkers for further experimentation. The approach was applied to two molecular-profiling studies. In the first study, PCA-assisted analysis of DNA microarray data revealed that discrete data structures exist in rat liver gene expression and correlated with blood clinical chemistry and liver pathological damage in response to a chemical toxicant diethylhexylphthalate, a peroxisome-proliferator-activator receptor agonist. Sixteen genes were then identified by shrunken centroid algorithm as the best candidate biomarkers for liver damage. Functional annotations of these genes revealed roles in acute phase response, lipid and fatty acid metabolism and they are functionally relevant to the observed toxicities. In the second study, 26 urine ions identified from a GC/MS spectrum, two of which were glucose fragment ions included as positive controls, showed robust changes with the development of diabetes in Zucker diabetic fatty rats. Further experiments are needed to define their chemical identities and establish functional relevancy to disease development.     

Parathyroid hormone (1-34) regulates integrin expression in vivo in rat osteoblasts.

Intermittent administration of parathyroid hormone (PTH) activates new sites of bone formation by stimulating osteoblast differentiation and function resulting in an increase in bone mass. Because integrins have been shown to play a crucial role in osteoblast differentiation and bone formation, in the present study, we evaluated whether human PTH (1-34) upon administration to rats, influenced integrin expression in osteoblastic cells isolated from the metaphysis and the diaphysis of rat long bones. Initial immunohistochemical evaluation of bone sections demonstrated that the osteoblasts expressed at least alphav, alpha2, alpha3, and alpha5beta1 integrins. Immunocolocalization studies for integrins and vinculin established that alphav, alpha2, and alpha5beta1, but not alpha3 integrins were present in the focal adhesion sites of osteoblasts attached to FN coated surfaces. Osteoprogenitor cells isolated from metaphyseal (but not diaphyseal) marrow of rats injected with intermittent PTH (1-34) exhibited greater alphav and reduced alpha2 levels, with no apparent changes in alpha3, and alpha5beta1 integrin levels, as assessed by immunohistochemistry, Northern, and Western blot analyses. However, these changes were not observed on the same cells treated with PTH in vitro. These observations suggest that integrin modulation by PTH is likely to be indirect and that selective phenotypic expression of integrin subtypes is part of the cascade of events that lead to PTH (1-34) mediated osteoblast differentiation.