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  • 1
    ISSN: 0730-2312
    Keywords: architectural transcription factor ; nuclear matrix ; osteoblast ; parathyroid hormone ; type I collagen ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In connective tissue, cell structure contributes to type I collagen expression. Differences in osteoblast microarchitecture may account for the two distinct cis elements regulating basal expression, in vivo and in vitro, of the rat type I collagen α1(I) polypeptide chain (COL1A1). The COL1A1 promoter conformation may be the penultimate culmination of osteoblast structure. Architectural transcription factors bind to the minor groove of AT-rich DNA and bend it, altering interactions between other trans-acting proteins. Similarly, nuclear matrix (NM) proteins bind to the minor groove of AT-rich matrix-attachment regions, regulating transcription by altering DNA structure. We propose that osteoblast NM architectural transcription factors link cell structure to promoter geometry and COL1A1 transcription. Our objective was to identify potential osteoblast NM architectural transcription factors near the in vitro and in vivo regulatory regions of the rat COL1A1 promoter. Nuclear protein-promoter interactions were analyzed by gel shift analysis and related techniques. NM extracts were derived from rat osteosarcoma cells and from rat bone. The NM protein, NMP4, and a soluble nuclear protein, NP, both bound to two homologous poly(dT) elements within the COL1A1 in vitro regulatory region and proximal to the in vivo regulatory element. These proteins bound within the minor groove and bent the DNA. Parathyroid hormone increased NP/NMP4 binding to both poly(dT) elements and decreased COL1A1 mRNA in the osteosarcoma cells. NP/NMP4-COL1A1 promoter interactions may represent a molecular pathway by which osteoblast structure is coupled to COL1A1 expression. J. Cell. Biochem. 69:336-352. © 1998 Wiley-Liss, Inc.
    Additional Material: 12 Ill.
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  • 2
    ISSN: 0730-2312
    Keywords: mechanical loading ; gene expression ; osteopontin ; myeloperoxidase ; rats ; differential display ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function. J. Cell. Biochem. 68:355-365, 1998. © 1998 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 67 (1997), S. 451-465 
    ISSN: 0730-2312
    Keywords: bone ; differentiation ; nuclear matrix ; osteoblast ; topoisomerase II ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: 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.
    Additional Material: 6 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 63 (1996), S. 374-383 
    ISSN: 0730-2312
    Keywords: tissue matrix ; primary spongiosa ; PTH-induced downregulation ; topoisomerase ; NuMA ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Parathyroid hormone (PTH) alters osteoblast morphology. How these changes in cell shape modify nuclear structure and ultimately gene expression is not known. Chronic exposure to rat PTH (1-34) [10 nM] attenuated the expression of 200, 190, and 160 kD proteins in the nuclear matrix-intermediate filament subfraction of the rat osteosarcoma cells, ROS 17/2.8 [Bidwell et al. (1994b): Endocrinology 134:1738-1744]. Here, we determined that these same PTH-responsive proteins were expressed in rat metaphyseal osteoblasts. We identified the 200 kD protein as a non-muscle myosin. Although the molecular weights, subcellular distribution, and half-lives of the 190 and 160 kD proteins were similar to topoisomerase II-α and -β, nuclear matrix enzymes that mediate DNA topology, the 190 and 160 kD proteins did not interact with topoisomerase antibodies. Nevertheless, the expression of topoisomerase II-α, and NuMA, a component of the nuclear core filaments, was also regulated by PTH in the osteosarcoma cells. The 190 kD protein was selectively expressed in bone cells as it was not observed in OK opossum kidney cells, H4 hepatoma cells, or NIH3T3 cells. PTH attenuated mRNA expression of the PTH receptor in our cell preparations. These results demonstrate that PTH selectively alters the expression of osteoblast membrane, cytoskeletal, and nucleoskeletal proteins. Topoisomerase II-α, NuMA, and the 190 and 160 kD proteins may direct the nuclear PTH signalling pathways to the target genes and play a structural role in osteoblast gene expression. © 1996 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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